38
ADRIA DEVELOPPEMENT Creac’h Gwen - F. 29196 QUIMPER Cedex - Tél. (33) 02.98.10.18.18 - Fax (33) 02.98.10.18.08 E-mail : [email protected] - Site web : http://www.adria.tm.fr ASSOCIATION LOI DE 1901 - N° SIRET 306 964 271 00036 - N° EXISTENCE 532900006329 - N°TVA FR4530696427100036 Oxoid Ltd, part of Thermo Fisher Scientific Wade Rd, Basingstoke, Hampshire RG24 8PW (UK) NF VALIDATION Validation of alternative analytical methods Application in food microbiology Summary report EN ISO validation study of the Listeria Precis TM method for the enumeration of Listeria monocytogenes in food products and environmental samples Quantitative method This report includes 38 pages, with 4 appendixes. Only copies including the totality of this report are authorised. Version 1 November 19, 2014 Cancel and replace the previous version which must be returned to ADRIA Développement or destroyed internally.

ÞÀ¿¸7 ú Y5zñLjÜ< /u +æü t¬Á ... - NF Validation · NF VALIDATION Validation of alternative analytical methods Application in food microbiology ... 500 – 1 000 1 000 –

  • Upload
    others

  • View
    3

  • Download
    0

Embed Size (px)

Citation preview

Page 1: ÞÀ¿¸7 ú Y5zñLjÜ< /u +æü t¬Á ... - NF Validation · NF VALIDATION Validation of alternative analytical methods Application in food microbiology ... 500 – 1 000 1 000 –

ADRIA DEVELOPPEMENT Creacrsquoh Gwen - F 29196 QUIMPER Cedex - Teacutel (33) 0298101818 - Fax (33) 0298101808

E-mail adriadeveloppementadriatmfr - Site web httpwwwadriatmfr

ASSOCIATION LOI DE 1901 - Ndeg SIRET 306 964 271 00036 - Ndeg EXISTENCE 532900006329 - NdegTVA FR4530696427100036

Oxoid Ltd part of

Thermo Fisher Scientific

Wade Rd Basingstoke

Hampshire RG24 8PW (UK)

NF VALIDATION

Validation of alternative analytical methods

Application in food microbiology

Summary report

EN ISO validation study of the

Listeria PrecisTM method for the

enumeration of Listeria monocytogenes in food products and environmental samples

Quantitative method

This report includes 38 pages with 4 appendixes

Only copies including the totality of this report are authorised

Version 1 November 19 2014

Cancel and replace the previous version

which must be returned to ADRIA Deacuteveloppement or destroyed internally

Thermo Fisher Scientific

ADRIA Deacuteveloppement 238 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

1 INTRODUCTION 4

11 Dates of the validation studies 4

12 Protocol and principle of the alternative method 4

13 Standard method 4

2 VALIDATION STUDY RESULTS 5

21 Initial validation (realised by ASEPT 2006) 5

211 Method Comparison Study 5

2111 Linearity 5

2112 Relative accuracy 9

2113 Detection limit (LOD) and quantification limit (LOQ) 17

2114 Relative Sensitivity 18

2115 SpecificitySelectivity 19

212 Practicability 20

213 Inter-laboratory study 21

2131 Organisation 21

2132 Verification of experimental parameters 21

2133 Results 23

214 Conclusion 29

22 Extension study (realised by ASEPT 2007) 30

Appendix 1 ndash Flow diagram of the Listeria PrecisTM method __________________________________ 31

Appendix 2 ndash ISO 11290-2 (1998) amp ISO 11290-2A1 (2004) Horizontal method for the detection and

enumeration of Listeria monocytogenes - Part 2 enumeration of Listeria monocytogenes in foods ____ 32

Appendix 3 ndash Mandelrsquos graphics _______________________________________________________ 33

Appendix 4 ndash Statistical results ________________________________________________________ 35

Thermo Fisher Scientific

ADRIA Deacuteveloppement 338 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

The modifications are highlighted

Quality assurance documents related to this study can be consulted upon

request from Oxoid part of Thermo Fisher Scientific

The technical protocol and the result interpretation were realised according to

the EN ISO 16140 and the AFNOR technical rules

o Company Oxoid Ltd

part of THERMO FISHER SCIENTIFIC

Wade Road Basingstoke

Hampshire RG24 8PW UK

o Expert Laboratory ADRIA Deacuteveloppement

ZA Creacrsquoh Gwen

F-29196 QUIMPER Cedex

o Studied method Listeria PrecisTM method for enumeration of

Listeria monocytogenes

o Validation standard NF EN ISO 16140 (October 2003) Food

microbiology ndash Protocol for the validation of

alternative methods

o Standard method ISO 11290-2 (1999) amp ISO 11290-2A1 (2004)

Horizontal method for the detection and

enumeration of Listeria monocytogenes - Part 2

enumeration of Listeria monocytogenes in foods

o Scope All human food products and environmental

samples

o Certification organism AFNOR Certification

Thermo Fisher Scientific

ADRIA Deacuteveloppement 438 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

1 INTRODUCTION

11 Dates of the validation studies

The Listeria PrecisTM method for enumeration of Listeria monocytogenes was

validated on September 15 2006 (certificate number UNI 0305 ndash 0906) for

food products and environmental samples

In 2007 an extension study was obtained for a new confirmation test OBIS

MONO

The alternative method was renewed in 2010 and 2014

12 Protocol and principle of the alternative method

The Listeria PrecisTM method is based on a specific chromogenic media for

Listeria monocytogenes enumeration

The protocol is described in Appendix 1

13 Standard method

The reference method is the ISO 11290-2 (1998) amp ISO 11290-2A1 (2004)

Horizontal method for the detection and enumeration of Listeria

monocytogenes - Part 2 enumeration of Listeria monocytogenes in foods

(See Appendix 2)

Analysis performed according to the COFRAC accreditation

Thermo Fisher Scientific

ADRIA Deacuteveloppement 538 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

2 VALIDATION STUDY RESULTS

21 Initial validation (realised by ASEPT 2006)

211 Method Comparison Study

2111 Linearity

Linearity is the ability of the method when used with a given matrix to give results that are in proportion

to the amount of analyte present in the sample that is an increase in analyte corresponds to a linear or

proportional increase in results

Food matrices and protocols

Six matrixstrain pairs were analysed with five contamination levels and two

replicates At least 50 analyses were performed by the alternative and the

standard methods

The contamination levels the samples and the inoculated strains are

presented in the table below

Table 1

Samples Strains Contamination levels

(CFUg)

Rillettes Listeria monocytogenes 4e 10 ndash 50

50 ndash 100

100 ndash 500

500 ndash 1 000

1 000 ndash 10 000

Raw milk Listeria monocytogenes 12a

Lettuce Listeria monocytogenes 12a

Smoked salmon Listeria monocytogenes 12b

Raw egg product Listeria monocytogenes 12b

Process water Listeria monocytogenes 12a

The samples were individually inoculated and were analysed in duplicate by

the alternative and the reference methods

Results

The bi-dimensional graphs are shown figure 1

Thermo Fisher Scientific

ADRIA Deacuteveloppement 638 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Figure 1 ndash Bi-dimensional graphs

Rillettes Raw milk

Smoked salmon Raw egg product

Lettuce Process water

Thermo Fisher Scientific

ADRIA Deacuteveloppement 738 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Statistical interpretations

Statistical interpretation results are provided in Table 2

Table 2 ndash Statistical interpretations

Matrix R Selected

regression

RobF Critical

value

P Correlation

coefficient

Regression equation

Rillettes 180 GMFR 609 541 4 09960 log (Alt) = 09887 log (Ref) + 01141

Lettuce 108 GMFR 000 541 100 09992 Log (Alt) = 10081 log (Ref) - 00257

Raw milk 102 GMFR 000 541 100 09997 Log (Alt) = 10188 log (Ref) - 00737

Smoked salmon 1048 OLS1 1806 541 263 09984 Log (Alt) = 10406 log (Ref) - 01418

Raw egg product 130 GMFR 548 541 49 09958 Log (Alt) = 09775 log (Ref) - 00161

Process water 184 GMFR 0079 541 969 09985 Log (Alt) = 09680 log (Ref) + 01317

GMFR = orthogonal linear regression

OLS1 = ordinary least-square linear regression

x ndash axis = reference method

y ndash axis = alternative method

OLS1 = ordinary least-square linear regression

x axis = alternative method

y axis = reference method

Statistical Interpretation

P gt 5 not significant 1 lt P lt 5 significant

P lt 1 highly significant

The regressions straight lines are shown figure 2

Thermo Fisher Scientific

ADRIA Deacuteveloppement 838 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Figure 2 ndash Regression straight lines

Lineacuteariteacute - Œuf

Process water Process water (2) Process water

Smoked salmon (GMFR) Smoked salmon (OLS 1)

Raw milk Raw egg product

Rillettes Lettuce

Thermo Fisher Scientific

ADRIA Deacuteveloppement 938 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Discussion

The linearity is accepted in all cases with

P values 5 and correlation coefficients superior to 099

For the matrix ldquoRillettesldquo the linearity test is significant (P = 4) with a high

correlation coefficient value (0996)

The Listeria PrecisTM method shows satisfying linearity

2112 Relative accuracy

The accuracy is the closeness of agreement between a test result and the accepted reference value

The bias is the difference between the expectation of the test results and an accepted reference value

Number and nature of the samples

The repartition per category and types is provided in table 3

Table 3 ndash Number and nature of the samples

Categories Types Number of samples

Analysed Exploited (1)

Meat products

Raw meat products 6 4

Ready to cook products 4 2

Ready to eat delicatessen 10 4

Total 20 10

Dairy products

Raw milk cheeses 7 4

Raw milks 4 3

Other dairy products 5 5

Total 16 12

Fishery products

Raw fishes 5 4

Processed fishes 7 2

Ready-to-eat products 6 4

Total 18 10

Vegetables

Raw vegetables 5 4

Frozen vegetables 3 3

Lettuce raw vegetables 5 5

Total 13 12

Egg products

Raw egg 5 5

Pastries 3 3

Pasteurised egg products 4 3

Total 12 11

Environmental samples

Wipes swabs 8 4

Process water 3 3

Dusts 3 3

Total 14 10

TOTAL 93 65

(1) Some data cannot be exploited because enumeration was under the detection level for at least one of the

method

Thermo Fisher Scientific

ADRIA Deacuteveloppement 1038 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Artificial contamination of the samples

Artificial contaminations were realised by spiking after performing injury

protocols on pure cultures or by cross-contaminations

56 samples were artificially contaminated 53 gave exploitable results among

a total of 65 samples 8150 of the samples were artificially contaminated

The injury protocol and the strains inoculated are provided in Table 4

Table 4

Stress Ndeg Injury protocol Strain Origin

A 45 min at 50 degC

48

LM -H123

LM-H170

Smoked salmon

Lettuce

Lettuce

B 30 min at 50 degC

and 2 h at ndash20degC

18

43

LM-O1

Raw milk (environment)

Environment

Egg product

C 30 min at 55degC

18

48

LM-H123

Raw milk

Smoked salmon

Lettuce

D 30 min at 50degC

et 2 h at ndash80degC

34

LM-H171

LM-O1

Rillettes

Lettuce

Egg product

E 30 min at 60 degC LM-O1 Egg product

Confirmatory tests

For the reference and the alternative methods confirmations were realised

by the tests described in the ISO 12290-2A1 standard For the alternative

method only one colony was confirmed while 5 colonies were tested for the

reference method

Results

Samples were analysed in duplicate by the reference and the alternative

methods

The contamination range is provided in table 5 per tested category

Thermo Fisher Scientific

ADRIA Deacuteveloppement 1138 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Table 5 ndash Contamination range

Food category Contamination level (log CFUg)

Meat products 100 ndash 404

Dairy products 148 ndash 408

Fishery products 100 ndash 384

Vegetables 170 ndash 511

Egg products 130 ndash 499

Environmental samples 170 ndash 511

All categories 100 ndash 511

Bi-dimensional graphs are given figure 3

Figure 3 ndash Bi-dimensional graphs

Graphe bi-dimensionnel Produits carneacutes

Fishery products

Vegetables

Egg products

Meat products

Thermo Fisher Scientific

ADRIA Deacuteveloppement 1238 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Statistical interpretation according to EN ISO16140 method

The results of the statistical interpretation are given in Table 6

Table 6 ndash Statistical interpretation results

Category n R Regression

used a t(a) b t(b) Critical T

P

Ordinate

at 0

Slope

at 1

Meat products 10 158 GMFR -0455 3321 115 3123 2306 001 001

Dairy products 12 065 GMFR -0027 0385 1002 0108 2228 092 071

Fishery products 10 130 GMFR -0305 1024 1034 0312 2306 076 034

Vegetables 12 086 GMFR -0015 0128 0993 0191 2228 085 090

Egg products 11 158 GMFR -0038 0866 1001 0090 2262 093 041

Environmental

samples 10 176 GMFR -0139 265 1034 231 2306 005 003

All products 65 118 GMFR -0182 3042 1040 2145 2000 004 000

All products

(estimated number) 65 118 GMFR -0182 3042 1040 2145 2000 004 000

All products (non

estimated results) 20 115 GMFR -0367 151 1138 1141 2101 027 015

Repeatability limits obtained for the alternative and reference methods as

well as bias obtained are presented in Table 7

Dairy products Environmental samples

Log (Ref) Log (Ref)

All categories

Thermo Fisher Scientific

ADRIA Deacuteveloppement 1338 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Table 7

Categories Bias

Repeatability limits

Rob r

Ref Alt

Meat products 0008 0314 0343

Dairy products -0031 0136 0130

Fishery products -0133 0220 0214

Vegetables -0041 0180 0319

Egg products -0026 0219 0206

Environmental samples -0007 0198 0119

All products -0032 0196 0215

Regression straight lines (graph and equation representations) for each food

category and for all products are presented figure 4

Thermo Fisher Scientific

ADRIA Deacuteveloppement 1438 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Figure 4 ndash Regression straight lines

Meat products

Fishery products

Dairy products

Vegetables

Thermo Fisher Scientific

ADRIA Deacuteveloppement 1538 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Environmental samples

Egg products

All products (estimated number)

Thermo Fisher Scientific

ADRIA Deacuteveloppement 1638 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

The hypotheses that the ordinates are close to 0 and the slopes close to 1

are approved in all cases except for

- Meat products (slope P = 1 ordinate P = 1)

- Environmental samples (ordinate P = 3)

- All products (ordinate P = 0)

The bias between the two methods vary from ndash 0133 to 0008 log CFU For

all categories the bias is ndash 0032 log CFU The repeatability limits of the

alternative and the reference method are similar For all products the values

are respectively 0215 and 0196 log CFU For all products these

interpretations are provided taking into account data with low contamination

level or not (estimated colonies number)

For all food products the regression straight line is the following

log (Alt) = 1040 log Ref ndash 0182

The Listeria PrecisTM method shows satisfying relative accuracy

All products

All products (Level 2)

Thermo Fisher Scientific

ADRIA Deacuteveloppement 1738 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

2113 Detection limit (LOD) and quantification limit (LOQ)

The critical level is the defined as the smallest amount which can be detected (not null) but not

quantified as an exact value Below this value it cannot be sure that the true value is not null

The detection limit is defined as being higher than the critical level because it involves a power the

probability 1- which has to be well over 50 for example 95

The quantification limit is defined as the smallest amount of analyte (that is the lowest actual number of

organisms) which can be measured and quantified with defined precision and accuracy under the

experimental conditions by the method under validation

Protocol

The detection limit of the alternative method was realised with pure cultures

Three different inoculation levels were tested with six replicates per level ie

18 analyses were done by the alternative method

Listeria monocytogenes 18 was used for the detection limit of the alternative

method

Quantification limit was calculated for six independent blank samples

determinations

Results

These data are intrinsic to the method used and are presented in the

following tables

Table 8 ndash Inoculation 1 ml on 3 plates

Level (CFUml) Positive samples number Standard deviation Bias

0 06

05 26 1211 0

1 36 0816 05

5 66 2483 85

Thermo Fisher Scientific

ADRIA Deacuteveloppement 1838 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Table 9 ndash Inoculation 01 ml on 1 plate

Level (CFUml) Positive samples number Standard deviation Bias

0 06

5 36 0548 05

10 46 1414 2

50 66 2258 55

Table 10

Formulas Obtained values

LC 165 S0 + X0 18

LOD 33 S0 + X0 32

LOQ 10 S0 + X0 87

2114 Relative Sensitivity

The relative sensitivity is defined as the ability of the alternative method to detect two different amounts

of analyte measured by the reference method within a given matrix at a specified average value or

over the whole measurement range that is it is the minimal quantity variation (increase of the analyte

concentration x) which gives a significant variation of the measured signal (response y)

These data are intrinsic to the used method and are obtained from the results

of the linearity study

Sensitivity patterns obtained for tested (matrixstrain) pairs are presented in

figure 5

Thermo Fisher Scientific

ADRIA Deacuteveloppement 1938 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Figure 5 ndash Sensitivity patterns for the tested (matrixstrain) pairs

2115 SpecificitySelectivity

The specificity is defined as the degree to which a method is affected (or not) by the other components

present in a multi-component sample That is the ability of a method to measure exactly a given

analyte or its amount within the sample without interference from non-target components such as a

matrix effect or background noise

The selectivity is defined as a measure of the degree of non-interference in the presence of non-target

analytes A method is selective if it can be used to detect the analyte under examination and that a

guarantee can be provided that the detected signal can only be a product by that specific analyte

The specificity and selectivity study was performed for the Listeria PrecisTM

detection method no additional test was realised for the enumeration

method The results were the following

- Among the Listeria monocytogenes strains tested 2 strains (Listeria

monocytogenes CIP 7834 and Listeria monocytogenes CIP 105459) gave

negative results These 2 strains were tested twice by running 48 h

incubation time of the compared agars Positive results were then

observed by the alternative method Pale characteristic colonies were

observed on the ISO agar by streaking the Half Fraser broth only

- No cross reaction was observed with the non-target strains except with

Listeria ivanovii which gave characteristic colonies on Brilliancetrade Listeria

Agar but the confirmatory tests provide a clear discrimination between

Listeria monocytogenes and Listeria ivanovi species

Rillettes

Lettuce

Raw milk

Smoked salmon

Raw egg

Process water

x (CFUg)

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2038 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

212 Practicability

1 Packaging Ready to use Brilliance Listeria agar plates are available

(x 10 plates)

2 Reagent volume

3 Storage conditionsndash

Shelf life of products

not opened

The plates are stored at 6 - 12degC in darkness conditions The

expiration date is mentioned on the package and on the plates It

is around 3 months

4 Modalities of use

after first use

5 Required

equipments No specific equipment is required

6 Reagents Ready to use plates

7 Training 05 day

8 Real time handling

Reference method

9 min for one sample

55 min for 10 samples

Alternative method

7 min for one sample

35 minutes for 10 samples

9 Time to result

Negative results

Reference method Day 2

Alternative method Day 2

Negative results after confirmation or positive results

Reference method Day 4 ndash Day 7

Alternative method Day 4 ndash Day 7

Positive results

Reference method Day 4 ndash Day 7

Alternative method Day 2 ndash Day 3 (with OBIS Mono Test)

or Day 4 ndash Day 7 (by the classical tests described in the

standardised method)

10 Technician skill Identical to that required for the reference method

11 Steps common to

the reference method Mother suspension revivification step and decimal dilutions

12 Traceability

13 Maintenance

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2138 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

213 Inter-laboratory study

2131 Organisation

12 laboratories participated to this study Pasteurised milk was inoculated by

Listeria monocytogenes 12a isolated from raw milk The targeted

inoculation levels were

- 0 CFUml

- 50 CFU ml

- 500 CFU ml

- 5 000 CFU ml

Each laboratory received eight flasks of 10 ml sample ie two flasks per

inoculation level Furthermore one non-inoculated sample has been added

to the package for total viable count microflora (NF ISO 4833 method)

Detailed instructions were transmitted to the collaborators by the expert

laboratory

Collaborative study laboratories and the expert laboratory carried out the

analyses with the alternative and reference methods

2132 Verification of experimental parameters

Strain stability during transport

In order to evaluate the Listeria monocytogenes strain stability during

transport bacterial count of all inoculated flasks were checked at different

time ie inoculation time after 24 h of conservation at 2deg - 8degC Results are

reported in table 11

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2238 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Table 11 ndash Listeria monocytogenes count

by ISO 11290-2A1 method (log CFUml)

Day of the analyses Level contamination Reference method

Day 0

(29052006)

1 45

2 440

3 4 500

Day 1

(30052006)

1 60

2 430

3 4 900

No evolution of the strain was observed after storage for 24 h at 2degC ndash 8degC

Contamination level

The real contamination levels are provided in table 12

Table 12 ndash Real contamination level (CFUml)

Target inoculation

(CFU ml) Samples Ndeg

Real contamination levels

(CFUml)

0 1 - 8 0

50 5 - 6 45

500 3 - 7 440

5000 2 - 4 4 500

Sample temperature at receipt

Measured temperatures at receipt are listed in table 13

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2338 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Table 13 - Sample temperature on receipt

Laboratories Receipt

day

Hour

receipt

Measured temperature

at receipt (degC)

Measured temperature by the

temperature probe (degC)

A

B

C

D

E

F

G

H

I

J

K

L

M

30052006

10h45

10h15

11h15

10h00

09h30

8h30

11h50

10h00

08h30

14h45

08h30

09h45

08h45

82

55

45

63

60

62

81

38

48

-

14

67

76

03

03

-13

03

04

-14

05

04

01

05

-13

04

Conclusion

The temperatures during the transport were correct for all the labs except for

Lab J which didnrsquot provide the information

All the labs received their packages with several broken flasks

Three labs (B D and L) received some damaged samples but proceeded to

analyses

All the labs started the analyses at Day 1 except Lab J

2133 Results

Aerobic mesophilic flora

The count of aerobic mesophilic flora varied from 94 102 CFUml to

33 103 CFUml

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2438 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Expert lab results

The results obtained by the expert lab are the following

Table 14

Target inoculation level (CFUml)

Reference method ISO 11290-2

Alternative method Listeria Precis Enumeration

Rep 1 Rep 2 Rep 1 Rep 2

lt 10 lt 10 lt 10 lt 10 lt10

50 50 (Ne) 60 (Ne) 50 (Ne) 30 (Ne)

500 470 390 490 500

5 000 4 700 5 100 5 900 3 400

Ne estimated number

The obtained results are in agreement with those expected

Collaborator Labs results

A synthesis of the results obtained by the collaborator labs is provided

Table 15 (CFUml) and Table 16 (log CFUml)

Table 15 ndash Synthesis of results obtained by the alternative

and the reference methods (CFUml)

Labs Level 1 Level 2 Level 2 bis Level 3

Reference method

Alternative method

Reference method

Alternative method

Reference method

Alternative method

Reference method

Alternative method

A 40 40 70 40 480 410 210 340 1000 300 400 300 4900 4600 4900 5100

B 40 15 40 20 350 300 360 450 350 650 800 600 4100 4700 5100 3700

C 50 20 20 70 440 430 480 450 300 650 300 500 4600 5400 4600 5000

D 40 60 40 40 490 460 600 540 250 450 600 1000 4900 4900 4400 4600

E 50 40 50 20 490 520 530 560 750 550 400 200 5400 4500 4100 5400

F 60 20 60 50 370 250 300 130 300 350 600 300 4400 4700 4900 5900

G 70 35 120 50 440 390 570 370 450 550 700 700 5000 4500 4000 4400

H 110 40 100 90 520 440 450 540 150 650 300 300 4200 4700 5600 4800

L 35 60 20 40 390 390 170 380 650 350 800 200 5300 4600 5600 5900

M 45 45 30 40 450 490 550 410 400 550 800 180 4900 4900 6400 4800

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2538 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Table 16 ndash Synthesis of results obtained by the alternative

and the reference methods (log CFUml)

Labs Level 1 Level 2 Level 2 bis Level 3

Reference method

Alternative method

Reference method

Alternative method

Reference method

Alternative method

Reference method

Alternative method

A 1602 1602 1845 1602 2681 2613 2322 2531 3000 2477 2602 2477 3690 3663 3690 3708

B 1602 1176 1602 1301 2544 2477 2556 2653 2544 2813 2903 2778 3613 3672 3708 3568

C 1699 1301 1301 1845 2643 2633 2681 2653 2477 2813 2477 2699 3663 3732 3663 3699

D 1602 1778 1602 1602 2690 2663 2778 2732 2398 2653 2778 3000 3690 3690 3643 3663

E 1699 1602 1699 1301 2690 2716 2724 2748 2875 2740 2602 2301 3732 3653 3613 3732

F 1778 1301 1778 1699 2568 2398 2477 2114 2477 2544 2778 2477 3643 3672 3690 3771

G 1845 1544 2079 1699 2643 2591 2756 2568 2653 2740 2845 2845 3699 3653 3602 3643

H 2041 1602 2000 1954 2716 2643 2653 2732 2176 2813 2477 2477 3623 3672 3748 3681

L 1544 1778 1301 1602 2591 2591 2230 2580 2813 2544 2903 2301 3724 3663 3748 3771

M 1653 1653 1477 1602 2653 2690 2740 2613 2602 2740 2903 2255 3690 3690 3806 3681

Level 2 results obtained taking account the 3 plates inoculated with the mother-suspension

Level 2 bis results obtained taking account the plates inoculated with 01 ml of the mother-suspension

Scrutiny of the measurement results for consistency

In order to identify the measurement results or laboratories that could be

inconsistent two graphical consistency techniques were realised Mandelrsquos

h-ank-statistics Mandel indicators h and k at 5 significance highlighted

some possible inconsistent data (See tables and graphics in Appendix 3 and

Appendix 4)

Mandelrsquos values Number of values observed over the threshold

Reference method Alternative method

h gt 1 Lab F Level 2 Lab F Level 2

h gt 5 Lab F Level 2

Lab B Levels 1 and 2

Lab A Level 2

Lab D Level 3

Lab F Level 2

Lab L Level 2

kgt 1 Lab F Level 3

k gt 5 Lab F Level 2 Lab C Level 2

Lab F Level 3

Lab L Levels 1 and 3

Lab M Level 1

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2638 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

The emphasized results appear (i) coherent with the targeted contamination

levels (ii) concordant between both methods

All the results were thus kept for the statistical analysis

Comparison of the trueness and precision characteristics of

the reference method and alternative methods

The statistical values are summarised hereafter

Level p

Reference method Alternative method

Repeatability ratio

Reproducibility ratio Median

Repeatability standard deviation

Reproducibility standard deviation

Median Repeatability

standard deviation

Reproducibility standard deviation

1 10 16519 02415 02425 16021 02045 02433 0847 1007

2 10 26551 00543 00659 26719 00914 01325 1682 2011

2 bis 10 26581 02607 02607 25951 01900 01953 0729 0749

3 10 18671 00378 00378 18671 00478 00611 1264 1617

Bias of the alternative method

In order to estimate the bias of the alternative method with regard to the reference method for every

level Dij and t are calculated according to the following equations

RefAlt ijYijYDij

Diffp

Dijit

)2(

)(median

If the t value is superior to 2 the alternative method is significantly biased

with regard to the reference method

The values are given in table 17

Table 17 ndash t (d) values obtained by level

Level Bias t (d) Conclusion

1 0068 114 Non significant bias

2 0021 082 Non significant bias

2 bis - 0037 035 Non significant bias

3 - 0037 043 Non significant bias

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2738 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

The statistical test concludes to a non significant bias for all the inoculation

levels The bias varies from ndash 0037 to 0068 log CFUml

Comparison of the repeatability standard deviations

If the ratio Srj Alt Srj Ref of the repeatability standard deviations of the

alternative method and the reference method is larger than 2 the precision

under repeatability conditions of the alternative method is considered to be

lower than that of the reference method If this ratio is smaller than 05 the

precision under repeatability conditions of the alternative method is

considered to be greater than that of the reference method

The ratio values are given in table 18

Table 18

Level

Repeatability standard deviation

Reference method Alternative method Alternative method

reference method ratio

1 0242 0205 0847

2 0054 0091 1682

2 bis 0261 0190 0729

3 0038 0048 1264

Level Repeatability limit

Reference method Alternative method

1 0676 0573

2 0152 0256

2 bis 0730 0532

3 0106 0134

The ratios of the repeatability standard deviations are below 2 in all cases

The precision under repeatability conditions of the alternative method is

equivalent to that of the reference method

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2838 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Comparison of the reproducibility standard deviations

If the ratio Srj Alt Srj Ref of the reproducibility standard deviations of the alternative method and the

reference method is larger than 2 the precision under reproducibility conditions of the alternative

method is considered to be lower than that of the reference method If this ratio is smaller than 05 the

precision under reproducibility conditions of the alternative method is considered to be greater than

that of the reference method

The ratio values are given in table 19

Table 19

Level

Reproducibility standard deviation

Reference method Alternative method Alternative method

reference method ratio

1 0242 0243 1007

2 0066 0133 2011

2 bis 0261 0195 0749

3 0038 0061 1617

Level Reproducibility limit

Reference method Alternative method

1 0676 0681

2 0185 0371

2 bis 0730 0547

3 0106 0171

The ratios of the reproducibility standard deviations are below or close to 2

The precision under reproducibility conditions of the alternative method is

equivalent to that of the reference method

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2938 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

214 Conclusion

Method comparison study

The Listeria PrecisTM enumeration method shows satisfying linearity and

relative accuracy

The alternative method shows satisfying inclusivity and exclusivity results

Two Listeria monocytogenes strains gave negative results with the

alternative method These strains grew weakly on the reference method

media (OampA Palcam and Oxford)

The limits of repeatability of the alternative method are similar to those of the

reference method

The bias between both methods are low between - 0133 Log CFUg and

- 0008 Log CFUg

The Listeria PrecisTM enumeration method offers the saving for confirmatory

tests

Positive results are obtained with the alternative method in 2-3 days (with

OBIS Mono test) or in 4-7 days (by the classical tests described in the

standardized method) against 4-7 days for the reference method

Inter-laboratory study

The bias between the reference and the alternative methods shows satisfying

and correct values

- level 1 (100 - 1 000 CFUg) bias = + 007 log CFUg

- level 2 (1 000 - 10 000 CFUg) bias = 002 log CFUg

- level 2 bis (1 000 - 10 000 CFUg) bias = - 004 log CFUg

- level 3 (10 000 - 100 000 CFUg) bias = - 004 log CFUg

The repeatability and the reproducibility of the alternative method look similar

to those of the reference method

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3038 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

22 Extension study (realised by ASEPT 2007)

An extension study was performed in 2007 for a new confirmatory test OBIS

MONO

150 Listeria monocytogenes strains and 100 non-target strains were isolated

onto TSAYE and BrillianceTM Listeria Agar plates All the Listeria

monocytogenes and the non-target strains gave the expected results

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3138 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Appendix 1 ndash Flow diagram of the Listeria PrecisTM method

x g sample + 9 x ml buffered peptone water

Revivification step for 1 h plusmn 5 min at 20degC plusmn 1degC

Streaking 1 ml onto 3 Brilliance Listeria Agar plates (mother suspension)

or 01 ml onto 1 Brilliance Listeria Agar plates (mother suspension and decimal dilutions)

Incubation for 48 h plusmn 3 h at 37degC plusmn 1degC

Confirmatory tests on at least one typical colony

- by the tests described in the ISO method

- by applying the OBIS mono test on an isolated colony

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3238 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Appendix 2 ndash ISO 11290-2 (1998) amp ISO 11290-2A1 (2004) Horizontal

method for the detection and enumeration of Listeria monocytogenes -

Part 2 enumeration of Listeria monocytogenes in foods

10 g cheese + 90 ml buffered peptone water or Half Fraser Broth (without supplements) (110 dilution)

Stomach 1 min 30 seconds

Keep at 20degC plusmn 2degC for 1 h plusmn 5 min

Decimal dilutions in 9 ml peptone salt tubes

Dilution 110

Dilution 1100

Dilution 11000

1 ml on 3 Chromogenic Listeria medium (ISO) plates (x 2) (-1)

01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-2)

01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-3)

01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-4)

Incubation at 37degC plusmn 1degC

24 h - 48 h

Numerate typical colonies at 24 h

(bluegreen surrounded by an opaque halo)

Numerate at 48 h

Streak typical colonies on TSYEA

(5 coloniesplate 2 successive dilutions)

Confirmation tests

Catalase

Gram stain

For confirmation of L monocytogenes

Haemolysis

Carbohydrate (rhamnose and xylose) utilisation

CAMP Test

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3338 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Appendix 3 ndash Mandelrsquos graphics

Laboratoire h Ref

Niveau 1 h Ref

Niveau 2 h Ref

Niveau 3 h Alt

Niveau 1 h Alt

Niveau 2 h Alt

Niveau 3 h5 h1 h5 h1

A -050 -015 083 062 -212 -039 198 299 -198 -299

B -261 -270 021 -077 -058 173 198 299 -198 -299

C -151 -031 -014 -015 -004 -005 198 299 -198 -299

D 038 040 -137 000 072 207 198 299 -198 -299

E -001 090 155 -052 056 -101 198 299 -198 -299

F -112 -321 -153 070 -325 023 198 299 -198 -299

G 042 -071 040 147 -009 176 198 299 -198 -299

H 169 046 -169 192 018 -083 198 299 -198 -299

L 009 -120 021 -077 -231 005 198 299 -198 -299

M 001 031 014 -032 004 -011 198 299 -198 -299

-300

-200

-100

000

100

200

300

A B C D E F G H L M

Vale

urs

h d

e M

an

del(

rob

uste

s)

Laboratoires

Meacutethode de reacutefeacuterence analyse robuste

h Ref Niveau 1

h Ref Niveau 2

h Ref Niveau 3

h 5=198

h 1=299

-300

-200

-100

000

100

200

300

A B C D E F G H L M

Vale

urs

de h

de M

an

del(

rob

uste

s)

Laboratoires

Meacutethode alternative analyse robuste

h Alt Niveau 1

h Alt Niveau 2

h Alt Niveau 2

h 1=299

h 5=198

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3438 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Laboratoire k Ref

Niveau 1 k Ref

Niveau 2 k Ref

Niveau 3 k Alt

Niveau 1 k Alt

Niveau 2 k Alt

Niveau 3 k1 k5

A 000 089 142 084 162 046 259 181

B 125 087 073 104 075 046 259 181

C 117 013 091 188 022 083 259 181

D 052 036 069 000 035 083 259 181

E 028 034 037 138 019 112 259 181

F 140 222 018 027 281 112 259 181

G 088 068 024 131 145 000 259 181

H 129 094 173 016 061 000 259 181

L 069 000 073 104 270 224 259 181

M 000 048 038 043 099 241 259 181

000

050

100

150

200

250

300

350

400

A B C D E F G H L M

Vale

urs

k d

e M

an

del(

rob

uste

s)

Laboratoires

Meacutethode de reacutefeacuterence analyse robuste

k Ref Niveau 1k Ref Niveau 2k Ref Niveau 3

k 1=259

k 5=181

000

050

100

150

200

250

300

A B C D E F G H L M

Vale

urs

k d

e M

an

del

(ro

bu

ste

s)

Laboratoires

Meacutethode alternative analyse robuste

k Alt Niveau 3

k Alt Niveau 1

k Alt Niveau 2

k 1=259

k 5=181

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3538 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Appendix 4 ndash Statistical results

Level 1

Nombre de laboratoires (p) 10

A 1602 1602 1602 000 0000 0000 -0495 0000 A 1845 1602 1724 017 0122 -0122 0621 0840 0122

B 1602 1176 1389 030 0213 -0213 -2613 1247 B 1602 1301 1452 021 0151 -0151 -0769 1041 0062

C 1699 1301 1500 028 0199 -0199 -1510 1165 C 1301 1845 1573 038 -0272 0272 -0148 1881 0073

D 1602 1778 1690 012 -0088 0088 0380 0516 D 1602 1602 1602 000 0000 0000 0000 0000 -0088

E 1699 1602 1651 007 0048 -0048 -0013 0284 E 1699 1301 1500 028 0199 -0199 -0522 1376 -0151

F 1778 1301 1540 034 0239 -0239 -1116 1397 F 1778 1699 1739 006 0040 -0040 0698 0274 0199

G 1845 1544 1695 021 0151 -0151 0425 0881 G 2079 1699 1889 027 0190 -0190 1467 1315 0194

H 2041 1602 1822 031 0220 -0220 1689 1286 H 2000 1954 1977 003 0023 -0023 1917 0158 0155

L 1544 1778 1661 017 -0117 0117 0092 0685 L 1301 1602 1452 021 -0151 0151 -0769 1041 -0210

M 1653 1653 1653 000 0000 0000 0013 0000 M 1477 1602 1540 009 -0062 0062 -0319 0432 -0114

m 165186 m 160206 mediane D 00678

Cn(2p) 1867 Cn(2p) 1867 Cp 16101

Cn(p) 16101 Cn(p) 16101 Qn(D) 0092926

n=2p n 20 n=2p n 20 Qdiff 01496169

f 11 f 11 t 114

l 55 l 55

n=p n 10 n=p n 10

f 6 f 6

l 15 l 15

Qn(2p) 0091465 Qn(2p) 007745

Qn(p) 0062469 Qn(p) 0121519

Qintra 017078 Qintra 014461

Qinter 010058 Qinter 019565

Sr(eacutecart-type de reacutepeacutetabiliteacute) 02415 Sr 02045

RSDr(coefficient de variation de la reacutepeacutetabiliteacute)1462 RSDr 1277

r(repeatability limit)) 0676 r 0573

SL(eacutecart-type interlaboratoire) -001905 SLlt0 SL 001737

SR(eacutecart-type de reproductibiliteacute) 02415 SR 02433

RSDR(coefficient de variation de la reproductibiliteacute)0146 RSDR 0152

R(limite de reproductibiliteacute)) 06762 R(limite de reproductibiliteacute))068123

h k h MoyenneLaboratoires

Niveau 1

si LaboratoiresDeviationMoyenne Dk DeviationsiAlt

Niveau 1

Ref

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3638 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Level 2

Nombre de laboratoires (p) 10

A 2681 2613 2647 005 0034 -0034 -0151 0891 A 2322 2531 2427 015 -0105 0105 -2119 1620 -0220

B 2544 2477 2511 005 0033 -0033 -2699 0872 B 2556 2653 2605 007 -0048 0048 -0581 0750 0094

C 2643 2633 2638 001 0005 -0005 -0311 0130 C 2681 2653 2667 002 0014 -0014 -0040 0217 0029

D 2690 2663 2676 002 0014 -0014 0400 0357 D 2778 2732 2755 003 0023 -0023 0721 0354 0079

E 2690 2716 2703 002 -0013 0013 0897 0336 E 2724 2748 2736 002 -0012 0012 0556 0185 0033

F 2568 2398 2483 012 0085 -0085 -3214 2217 F 2477 2114 2296 026 0182 -0182 -3255 2811 -0188

G 2643 2591 2617 004 0026 -0026 -0707 0682 G 2756 2568 2662 013 0094 -0094 -0085 1453 0045

H 2716 2643 2680 005 0036 -0036 0460 0945 H 2653 2732 2693 006 -0040 0040 0181 0613 0013

L 2591 2591 2591 000 0000 0000 -1196 0000 L 2230 2580 2405 025 -0175 0175 -2307 2704 -0186

M 2653 2690 2672 003 -0018 0018 0311 0481 M 2740 2613 2677 009 0064 -0064 0040 0987 0005

m 2655082 m 26719001 mediane D 00209

Cn(2p) 1867 Cn(2p) 1867 Cp 16101

Cn(p) 16101 Cn(p) 16101 Qn(D) 0039911

n=2p n 20 n=2p n 20 Qdiff 00642591

f 11 f 11 t 082

l 55 l 55

n=p n 10 n=p n 10

f 6 f 6

l 15 l 15

Qn(2p) 0020570 Qn(2p) 00345985

Qn(p) 00332437 Qn(p) 00718158

Qintra 003841 Qintra 006460

Qinter 005352 Qinter 011563

Sr(eacutecart-type de reacutepeacutetabiliteacute) 00543 Sr 00914

RSDr(coefficient de variation de la reacutepeacutetabiliteacute)205 RSDr 342

r(repeatability limit)) 0152 r 0256

SL(eacutecart-type interlaboratoire) 000139 SL 000920

SR(eacutecart-type de reproductibiliteacute) 00659 SR 01325

RSDR(coefficient de variation de la reproductibiliteacute)0025 RSDR 0050

R(limite de reproductibiliteacute)) 018446 R(limite de reproductibiliteacute))03708618

Laboratoires

Niveau 2

Moyenne si Laboratoires

Niveau 2

DRef Alt Moyenne si Deviation h Deviation h k k

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3738 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Level 2 bis

Nombre de laboratoires (p) 10

A 3000 2477 2739 037 0261 -0261 0832 1418 A 2602 2477 2540 009 0062 -0062 -0391 0465 -0199

B 2544 2813 2678 019 -0134 0134 0211 0729 B 2903 2778 2841 009 0062 -0062 1732 0465 0162

C 2477 2813 2645 024 -0168 0168 -0135 0911 C 2477 2699 2588 016 -0111 0111 -0049 0826 -0057

D 2398 2653 2526 018 -0128 0128 -1370 0692 D 2778 3000 2889 016 -0111 0111 2074 0826 0363

E 2875 2740 2808 010 0067 -0067 1547 0365 E 2602 2301 2452 021 0151 -0151 -1012 1120 -0356

F 2477 2544 2511 005 -0033 0033 -1525 0182 F 2778 2477 2628 021 0151 -0151 0230 1120 0117

G 2653 2740 2697 006 -0044 0044 0400 0236 G 2845 2845 2845 000 0000 0000 1764 0000 0148

H 2176 2813 2495 045 -0318 0318 -1692 1727 H 2477 2477 2477 000 0000 0000 -0832 0000 -0017

L 2813 2544 2678 019 0134 -0134 0211 0729 L 2903 2301 2602 043 0301 -0301 0049 2241 -0076

M 2602 2740 2671 010 -0069 0069 0135 0375 M 2903 2255 2579 046 0324 -0324 -0112 2411 -0092

m 2658114 m 259505 mediane D -00372

Cn(2p) 1867 Cn(2p) 1867 Cp 16101

Cn(p) 16101 Cn(p) 16101 Qn(D) 01656915

n=2p n 20 n=2p n 20 Qdiff 02667753

f 11 f 11 t 035

l 55 l 55

n=p n 10 n=p n 10

f 6 f 6

l 15 l 15

Qn(2p) 0098745 Qn(2p) 007195

Qn(p) 006007 Qn(p) 008805

Qintra 018437 Qintra 013435

Qinter 009672 Qinter 014176

Sr(eacutecart-type de reacutepeacutetabiliteacute) 02607 Sr 01900

RSDr(coefficient de variation de la reacutepeacutetabiliteacute)981 RSDr 732

r(repeatability limit)) 0730 r 0532

SL(eacutecart-type interlaboratoire) -002464 SLlt0 SL 000205

SR(eacutecart-type de reproductibiliteacute) 02607 SR 01953

RSDR(coefficient de variation de la reproductibiliteacute)0098 RSDR 0075

R(limite de reproductibiliteacute)) 072996 R(limite de reproductibiliteacute))054686

Laboratoires

Niveau 2 bis

Moyenne si Laboratoires

Niveau 2 bis

DRef Alt Moyenne si Deviation h Deviation h k k

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3838 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Level 3

Nombre de laboratoires (p) 10

A 3690 3663 3676 002 0014 -0014 -0311 0513 A 3690 3708 3699 001 -0009 0009 0354 0257 0022

B 3613 3672 3642 004 -0030 0030 -1851 1109 B 3708 3568 3638 010 0070 -0070 -0844 2061 -0005

C 3663 3732 3698 005 -0035 0035 0645 1302 C 3663 3699 3681 003 -0018 0018 0000 0536 -0017

D 3690 3690 3690 000 0000 0000 0311 0000 D 3643 3663 3653 001 -0010 0010 -0545 0286 -0037

E 3732 3653 3693 006 0040 -0040 0429 1480 E 3613 3732 3673 008 -0060 0060 -0162 1769 -0020

F 3643 3672 3658 002 -0014 0014 -1157 0536 F 3690 3771 3731 006 -0040 0040 0975 1193 0073

G 3699 3653 3676 003 0023 -0023 -0328 0856 G 3602 3643 3623 003 -0021 0021 -1141 0612 -0053

H 3623 3672 3648 003 -0024 0024 -1615 0913 H 3748 3681 3715 005 0033 -0033 0664 0990 0067

L 3724 3663 3694 004 0031 -0031 0461 1150 L 3748 3771 3760 002 -0011 0011 1544 0335 0066

M 3690 3690 3690 000 0000 0000 0311 0000 M 3806 3681 3744 009 0062 -0062 1234 1848 0054

m 368334 m 368086 mediane D 00089

Cn(2p) 1867 Cn(2p) 1867 Cp 16101

Cn(p) 16101 Cn(p) 16101 Qn(D) 00325359

n=2p n 20 n=2p n 20 Qdiff 00523852

f 11 f 11 t 043

l 55 l 55

n=p n 10 n=p n 10

f 6 f 6

l 15 l 15

Qn(2p) 0014323 Qn(2p) 001811

Qn(p) 0013719 Qn(p) 003164

Qintra 002674 Qintra 003381

Qinter 002209 Qinter 005094

Sr(eacutecart-type de reacutepeacutetabiliteacute) 00378 Sr 00478

RSDr(coefficient de variation de la reacutepeacutetabiliteacute)103 RSDr 130

r(repeatability limit)) 0106 r 0134

SL(eacutecart-type interlaboratoire) -000023 SLlt0 SL 000145

SR(eacutecart-type de reproductibiliteacute) 00378 SR 00611

RSDR(coefficient de variation de la reproductibiliteacute)0010 RSDR 0017

R(limite de reproductibiliteacute)) 0105894 R(limite de reproductibiliteacute))017119

Ref Alt DLaboratoires

Niveau 3

Moyenne si Deviation h k Laboratoires

Niveau 3

Moyenne si Deviation h k

Page 2: ÞÀ¿¸7 ú Y5zñLjÜ< /u +æü t¬Á ... - NF Validation · NF VALIDATION Validation of alternative analytical methods Application in food microbiology ... 500 – 1 000 1 000 –

Thermo Fisher Scientific

ADRIA Deacuteveloppement 238 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

1 INTRODUCTION 4

11 Dates of the validation studies 4

12 Protocol and principle of the alternative method 4

13 Standard method 4

2 VALIDATION STUDY RESULTS 5

21 Initial validation (realised by ASEPT 2006) 5

211 Method Comparison Study 5

2111 Linearity 5

2112 Relative accuracy 9

2113 Detection limit (LOD) and quantification limit (LOQ) 17

2114 Relative Sensitivity 18

2115 SpecificitySelectivity 19

212 Practicability 20

213 Inter-laboratory study 21

2131 Organisation 21

2132 Verification of experimental parameters 21

2133 Results 23

214 Conclusion 29

22 Extension study (realised by ASEPT 2007) 30

Appendix 1 ndash Flow diagram of the Listeria PrecisTM method __________________________________ 31

Appendix 2 ndash ISO 11290-2 (1998) amp ISO 11290-2A1 (2004) Horizontal method for the detection and

enumeration of Listeria monocytogenes - Part 2 enumeration of Listeria monocytogenes in foods ____ 32

Appendix 3 ndash Mandelrsquos graphics _______________________________________________________ 33

Appendix 4 ndash Statistical results ________________________________________________________ 35

Thermo Fisher Scientific

ADRIA Deacuteveloppement 338 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

The modifications are highlighted

Quality assurance documents related to this study can be consulted upon

request from Oxoid part of Thermo Fisher Scientific

The technical protocol and the result interpretation were realised according to

the EN ISO 16140 and the AFNOR technical rules

o Company Oxoid Ltd

part of THERMO FISHER SCIENTIFIC

Wade Road Basingstoke

Hampshire RG24 8PW UK

o Expert Laboratory ADRIA Deacuteveloppement

ZA Creacrsquoh Gwen

F-29196 QUIMPER Cedex

o Studied method Listeria PrecisTM method for enumeration of

Listeria monocytogenes

o Validation standard NF EN ISO 16140 (October 2003) Food

microbiology ndash Protocol for the validation of

alternative methods

o Standard method ISO 11290-2 (1999) amp ISO 11290-2A1 (2004)

Horizontal method for the detection and

enumeration of Listeria monocytogenes - Part 2

enumeration of Listeria monocytogenes in foods

o Scope All human food products and environmental

samples

o Certification organism AFNOR Certification

Thermo Fisher Scientific

ADRIA Deacuteveloppement 438 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

1 INTRODUCTION

11 Dates of the validation studies

The Listeria PrecisTM method for enumeration of Listeria monocytogenes was

validated on September 15 2006 (certificate number UNI 0305 ndash 0906) for

food products and environmental samples

In 2007 an extension study was obtained for a new confirmation test OBIS

MONO

The alternative method was renewed in 2010 and 2014

12 Protocol and principle of the alternative method

The Listeria PrecisTM method is based on a specific chromogenic media for

Listeria monocytogenes enumeration

The protocol is described in Appendix 1

13 Standard method

The reference method is the ISO 11290-2 (1998) amp ISO 11290-2A1 (2004)

Horizontal method for the detection and enumeration of Listeria

monocytogenes - Part 2 enumeration of Listeria monocytogenes in foods

(See Appendix 2)

Analysis performed according to the COFRAC accreditation

Thermo Fisher Scientific

ADRIA Deacuteveloppement 538 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

2 VALIDATION STUDY RESULTS

21 Initial validation (realised by ASEPT 2006)

211 Method Comparison Study

2111 Linearity

Linearity is the ability of the method when used with a given matrix to give results that are in proportion

to the amount of analyte present in the sample that is an increase in analyte corresponds to a linear or

proportional increase in results

Food matrices and protocols

Six matrixstrain pairs were analysed with five contamination levels and two

replicates At least 50 analyses were performed by the alternative and the

standard methods

The contamination levels the samples and the inoculated strains are

presented in the table below

Table 1

Samples Strains Contamination levels

(CFUg)

Rillettes Listeria monocytogenes 4e 10 ndash 50

50 ndash 100

100 ndash 500

500 ndash 1 000

1 000 ndash 10 000

Raw milk Listeria monocytogenes 12a

Lettuce Listeria monocytogenes 12a

Smoked salmon Listeria monocytogenes 12b

Raw egg product Listeria monocytogenes 12b

Process water Listeria monocytogenes 12a

The samples were individually inoculated and were analysed in duplicate by

the alternative and the reference methods

Results

The bi-dimensional graphs are shown figure 1

Thermo Fisher Scientific

ADRIA Deacuteveloppement 638 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Figure 1 ndash Bi-dimensional graphs

Rillettes Raw milk

Smoked salmon Raw egg product

Lettuce Process water

Thermo Fisher Scientific

ADRIA Deacuteveloppement 738 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Statistical interpretations

Statistical interpretation results are provided in Table 2

Table 2 ndash Statistical interpretations

Matrix R Selected

regression

RobF Critical

value

P Correlation

coefficient

Regression equation

Rillettes 180 GMFR 609 541 4 09960 log (Alt) = 09887 log (Ref) + 01141

Lettuce 108 GMFR 000 541 100 09992 Log (Alt) = 10081 log (Ref) - 00257

Raw milk 102 GMFR 000 541 100 09997 Log (Alt) = 10188 log (Ref) - 00737

Smoked salmon 1048 OLS1 1806 541 263 09984 Log (Alt) = 10406 log (Ref) - 01418

Raw egg product 130 GMFR 548 541 49 09958 Log (Alt) = 09775 log (Ref) - 00161

Process water 184 GMFR 0079 541 969 09985 Log (Alt) = 09680 log (Ref) + 01317

GMFR = orthogonal linear regression

OLS1 = ordinary least-square linear regression

x ndash axis = reference method

y ndash axis = alternative method

OLS1 = ordinary least-square linear regression

x axis = alternative method

y axis = reference method

Statistical Interpretation

P gt 5 not significant 1 lt P lt 5 significant

P lt 1 highly significant

The regressions straight lines are shown figure 2

Thermo Fisher Scientific

ADRIA Deacuteveloppement 838 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Figure 2 ndash Regression straight lines

Lineacuteariteacute - Œuf

Process water Process water (2) Process water

Smoked salmon (GMFR) Smoked salmon (OLS 1)

Raw milk Raw egg product

Rillettes Lettuce

Thermo Fisher Scientific

ADRIA Deacuteveloppement 938 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Discussion

The linearity is accepted in all cases with

P values 5 and correlation coefficients superior to 099

For the matrix ldquoRillettesldquo the linearity test is significant (P = 4) with a high

correlation coefficient value (0996)

The Listeria PrecisTM method shows satisfying linearity

2112 Relative accuracy

The accuracy is the closeness of agreement between a test result and the accepted reference value

The bias is the difference between the expectation of the test results and an accepted reference value

Number and nature of the samples

The repartition per category and types is provided in table 3

Table 3 ndash Number and nature of the samples

Categories Types Number of samples

Analysed Exploited (1)

Meat products

Raw meat products 6 4

Ready to cook products 4 2

Ready to eat delicatessen 10 4

Total 20 10

Dairy products

Raw milk cheeses 7 4

Raw milks 4 3

Other dairy products 5 5

Total 16 12

Fishery products

Raw fishes 5 4

Processed fishes 7 2

Ready-to-eat products 6 4

Total 18 10

Vegetables

Raw vegetables 5 4

Frozen vegetables 3 3

Lettuce raw vegetables 5 5

Total 13 12

Egg products

Raw egg 5 5

Pastries 3 3

Pasteurised egg products 4 3

Total 12 11

Environmental samples

Wipes swabs 8 4

Process water 3 3

Dusts 3 3

Total 14 10

TOTAL 93 65

(1) Some data cannot be exploited because enumeration was under the detection level for at least one of the

method

Thermo Fisher Scientific

ADRIA Deacuteveloppement 1038 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Artificial contamination of the samples

Artificial contaminations were realised by spiking after performing injury

protocols on pure cultures or by cross-contaminations

56 samples were artificially contaminated 53 gave exploitable results among

a total of 65 samples 8150 of the samples were artificially contaminated

The injury protocol and the strains inoculated are provided in Table 4

Table 4

Stress Ndeg Injury protocol Strain Origin

A 45 min at 50 degC

48

LM -H123

LM-H170

Smoked salmon

Lettuce

Lettuce

B 30 min at 50 degC

and 2 h at ndash20degC

18

43

LM-O1

Raw milk (environment)

Environment

Egg product

C 30 min at 55degC

18

48

LM-H123

Raw milk

Smoked salmon

Lettuce

D 30 min at 50degC

et 2 h at ndash80degC

34

LM-H171

LM-O1

Rillettes

Lettuce

Egg product

E 30 min at 60 degC LM-O1 Egg product

Confirmatory tests

For the reference and the alternative methods confirmations were realised

by the tests described in the ISO 12290-2A1 standard For the alternative

method only one colony was confirmed while 5 colonies were tested for the

reference method

Results

Samples were analysed in duplicate by the reference and the alternative

methods

The contamination range is provided in table 5 per tested category

Thermo Fisher Scientific

ADRIA Deacuteveloppement 1138 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Table 5 ndash Contamination range

Food category Contamination level (log CFUg)

Meat products 100 ndash 404

Dairy products 148 ndash 408

Fishery products 100 ndash 384

Vegetables 170 ndash 511

Egg products 130 ndash 499

Environmental samples 170 ndash 511

All categories 100 ndash 511

Bi-dimensional graphs are given figure 3

Figure 3 ndash Bi-dimensional graphs

Graphe bi-dimensionnel Produits carneacutes

Fishery products

Vegetables

Egg products

Meat products

Thermo Fisher Scientific

ADRIA Deacuteveloppement 1238 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Statistical interpretation according to EN ISO16140 method

The results of the statistical interpretation are given in Table 6

Table 6 ndash Statistical interpretation results

Category n R Regression

used a t(a) b t(b) Critical T

P

Ordinate

at 0

Slope

at 1

Meat products 10 158 GMFR -0455 3321 115 3123 2306 001 001

Dairy products 12 065 GMFR -0027 0385 1002 0108 2228 092 071

Fishery products 10 130 GMFR -0305 1024 1034 0312 2306 076 034

Vegetables 12 086 GMFR -0015 0128 0993 0191 2228 085 090

Egg products 11 158 GMFR -0038 0866 1001 0090 2262 093 041

Environmental

samples 10 176 GMFR -0139 265 1034 231 2306 005 003

All products 65 118 GMFR -0182 3042 1040 2145 2000 004 000

All products

(estimated number) 65 118 GMFR -0182 3042 1040 2145 2000 004 000

All products (non

estimated results) 20 115 GMFR -0367 151 1138 1141 2101 027 015

Repeatability limits obtained for the alternative and reference methods as

well as bias obtained are presented in Table 7

Dairy products Environmental samples

Log (Ref) Log (Ref)

All categories

Thermo Fisher Scientific

ADRIA Deacuteveloppement 1338 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Table 7

Categories Bias

Repeatability limits

Rob r

Ref Alt

Meat products 0008 0314 0343

Dairy products -0031 0136 0130

Fishery products -0133 0220 0214

Vegetables -0041 0180 0319

Egg products -0026 0219 0206

Environmental samples -0007 0198 0119

All products -0032 0196 0215

Regression straight lines (graph and equation representations) for each food

category and for all products are presented figure 4

Thermo Fisher Scientific

ADRIA Deacuteveloppement 1438 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Figure 4 ndash Regression straight lines

Meat products

Fishery products

Dairy products

Vegetables

Thermo Fisher Scientific

ADRIA Deacuteveloppement 1538 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Environmental samples

Egg products

All products (estimated number)

Thermo Fisher Scientific

ADRIA Deacuteveloppement 1638 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

The hypotheses that the ordinates are close to 0 and the slopes close to 1

are approved in all cases except for

- Meat products (slope P = 1 ordinate P = 1)

- Environmental samples (ordinate P = 3)

- All products (ordinate P = 0)

The bias between the two methods vary from ndash 0133 to 0008 log CFU For

all categories the bias is ndash 0032 log CFU The repeatability limits of the

alternative and the reference method are similar For all products the values

are respectively 0215 and 0196 log CFU For all products these

interpretations are provided taking into account data with low contamination

level or not (estimated colonies number)

For all food products the regression straight line is the following

log (Alt) = 1040 log Ref ndash 0182

The Listeria PrecisTM method shows satisfying relative accuracy

All products

All products (Level 2)

Thermo Fisher Scientific

ADRIA Deacuteveloppement 1738 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

2113 Detection limit (LOD) and quantification limit (LOQ)

The critical level is the defined as the smallest amount which can be detected (not null) but not

quantified as an exact value Below this value it cannot be sure that the true value is not null

The detection limit is defined as being higher than the critical level because it involves a power the

probability 1- which has to be well over 50 for example 95

The quantification limit is defined as the smallest amount of analyte (that is the lowest actual number of

organisms) which can be measured and quantified with defined precision and accuracy under the

experimental conditions by the method under validation

Protocol

The detection limit of the alternative method was realised with pure cultures

Three different inoculation levels were tested with six replicates per level ie

18 analyses were done by the alternative method

Listeria monocytogenes 18 was used for the detection limit of the alternative

method

Quantification limit was calculated for six independent blank samples

determinations

Results

These data are intrinsic to the method used and are presented in the

following tables

Table 8 ndash Inoculation 1 ml on 3 plates

Level (CFUml) Positive samples number Standard deviation Bias

0 06

05 26 1211 0

1 36 0816 05

5 66 2483 85

Thermo Fisher Scientific

ADRIA Deacuteveloppement 1838 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Table 9 ndash Inoculation 01 ml on 1 plate

Level (CFUml) Positive samples number Standard deviation Bias

0 06

5 36 0548 05

10 46 1414 2

50 66 2258 55

Table 10

Formulas Obtained values

LC 165 S0 + X0 18

LOD 33 S0 + X0 32

LOQ 10 S0 + X0 87

2114 Relative Sensitivity

The relative sensitivity is defined as the ability of the alternative method to detect two different amounts

of analyte measured by the reference method within a given matrix at a specified average value or

over the whole measurement range that is it is the minimal quantity variation (increase of the analyte

concentration x) which gives a significant variation of the measured signal (response y)

These data are intrinsic to the used method and are obtained from the results

of the linearity study

Sensitivity patterns obtained for tested (matrixstrain) pairs are presented in

figure 5

Thermo Fisher Scientific

ADRIA Deacuteveloppement 1938 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Figure 5 ndash Sensitivity patterns for the tested (matrixstrain) pairs

2115 SpecificitySelectivity

The specificity is defined as the degree to which a method is affected (or not) by the other components

present in a multi-component sample That is the ability of a method to measure exactly a given

analyte or its amount within the sample without interference from non-target components such as a

matrix effect or background noise

The selectivity is defined as a measure of the degree of non-interference in the presence of non-target

analytes A method is selective if it can be used to detect the analyte under examination and that a

guarantee can be provided that the detected signal can only be a product by that specific analyte

The specificity and selectivity study was performed for the Listeria PrecisTM

detection method no additional test was realised for the enumeration

method The results were the following

- Among the Listeria monocytogenes strains tested 2 strains (Listeria

monocytogenes CIP 7834 and Listeria monocytogenes CIP 105459) gave

negative results These 2 strains were tested twice by running 48 h

incubation time of the compared agars Positive results were then

observed by the alternative method Pale characteristic colonies were

observed on the ISO agar by streaking the Half Fraser broth only

- No cross reaction was observed with the non-target strains except with

Listeria ivanovii which gave characteristic colonies on Brilliancetrade Listeria

Agar but the confirmatory tests provide a clear discrimination between

Listeria monocytogenes and Listeria ivanovi species

Rillettes

Lettuce

Raw milk

Smoked salmon

Raw egg

Process water

x (CFUg)

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2038 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

212 Practicability

1 Packaging Ready to use Brilliance Listeria agar plates are available

(x 10 plates)

2 Reagent volume

3 Storage conditionsndash

Shelf life of products

not opened

The plates are stored at 6 - 12degC in darkness conditions The

expiration date is mentioned on the package and on the plates It

is around 3 months

4 Modalities of use

after first use

5 Required

equipments No specific equipment is required

6 Reagents Ready to use plates

7 Training 05 day

8 Real time handling

Reference method

9 min for one sample

55 min for 10 samples

Alternative method

7 min for one sample

35 minutes for 10 samples

9 Time to result

Negative results

Reference method Day 2

Alternative method Day 2

Negative results after confirmation or positive results

Reference method Day 4 ndash Day 7

Alternative method Day 4 ndash Day 7

Positive results

Reference method Day 4 ndash Day 7

Alternative method Day 2 ndash Day 3 (with OBIS Mono Test)

or Day 4 ndash Day 7 (by the classical tests described in the

standardised method)

10 Technician skill Identical to that required for the reference method

11 Steps common to

the reference method Mother suspension revivification step and decimal dilutions

12 Traceability

13 Maintenance

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2138 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

213 Inter-laboratory study

2131 Organisation

12 laboratories participated to this study Pasteurised milk was inoculated by

Listeria monocytogenes 12a isolated from raw milk The targeted

inoculation levels were

- 0 CFUml

- 50 CFU ml

- 500 CFU ml

- 5 000 CFU ml

Each laboratory received eight flasks of 10 ml sample ie two flasks per

inoculation level Furthermore one non-inoculated sample has been added

to the package for total viable count microflora (NF ISO 4833 method)

Detailed instructions were transmitted to the collaborators by the expert

laboratory

Collaborative study laboratories and the expert laboratory carried out the

analyses with the alternative and reference methods

2132 Verification of experimental parameters

Strain stability during transport

In order to evaluate the Listeria monocytogenes strain stability during

transport bacterial count of all inoculated flasks were checked at different

time ie inoculation time after 24 h of conservation at 2deg - 8degC Results are

reported in table 11

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2238 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Table 11 ndash Listeria monocytogenes count

by ISO 11290-2A1 method (log CFUml)

Day of the analyses Level contamination Reference method

Day 0

(29052006)

1 45

2 440

3 4 500

Day 1

(30052006)

1 60

2 430

3 4 900

No evolution of the strain was observed after storage for 24 h at 2degC ndash 8degC

Contamination level

The real contamination levels are provided in table 12

Table 12 ndash Real contamination level (CFUml)

Target inoculation

(CFU ml) Samples Ndeg

Real contamination levels

(CFUml)

0 1 - 8 0

50 5 - 6 45

500 3 - 7 440

5000 2 - 4 4 500

Sample temperature at receipt

Measured temperatures at receipt are listed in table 13

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2338 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Table 13 - Sample temperature on receipt

Laboratories Receipt

day

Hour

receipt

Measured temperature

at receipt (degC)

Measured temperature by the

temperature probe (degC)

A

B

C

D

E

F

G

H

I

J

K

L

M

30052006

10h45

10h15

11h15

10h00

09h30

8h30

11h50

10h00

08h30

14h45

08h30

09h45

08h45

82

55

45

63

60

62

81

38

48

-

14

67

76

03

03

-13

03

04

-14

05

04

01

05

-13

04

Conclusion

The temperatures during the transport were correct for all the labs except for

Lab J which didnrsquot provide the information

All the labs received their packages with several broken flasks

Three labs (B D and L) received some damaged samples but proceeded to

analyses

All the labs started the analyses at Day 1 except Lab J

2133 Results

Aerobic mesophilic flora

The count of aerobic mesophilic flora varied from 94 102 CFUml to

33 103 CFUml

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2438 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Expert lab results

The results obtained by the expert lab are the following

Table 14

Target inoculation level (CFUml)

Reference method ISO 11290-2

Alternative method Listeria Precis Enumeration

Rep 1 Rep 2 Rep 1 Rep 2

lt 10 lt 10 lt 10 lt 10 lt10

50 50 (Ne) 60 (Ne) 50 (Ne) 30 (Ne)

500 470 390 490 500

5 000 4 700 5 100 5 900 3 400

Ne estimated number

The obtained results are in agreement with those expected

Collaborator Labs results

A synthesis of the results obtained by the collaborator labs is provided

Table 15 (CFUml) and Table 16 (log CFUml)

Table 15 ndash Synthesis of results obtained by the alternative

and the reference methods (CFUml)

Labs Level 1 Level 2 Level 2 bis Level 3

Reference method

Alternative method

Reference method

Alternative method

Reference method

Alternative method

Reference method

Alternative method

A 40 40 70 40 480 410 210 340 1000 300 400 300 4900 4600 4900 5100

B 40 15 40 20 350 300 360 450 350 650 800 600 4100 4700 5100 3700

C 50 20 20 70 440 430 480 450 300 650 300 500 4600 5400 4600 5000

D 40 60 40 40 490 460 600 540 250 450 600 1000 4900 4900 4400 4600

E 50 40 50 20 490 520 530 560 750 550 400 200 5400 4500 4100 5400

F 60 20 60 50 370 250 300 130 300 350 600 300 4400 4700 4900 5900

G 70 35 120 50 440 390 570 370 450 550 700 700 5000 4500 4000 4400

H 110 40 100 90 520 440 450 540 150 650 300 300 4200 4700 5600 4800

L 35 60 20 40 390 390 170 380 650 350 800 200 5300 4600 5600 5900

M 45 45 30 40 450 490 550 410 400 550 800 180 4900 4900 6400 4800

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2538 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Table 16 ndash Synthesis of results obtained by the alternative

and the reference methods (log CFUml)

Labs Level 1 Level 2 Level 2 bis Level 3

Reference method

Alternative method

Reference method

Alternative method

Reference method

Alternative method

Reference method

Alternative method

A 1602 1602 1845 1602 2681 2613 2322 2531 3000 2477 2602 2477 3690 3663 3690 3708

B 1602 1176 1602 1301 2544 2477 2556 2653 2544 2813 2903 2778 3613 3672 3708 3568

C 1699 1301 1301 1845 2643 2633 2681 2653 2477 2813 2477 2699 3663 3732 3663 3699

D 1602 1778 1602 1602 2690 2663 2778 2732 2398 2653 2778 3000 3690 3690 3643 3663

E 1699 1602 1699 1301 2690 2716 2724 2748 2875 2740 2602 2301 3732 3653 3613 3732

F 1778 1301 1778 1699 2568 2398 2477 2114 2477 2544 2778 2477 3643 3672 3690 3771

G 1845 1544 2079 1699 2643 2591 2756 2568 2653 2740 2845 2845 3699 3653 3602 3643

H 2041 1602 2000 1954 2716 2643 2653 2732 2176 2813 2477 2477 3623 3672 3748 3681

L 1544 1778 1301 1602 2591 2591 2230 2580 2813 2544 2903 2301 3724 3663 3748 3771

M 1653 1653 1477 1602 2653 2690 2740 2613 2602 2740 2903 2255 3690 3690 3806 3681

Level 2 results obtained taking account the 3 plates inoculated with the mother-suspension

Level 2 bis results obtained taking account the plates inoculated with 01 ml of the mother-suspension

Scrutiny of the measurement results for consistency

In order to identify the measurement results or laboratories that could be

inconsistent two graphical consistency techniques were realised Mandelrsquos

h-ank-statistics Mandel indicators h and k at 5 significance highlighted

some possible inconsistent data (See tables and graphics in Appendix 3 and

Appendix 4)

Mandelrsquos values Number of values observed over the threshold

Reference method Alternative method

h gt 1 Lab F Level 2 Lab F Level 2

h gt 5 Lab F Level 2

Lab B Levels 1 and 2

Lab A Level 2

Lab D Level 3

Lab F Level 2

Lab L Level 2

kgt 1 Lab F Level 3

k gt 5 Lab F Level 2 Lab C Level 2

Lab F Level 3

Lab L Levels 1 and 3

Lab M Level 1

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2638 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

The emphasized results appear (i) coherent with the targeted contamination

levels (ii) concordant between both methods

All the results were thus kept for the statistical analysis

Comparison of the trueness and precision characteristics of

the reference method and alternative methods

The statistical values are summarised hereafter

Level p

Reference method Alternative method

Repeatability ratio

Reproducibility ratio Median

Repeatability standard deviation

Reproducibility standard deviation

Median Repeatability

standard deviation

Reproducibility standard deviation

1 10 16519 02415 02425 16021 02045 02433 0847 1007

2 10 26551 00543 00659 26719 00914 01325 1682 2011

2 bis 10 26581 02607 02607 25951 01900 01953 0729 0749

3 10 18671 00378 00378 18671 00478 00611 1264 1617

Bias of the alternative method

In order to estimate the bias of the alternative method with regard to the reference method for every

level Dij and t are calculated according to the following equations

RefAlt ijYijYDij

Diffp

Dijit

)2(

)(median

If the t value is superior to 2 the alternative method is significantly biased

with regard to the reference method

The values are given in table 17

Table 17 ndash t (d) values obtained by level

Level Bias t (d) Conclusion

1 0068 114 Non significant bias

2 0021 082 Non significant bias

2 bis - 0037 035 Non significant bias

3 - 0037 043 Non significant bias

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2738 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

The statistical test concludes to a non significant bias for all the inoculation

levels The bias varies from ndash 0037 to 0068 log CFUml

Comparison of the repeatability standard deviations

If the ratio Srj Alt Srj Ref of the repeatability standard deviations of the

alternative method and the reference method is larger than 2 the precision

under repeatability conditions of the alternative method is considered to be

lower than that of the reference method If this ratio is smaller than 05 the

precision under repeatability conditions of the alternative method is

considered to be greater than that of the reference method

The ratio values are given in table 18

Table 18

Level

Repeatability standard deviation

Reference method Alternative method Alternative method

reference method ratio

1 0242 0205 0847

2 0054 0091 1682

2 bis 0261 0190 0729

3 0038 0048 1264

Level Repeatability limit

Reference method Alternative method

1 0676 0573

2 0152 0256

2 bis 0730 0532

3 0106 0134

The ratios of the repeatability standard deviations are below 2 in all cases

The precision under repeatability conditions of the alternative method is

equivalent to that of the reference method

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2838 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Comparison of the reproducibility standard deviations

If the ratio Srj Alt Srj Ref of the reproducibility standard deviations of the alternative method and the

reference method is larger than 2 the precision under reproducibility conditions of the alternative

method is considered to be lower than that of the reference method If this ratio is smaller than 05 the

precision under reproducibility conditions of the alternative method is considered to be greater than

that of the reference method

The ratio values are given in table 19

Table 19

Level

Reproducibility standard deviation

Reference method Alternative method Alternative method

reference method ratio

1 0242 0243 1007

2 0066 0133 2011

2 bis 0261 0195 0749

3 0038 0061 1617

Level Reproducibility limit

Reference method Alternative method

1 0676 0681

2 0185 0371

2 bis 0730 0547

3 0106 0171

The ratios of the reproducibility standard deviations are below or close to 2

The precision under reproducibility conditions of the alternative method is

equivalent to that of the reference method

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2938 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

214 Conclusion

Method comparison study

The Listeria PrecisTM enumeration method shows satisfying linearity and

relative accuracy

The alternative method shows satisfying inclusivity and exclusivity results

Two Listeria monocytogenes strains gave negative results with the

alternative method These strains grew weakly on the reference method

media (OampA Palcam and Oxford)

The limits of repeatability of the alternative method are similar to those of the

reference method

The bias between both methods are low between - 0133 Log CFUg and

- 0008 Log CFUg

The Listeria PrecisTM enumeration method offers the saving for confirmatory

tests

Positive results are obtained with the alternative method in 2-3 days (with

OBIS Mono test) or in 4-7 days (by the classical tests described in the

standardized method) against 4-7 days for the reference method

Inter-laboratory study

The bias between the reference and the alternative methods shows satisfying

and correct values

- level 1 (100 - 1 000 CFUg) bias = + 007 log CFUg

- level 2 (1 000 - 10 000 CFUg) bias = 002 log CFUg

- level 2 bis (1 000 - 10 000 CFUg) bias = - 004 log CFUg

- level 3 (10 000 - 100 000 CFUg) bias = - 004 log CFUg

The repeatability and the reproducibility of the alternative method look similar

to those of the reference method

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3038 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

22 Extension study (realised by ASEPT 2007)

An extension study was performed in 2007 for a new confirmatory test OBIS

MONO

150 Listeria monocytogenes strains and 100 non-target strains were isolated

onto TSAYE and BrillianceTM Listeria Agar plates All the Listeria

monocytogenes and the non-target strains gave the expected results

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3138 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Appendix 1 ndash Flow diagram of the Listeria PrecisTM method

x g sample + 9 x ml buffered peptone water

Revivification step for 1 h plusmn 5 min at 20degC plusmn 1degC

Streaking 1 ml onto 3 Brilliance Listeria Agar plates (mother suspension)

or 01 ml onto 1 Brilliance Listeria Agar plates (mother suspension and decimal dilutions)

Incubation for 48 h plusmn 3 h at 37degC plusmn 1degC

Confirmatory tests on at least one typical colony

- by the tests described in the ISO method

- by applying the OBIS mono test on an isolated colony

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3238 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Appendix 2 ndash ISO 11290-2 (1998) amp ISO 11290-2A1 (2004) Horizontal

method for the detection and enumeration of Listeria monocytogenes -

Part 2 enumeration of Listeria monocytogenes in foods

10 g cheese + 90 ml buffered peptone water or Half Fraser Broth (without supplements) (110 dilution)

Stomach 1 min 30 seconds

Keep at 20degC plusmn 2degC for 1 h plusmn 5 min

Decimal dilutions in 9 ml peptone salt tubes

Dilution 110

Dilution 1100

Dilution 11000

1 ml on 3 Chromogenic Listeria medium (ISO) plates (x 2) (-1)

01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-2)

01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-3)

01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-4)

Incubation at 37degC plusmn 1degC

24 h - 48 h

Numerate typical colonies at 24 h

(bluegreen surrounded by an opaque halo)

Numerate at 48 h

Streak typical colonies on TSYEA

(5 coloniesplate 2 successive dilutions)

Confirmation tests

Catalase

Gram stain

For confirmation of L monocytogenes

Haemolysis

Carbohydrate (rhamnose and xylose) utilisation

CAMP Test

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3338 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Appendix 3 ndash Mandelrsquos graphics

Laboratoire h Ref

Niveau 1 h Ref

Niveau 2 h Ref

Niveau 3 h Alt

Niveau 1 h Alt

Niveau 2 h Alt

Niveau 3 h5 h1 h5 h1

A -050 -015 083 062 -212 -039 198 299 -198 -299

B -261 -270 021 -077 -058 173 198 299 -198 -299

C -151 -031 -014 -015 -004 -005 198 299 -198 -299

D 038 040 -137 000 072 207 198 299 -198 -299

E -001 090 155 -052 056 -101 198 299 -198 -299

F -112 -321 -153 070 -325 023 198 299 -198 -299

G 042 -071 040 147 -009 176 198 299 -198 -299

H 169 046 -169 192 018 -083 198 299 -198 -299

L 009 -120 021 -077 -231 005 198 299 -198 -299

M 001 031 014 -032 004 -011 198 299 -198 -299

-300

-200

-100

000

100

200

300

A B C D E F G H L M

Vale

urs

h d

e M

an

del(

rob

uste

s)

Laboratoires

Meacutethode de reacutefeacuterence analyse robuste

h Ref Niveau 1

h Ref Niveau 2

h Ref Niveau 3

h 5=198

h 1=299

-300

-200

-100

000

100

200

300

A B C D E F G H L M

Vale

urs

de h

de M

an

del(

rob

uste

s)

Laboratoires

Meacutethode alternative analyse robuste

h Alt Niveau 1

h Alt Niveau 2

h Alt Niveau 2

h 1=299

h 5=198

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3438 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Laboratoire k Ref

Niveau 1 k Ref

Niveau 2 k Ref

Niveau 3 k Alt

Niveau 1 k Alt

Niveau 2 k Alt

Niveau 3 k1 k5

A 000 089 142 084 162 046 259 181

B 125 087 073 104 075 046 259 181

C 117 013 091 188 022 083 259 181

D 052 036 069 000 035 083 259 181

E 028 034 037 138 019 112 259 181

F 140 222 018 027 281 112 259 181

G 088 068 024 131 145 000 259 181

H 129 094 173 016 061 000 259 181

L 069 000 073 104 270 224 259 181

M 000 048 038 043 099 241 259 181

000

050

100

150

200

250

300

350

400

A B C D E F G H L M

Vale

urs

k d

e M

an

del(

rob

uste

s)

Laboratoires

Meacutethode de reacutefeacuterence analyse robuste

k Ref Niveau 1k Ref Niveau 2k Ref Niveau 3

k 1=259

k 5=181

000

050

100

150

200

250

300

A B C D E F G H L M

Vale

urs

k d

e M

an

del

(ro

bu

ste

s)

Laboratoires

Meacutethode alternative analyse robuste

k Alt Niveau 3

k Alt Niveau 1

k Alt Niveau 2

k 1=259

k 5=181

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3538 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Appendix 4 ndash Statistical results

Level 1

Nombre de laboratoires (p) 10

A 1602 1602 1602 000 0000 0000 -0495 0000 A 1845 1602 1724 017 0122 -0122 0621 0840 0122

B 1602 1176 1389 030 0213 -0213 -2613 1247 B 1602 1301 1452 021 0151 -0151 -0769 1041 0062

C 1699 1301 1500 028 0199 -0199 -1510 1165 C 1301 1845 1573 038 -0272 0272 -0148 1881 0073

D 1602 1778 1690 012 -0088 0088 0380 0516 D 1602 1602 1602 000 0000 0000 0000 0000 -0088

E 1699 1602 1651 007 0048 -0048 -0013 0284 E 1699 1301 1500 028 0199 -0199 -0522 1376 -0151

F 1778 1301 1540 034 0239 -0239 -1116 1397 F 1778 1699 1739 006 0040 -0040 0698 0274 0199

G 1845 1544 1695 021 0151 -0151 0425 0881 G 2079 1699 1889 027 0190 -0190 1467 1315 0194

H 2041 1602 1822 031 0220 -0220 1689 1286 H 2000 1954 1977 003 0023 -0023 1917 0158 0155

L 1544 1778 1661 017 -0117 0117 0092 0685 L 1301 1602 1452 021 -0151 0151 -0769 1041 -0210

M 1653 1653 1653 000 0000 0000 0013 0000 M 1477 1602 1540 009 -0062 0062 -0319 0432 -0114

m 165186 m 160206 mediane D 00678

Cn(2p) 1867 Cn(2p) 1867 Cp 16101

Cn(p) 16101 Cn(p) 16101 Qn(D) 0092926

n=2p n 20 n=2p n 20 Qdiff 01496169

f 11 f 11 t 114

l 55 l 55

n=p n 10 n=p n 10

f 6 f 6

l 15 l 15

Qn(2p) 0091465 Qn(2p) 007745

Qn(p) 0062469 Qn(p) 0121519

Qintra 017078 Qintra 014461

Qinter 010058 Qinter 019565

Sr(eacutecart-type de reacutepeacutetabiliteacute) 02415 Sr 02045

RSDr(coefficient de variation de la reacutepeacutetabiliteacute)1462 RSDr 1277

r(repeatability limit)) 0676 r 0573

SL(eacutecart-type interlaboratoire) -001905 SLlt0 SL 001737

SR(eacutecart-type de reproductibiliteacute) 02415 SR 02433

RSDR(coefficient de variation de la reproductibiliteacute)0146 RSDR 0152

R(limite de reproductibiliteacute)) 06762 R(limite de reproductibiliteacute))068123

h k h MoyenneLaboratoires

Niveau 1

si LaboratoiresDeviationMoyenne Dk DeviationsiAlt

Niveau 1

Ref

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3638 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Level 2

Nombre de laboratoires (p) 10

A 2681 2613 2647 005 0034 -0034 -0151 0891 A 2322 2531 2427 015 -0105 0105 -2119 1620 -0220

B 2544 2477 2511 005 0033 -0033 -2699 0872 B 2556 2653 2605 007 -0048 0048 -0581 0750 0094

C 2643 2633 2638 001 0005 -0005 -0311 0130 C 2681 2653 2667 002 0014 -0014 -0040 0217 0029

D 2690 2663 2676 002 0014 -0014 0400 0357 D 2778 2732 2755 003 0023 -0023 0721 0354 0079

E 2690 2716 2703 002 -0013 0013 0897 0336 E 2724 2748 2736 002 -0012 0012 0556 0185 0033

F 2568 2398 2483 012 0085 -0085 -3214 2217 F 2477 2114 2296 026 0182 -0182 -3255 2811 -0188

G 2643 2591 2617 004 0026 -0026 -0707 0682 G 2756 2568 2662 013 0094 -0094 -0085 1453 0045

H 2716 2643 2680 005 0036 -0036 0460 0945 H 2653 2732 2693 006 -0040 0040 0181 0613 0013

L 2591 2591 2591 000 0000 0000 -1196 0000 L 2230 2580 2405 025 -0175 0175 -2307 2704 -0186

M 2653 2690 2672 003 -0018 0018 0311 0481 M 2740 2613 2677 009 0064 -0064 0040 0987 0005

m 2655082 m 26719001 mediane D 00209

Cn(2p) 1867 Cn(2p) 1867 Cp 16101

Cn(p) 16101 Cn(p) 16101 Qn(D) 0039911

n=2p n 20 n=2p n 20 Qdiff 00642591

f 11 f 11 t 082

l 55 l 55

n=p n 10 n=p n 10

f 6 f 6

l 15 l 15

Qn(2p) 0020570 Qn(2p) 00345985

Qn(p) 00332437 Qn(p) 00718158

Qintra 003841 Qintra 006460

Qinter 005352 Qinter 011563

Sr(eacutecart-type de reacutepeacutetabiliteacute) 00543 Sr 00914

RSDr(coefficient de variation de la reacutepeacutetabiliteacute)205 RSDr 342

r(repeatability limit)) 0152 r 0256

SL(eacutecart-type interlaboratoire) 000139 SL 000920

SR(eacutecart-type de reproductibiliteacute) 00659 SR 01325

RSDR(coefficient de variation de la reproductibiliteacute)0025 RSDR 0050

R(limite de reproductibiliteacute)) 018446 R(limite de reproductibiliteacute))03708618

Laboratoires

Niveau 2

Moyenne si Laboratoires

Niveau 2

DRef Alt Moyenne si Deviation h Deviation h k k

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3738 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Level 2 bis

Nombre de laboratoires (p) 10

A 3000 2477 2739 037 0261 -0261 0832 1418 A 2602 2477 2540 009 0062 -0062 -0391 0465 -0199

B 2544 2813 2678 019 -0134 0134 0211 0729 B 2903 2778 2841 009 0062 -0062 1732 0465 0162

C 2477 2813 2645 024 -0168 0168 -0135 0911 C 2477 2699 2588 016 -0111 0111 -0049 0826 -0057

D 2398 2653 2526 018 -0128 0128 -1370 0692 D 2778 3000 2889 016 -0111 0111 2074 0826 0363

E 2875 2740 2808 010 0067 -0067 1547 0365 E 2602 2301 2452 021 0151 -0151 -1012 1120 -0356

F 2477 2544 2511 005 -0033 0033 -1525 0182 F 2778 2477 2628 021 0151 -0151 0230 1120 0117

G 2653 2740 2697 006 -0044 0044 0400 0236 G 2845 2845 2845 000 0000 0000 1764 0000 0148

H 2176 2813 2495 045 -0318 0318 -1692 1727 H 2477 2477 2477 000 0000 0000 -0832 0000 -0017

L 2813 2544 2678 019 0134 -0134 0211 0729 L 2903 2301 2602 043 0301 -0301 0049 2241 -0076

M 2602 2740 2671 010 -0069 0069 0135 0375 M 2903 2255 2579 046 0324 -0324 -0112 2411 -0092

m 2658114 m 259505 mediane D -00372

Cn(2p) 1867 Cn(2p) 1867 Cp 16101

Cn(p) 16101 Cn(p) 16101 Qn(D) 01656915

n=2p n 20 n=2p n 20 Qdiff 02667753

f 11 f 11 t 035

l 55 l 55

n=p n 10 n=p n 10

f 6 f 6

l 15 l 15

Qn(2p) 0098745 Qn(2p) 007195

Qn(p) 006007 Qn(p) 008805

Qintra 018437 Qintra 013435

Qinter 009672 Qinter 014176

Sr(eacutecart-type de reacutepeacutetabiliteacute) 02607 Sr 01900

RSDr(coefficient de variation de la reacutepeacutetabiliteacute)981 RSDr 732

r(repeatability limit)) 0730 r 0532

SL(eacutecart-type interlaboratoire) -002464 SLlt0 SL 000205

SR(eacutecart-type de reproductibiliteacute) 02607 SR 01953

RSDR(coefficient de variation de la reproductibiliteacute)0098 RSDR 0075

R(limite de reproductibiliteacute)) 072996 R(limite de reproductibiliteacute))054686

Laboratoires

Niveau 2 bis

Moyenne si Laboratoires

Niveau 2 bis

DRef Alt Moyenne si Deviation h Deviation h k k

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3838 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Level 3

Nombre de laboratoires (p) 10

A 3690 3663 3676 002 0014 -0014 -0311 0513 A 3690 3708 3699 001 -0009 0009 0354 0257 0022

B 3613 3672 3642 004 -0030 0030 -1851 1109 B 3708 3568 3638 010 0070 -0070 -0844 2061 -0005

C 3663 3732 3698 005 -0035 0035 0645 1302 C 3663 3699 3681 003 -0018 0018 0000 0536 -0017

D 3690 3690 3690 000 0000 0000 0311 0000 D 3643 3663 3653 001 -0010 0010 -0545 0286 -0037

E 3732 3653 3693 006 0040 -0040 0429 1480 E 3613 3732 3673 008 -0060 0060 -0162 1769 -0020

F 3643 3672 3658 002 -0014 0014 -1157 0536 F 3690 3771 3731 006 -0040 0040 0975 1193 0073

G 3699 3653 3676 003 0023 -0023 -0328 0856 G 3602 3643 3623 003 -0021 0021 -1141 0612 -0053

H 3623 3672 3648 003 -0024 0024 -1615 0913 H 3748 3681 3715 005 0033 -0033 0664 0990 0067

L 3724 3663 3694 004 0031 -0031 0461 1150 L 3748 3771 3760 002 -0011 0011 1544 0335 0066

M 3690 3690 3690 000 0000 0000 0311 0000 M 3806 3681 3744 009 0062 -0062 1234 1848 0054

m 368334 m 368086 mediane D 00089

Cn(2p) 1867 Cn(2p) 1867 Cp 16101

Cn(p) 16101 Cn(p) 16101 Qn(D) 00325359

n=2p n 20 n=2p n 20 Qdiff 00523852

f 11 f 11 t 043

l 55 l 55

n=p n 10 n=p n 10

f 6 f 6

l 15 l 15

Qn(2p) 0014323 Qn(2p) 001811

Qn(p) 0013719 Qn(p) 003164

Qintra 002674 Qintra 003381

Qinter 002209 Qinter 005094

Sr(eacutecart-type de reacutepeacutetabiliteacute) 00378 Sr 00478

RSDr(coefficient de variation de la reacutepeacutetabiliteacute)103 RSDr 130

r(repeatability limit)) 0106 r 0134

SL(eacutecart-type interlaboratoire) -000023 SLlt0 SL 000145

SR(eacutecart-type de reproductibiliteacute) 00378 SR 00611

RSDR(coefficient de variation de la reproductibiliteacute)0010 RSDR 0017

R(limite de reproductibiliteacute)) 0105894 R(limite de reproductibiliteacute))017119

Ref Alt DLaboratoires

Niveau 3

Moyenne si Deviation h k Laboratoires

Niveau 3

Moyenne si Deviation h k

Page 3: ÞÀ¿¸7 ú Y5zñLjÜ< /u +æü t¬Á ... - NF Validation · NF VALIDATION Validation of alternative analytical methods Application in food microbiology ... 500 – 1 000 1 000 –

Thermo Fisher Scientific

ADRIA Deacuteveloppement 338 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

The modifications are highlighted

Quality assurance documents related to this study can be consulted upon

request from Oxoid part of Thermo Fisher Scientific

The technical protocol and the result interpretation were realised according to

the EN ISO 16140 and the AFNOR technical rules

o Company Oxoid Ltd

part of THERMO FISHER SCIENTIFIC

Wade Road Basingstoke

Hampshire RG24 8PW UK

o Expert Laboratory ADRIA Deacuteveloppement

ZA Creacrsquoh Gwen

F-29196 QUIMPER Cedex

o Studied method Listeria PrecisTM method for enumeration of

Listeria monocytogenes

o Validation standard NF EN ISO 16140 (October 2003) Food

microbiology ndash Protocol for the validation of

alternative methods

o Standard method ISO 11290-2 (1999) amp ISO 11290-2A1 (2004)

Horizontal method for the detection and

enumeration of Listeria monocytogenes - Part 2

enumeration of Listeria monocytogenes in foods

o Scope All human food products and environmental

samples

o Certification organism AFNOR Certification

Thermo Fisher Scientific

ADRIA Deacuteveloppement 438 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

1 INTRODUCTION

11 Dates of the validation studies

The Listeria PrecisTM method for enumeration of Listeria monocytogenes was

validated on September 15 2006 (certificate number UNI 0305 ndash 0906) for

food products and environmental samples

In 2007 an extension study was obtained for a new confirmation test OBIS

MONO

The alternative method was renewed in 2010 and 2014

12 Protocol and principle of the alternative method

The Listeria PrecisTM method is based on a specific chromogenic media for

Listeria monocytogenes enumeration

The protocol is described in Appendix 1

13 Standard method

The reference method is the ISO 11290-2 (1998) amp ISO 11290-2A1 (2004)

Horizontal method for the detection and enumeration of Listeria

monocytogenes - Part 2 enumeration of Listeria monocytogenes in foods

(See Appendix 2)

Analysis performed according to the COFRAC accreditation

Thermo Fisher Scientific

ADRIA Deacuteveloppement 538 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

2 VALIDATION STUDY RESULTS

21 Initial validation (realised by ASEPT 2006)

211 Method Comparison Study

2111 Linearity

Linearity is the ability of the method when used with a given matrix to give results that are in proportion

to the amount of analyte present in the sample that is an increase in analyte corresponds to a linear or

proportional increase in results

Food matrices and protocols

Six matrixstrain pairs were analysed with five contamination levels and two

replicates At least 50 analyses were performed by the alternative and the

standard methods

The contamination levels the samples and the inoculated strains are

presented in the table below

Table 1

Samples Strains Contamination levels

(CFUg)

Rillettes Listeria monocytogenes 4e 10 ndash 50

50 ndash 100

100 ndash 500

500 ndash 1 000

1 000 ndash 10 000

Raw milk Listeria monocytogenes 12a

Lettuce Listeria monocytogenes 12a

Smoked salmon Listeria monocytogenes 12b

Raw egg product Listeria monocytogenes 12b

Process water Listeria monocytogenes 12a

The samples were individually inoculated and were analysed in duplicate by

the alternative and the reference methods

Results

The bi-dimensional graphs are shown figure 1

Thermo Fisher Scientific

ADRIA Deacuteveloppement 638 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Figure 1 ndash Bi-dimensional graphs

Rillettes Raw milk

Smoked salmon Raw egg product

Lettuce Process water

Thermo Fisher Scientific

ADRIA Deacuteveloppement 738 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Statistical interpretations

Statistical interpretation results are provided in Table 2

Table 2 ndash Statistical interpretations

Matrix R Selected

regression

RobF Critical

value

P Correlation

coefficient

Regression equation

Rillettes 180 GMFR 609 541 4 09960 log (Alt) = 09887 log (Ref) + 01141

Lettuce 108 GMFR 000 541 100 09992 Log (Alt) = 10081 log (Ref) - 00257

Raw milk 102 GMFR 000 541 100 09997 Log (Alt) = 10188 log (Ref) - 00737

Smoked salmon 1048 OLS1 1806 541 263 09984 Log (Alt) = 10406 log (Ref) - 01418

Raw egg product 130 GMFR 548 541 49 09958 Log (Alt) = 09775 log (Ref) - 00161

Process water 184 GMFR 0079 541 969 09985 Log (Alt) = 09680 log (Ref) + 01317

GMFR = orthogonal linear regression

OLS1 = ordinary least-square linear regression

x ndash axis = reference method

y ndash axis = alternative method

OLS1 = ordinary least-square linear regression

x axis = alternative method

y axis = reference method

Statistical Interpretation

P gt 5 not significant 1 lt P lt 5 significant

P lt 1 highly significant

The regressions straight lines are shown figure 2

Thermo Fisher Scientific

ADRIA Deacuteveloppement 838 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Figure 2 ndash Regression straight lines

Lineacuteariteacute - Œuf

Process water Process water (2) Process water

Smoked salmon (GMFR) Smoked salmon (OLS 1)

Raw milk Raw egg product

Rillettes Lettuce

Thermo Fisher Scientific

ADRIA Deacuteveloppement 938 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Discussion

The linearity is accepted in all cases with

P values 5 and correlation coefficients superior to 099

For the matrix ldquoRillettesldquo the linearity test is significant (P = 4) with a high

correlation coefficient value (0996)

The Listeria PrecisTM method shows satisfying linearity

2112 Relative accuracy

The accuracy is the closeness of agreement between a test result and the accepted reference value

The bias is the difference between the expectation of the test results and an accepted reference value

Number and nature of the samples

The repartition per category and types is provided in table 3

Table 3 ndash Number and nature of the samples

Categories Types Number of samples

Analysed Exploited (1)

Meat products

Raw meat products 6 4

Ready to cook products 4 2

Ready to eat delicatessen 10 4

Total 20 10

Dairy products

Raw milk cheeses 7 4

Raw milks 4 3

Other dairy products 5 5

Total 16 12

Fishery products

Raw fishes 5 4

Processed fishes 7 2

Ready-to-eat products 6 4

Total 18 10

Vegetables

Raw vegetables 5 4

Frozen vegetables 3 3

Lettuce raw vegetables 5 5

Total 13 12

Egg products

Raw egg 5 5

Pastries 3 3

Pasteurised egg products 4 3

Total 12 11

Environmental samples

Wipes swabs 8 4

Process water 3 3

Dusts 3 3

Total 14 10

TOTAL 93 65

(1) Some data cannot be exploited because enumeration was under the detection level for at least one of the

method

Thermo Fisher Scientific

ADRIA Deacuteveloppement 1038 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Artificial contamination of the samples

Artificial contaminations were realised by spiking after performing injury

protocols on pure cultures or by cross-contaminations

56 samples were artificially contaminated 53 gave exploitable results among

a total of 65 samples 8150 of the samples were artificially contaminated

The injury protocol and the strains inoculated are provided in Table 4

Table 4

Stress Ndeg Injury protocol Strain Origin

A 45 min at 50 degC

48

LM -H123

LM-H170

Smoked salmon

Lettuce

Lettuce

B 30 min at 50 degC

and 2 h at ndash20degC

18

43

LM-O1

Raw milk (environment)

Environment

Egg product

C 30 min at 55degC

18

48

LM-H123

Raw milk

Smoked salmon

Lettuce

D 30 min at 50degC

et 2 h at ndash80degC

34

LM-H171

LM-O1

Rillettes

Lettuce

Egg product

E 30 min at 60 degC LM-O1 Egg product

Confirmatory tests

For the reference and the alternative methods confirmations were realised

by the tests described in the ISO 12290-2A1 standard For the alternative

method only one colony was confirmed while 5 colonies were tested for the

reference method

Results

Samples were analysed in duplicate by the reference and the alternative

methods

The contamination range is provided in table 5 per tested category

Thermo Fisher Scientific

ADRIA Deacuteveloppement 1138 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Table 5 ndash Contamination range

Food category Contamination level (log CFUg)

Meat products 100 ndash 404

Dairy products 148 ndash 408

Fishery products 100 ndash 384

Vegetables 170 ndash 511

Egg products 130 ndash 499

Environmental samples 170 ndash 511

All categories 100 ndash 511

Bi-dimensional graphs are given figure 3

Figure 3 ndash Bi-dimensional graphs

Graphe bi-dimensionnel Produits carneacutes

Fishery products

Vegetables

Egg products

Meat products

Thermo Fisher Scientific

ADRIA Deacuteveloppement 1238 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Statistical interpretation according to EN ISO16140 method

The results of the statistical interpretation are given in Table 6

Table 6 ndash Statistical interpretation results

Category n R Regression

used a t(a) b t(b) Critical T

P

Ordinate

at 0

Slope

at 1

Meat products 10 158 GMFR -0455 3321 115 3123 2306 001 001

Dairy products 12 065 GMFR -0027 0385 1002 0108 2228 092 071

Fishery products 10 130 GMFR -0305 1024 1034 0312 2306 076 034

Vegetables 12 086 GMFR -0015 0128 0993 0191 2228 085 090

Egg products 11 158 GMFR -0038 0866 1001 0090 2262 093 041

Environmental

samples 10 176 GMFR -0139 265 1034 231 2306 005 003

All products 65 118 GMFR -0182 3042 1040 2145 2000 004 000

All products

(estimated number) 65 118 GMFR -0182 3042 1040 2145 2000 004 000

All products (non

estimated results) 20 115 GMFR -0367 151 1138 1141 2101 027 015

Repeatability limits obtained for the alternative and reference methods as

well as bias obtained are presented in Table 7

Dairy products Environmental samples

Log (Ref) Log (Ref)

All categories

Thermo Fisher Scientific

ADRIA Deacuteveloppement 1338 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Table 7

Categories Bias

Repeatability limits

Rob r

Ref Alt

Meat products 0008 0314 0343

Dairy products -0031 0136 0130

Fishery products -0133 0220 0214

Vegetables -0041 0180 0319

Egg products -0026 0219 0206

Environmental samples -0007 0198 0119

All products -0032 0196 0215

Regression straight lines (graph and equation representations) for each food

category and for all products are presented figure 4

Thermo Fisher Scientific

ADRIA Deacuteveloppement 1438 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Figure 4 ndash Regression straight lines

Meat products

Fishery products

Dairy products

Vegetables

Thermo Fisher Scientific

ADRIA Deacuteveloppement 1538 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Environmental samples

Egg products

All products (estimated number)

Thermo Fisher Scientific

ADRIA Deacuteveloppement 1638 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

The hypotheses that the ordinates are close to 0 and the slopes close to 1

are approved in all cases except for

- Meat products (slope P = 1 ordinate P = 1)

- Environmental samples (ordinate P = 3)

- All products (ordinate P = 0)

The bias between the two methods vary from ndash 0133 to 0008 log CFU For

all categories the bias is ndash 0032 log CFU The repeatability limits of the

alternative and the reference method are similar For all products the values

are respectively 0215 and 0196 log CFU For all products these

interpretations are provided taking into account data with low contamination

level or not (estimated colonies number)

For all food products the regression straight line is the following

log (Alt) = 1040 log Ref ndash 0182

The Listeria PrecisTM method shows satisfying relative accuracy

All products

All products (Level 2)

Thermo Fisher Scientific

ADRIA Deacuteveloppement 1738 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

2113 Detection limit (LOD) and quantification limit (LOQ)

The critical level is the defined as the smallest amount which can be detected (not null) but not

quantified as an exact value Below this value it cannot be sure that the true value is not null

The detection limit is defined as being higher than the critical level because it involves a power the

probability 1- which has to be well over 50 for example 95

The quantification limit is defined as the smallest amount of analyte (that is the lowest actual number of

organisms) which can be measured and quantified with defined precision and accuracy under the

experimental conditions by the method under validation

Protocol

The detection limit of the alternative method was realised with pure cultures

Three different inoculation levels were tested with six replicates per level ie

18 analyses were done by the alternative method

Listeria monocytogenes 18 was used for the detection limit of the alternative

method

Quantification limit was calculated for six independent blank samples

determinations

Results

These data are intrinsic to the method used and are presented in the

following tables

Table 8 ndash Inoculation 1 ml on 3 plates

Level (CFUml) Positive samples number Standard deviation Bias

0 06

05 26 1211 0

1 36 0816 05

5 66 2483 85

Thermo Fisher Scientific

ADRIA Deacuteveloppement 1838 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Table 9 ndash Inoculation 01 ml on 1 plate

Level (CFUml) Positive samples number Standard deviation Bias

0 06

5 36 0548 05

10 46 1414 2

50 66 2258 55

Table 10

Formulas Obtained values

LC 165 S0 + X0 18

LOD 33 S0 + X0 32

LOQ 10 S0 + X0 87

2114 Relative Sensitivity

The relative sensitivity is defined as the ability of the alternative method to detect two different amounts

of analyte measured by the reference method within a given matrix at a specified average value or

over the whole measurement range that is it is the minimal quantity variation (increase of the analyte

concentration x) which gives a significant variation of the measured signal (response y)

These data are intrinsic to the used method and are obtained from the results

of the linearity study

Sensitivity patterns obtained for tested (matrixstrain) pairs are presented in

figure 5

Thermo Fisher Scientific

ADRIA Deacuteveloppement 1938 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Figure 5 ndash Sensitivity patterns for the tested (matrixstrain) pairs

2115 SpecificitySelectivity

The specificity is defined as the degree to which a method is affected (or not) by the other components

present in a multi-component sample That is the ability of a method to measure exactly a given

analyte or its amount within the sample without interference from non-target components such as a

matrix effect or background noise

The selectivity is defined as a measure of the degree of non-interference in the presence of non-target

analytes A method is selective if it can be used to detect the analyte under examination and that a

guarantee can be provided that the detected signal can only be a product by that specific analyte

The specificity and selectivity study was performed for the Listeria PrecisTM

detection method no additional test was realised for the enumeration

method The results were the following

- Among the Listeria monocytogenes strains tested 2 strains (Listeria

monocytogenes CIP 7834 and Listeria monocytogenes CIP 105459) gave

negative results These 2 strains were tested twice by running 48 h

incubation time of the compared agars Positive results were then

observed by the alternative method Pale characteristic colonies were

observed on the ISO agar by streaking the Half Fraser broth only

- No cross reaction was observed with the non-target strains except with

Listeria ivanovii which gave characteristic colonies on Brilliancetrade Listeria

Agar but the confirmatory tests provide a clear discrimination between

Listeria monocytogenes and Listeria ivanovi species

Rillettes

Lettuce

Raw milk

Smoked salmon

Raw egg

Process water

x (CFUg)

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2038 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

212 Practicability

1 Packaging Ready to use Brilliance Listeria agar plates are available

(x 10 plates)

2 Reagent volume

3 Storage conditionsndash

Shelf life of products

not opened

The plates are stored at 6 - 12degC in darkness conditions The

expiration date is mentioned on the package and on the plates It

is around 3 months

4 Modalities of use

after first use

5 Required

equipments No specific equipment is required

6 Reagents Ready to use plates

7 Training 05 day

8 Real time handling

Reference method

9 min for one sample

55 min for 10 samples

Alternative method

7 min for one sample

35 minutes for 10 samples

9 Time to result

Negative results

Reference method Day 2

Alternative method Day 2

Negative results after confirmation or positive results

Reference method Day 4 ndash Day 7

Alternative method Day 4 ndash Day 7

Positive results

Reference method Day 4 ndash Day 7

Alternative method Day 2 ndash Day 3 (with OBIS Mono Test)

or Day 4 ndash Day 7 (by the classical tests described in the

standardised method)

10 Technician skill Identical to that required for the reference method

11 Steps common to

the reference method Mother suspension revivification step and decimal dilutions

12 Traceability

13 Maintenance

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2138 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

213 Inter-laboratory study

2131 Organisation

12 laboratories participated to this study Pasteurised milk was inoculated by

Listeria monocytogenes 12a isolated from raw milk The targeted

inoculation levels were

- 0 CFUml

- 50 CFU ml

- 500 CFU ml

- 5 000 CFU ml

Each laboratory received eight flasks of 10 ml sample ie two flasks per

inoculation level Furthermore one non-inoculated sample has been added

to the package for total viable count microflora (NF ISO 4833 method)

Detailed instructions were transmitted to the collaborators by the expert

laboratory

Collaborative study laboratories and the expert laboratory carried out the

analyses with the alternative and reference methods

2132 Verification of experimental parameters

Strain stability during transport

In order to evaluate the Listeria monocytogenes strain stability during

transport bacterial count of all inoculated flasks were checked at different

time ie inoculation time after 24 h of conservation at 2deg - 8degC Results are

reported in table 11

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2238 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Table 11 ndash Listeria monocytogenes count

by ISO 11290-2A1 method (log CFUml)

Day of the analyses Level contamination Reference method

Day 0

(29052006)

1 45

2 440

3 4 500

Day 1

(30052006)

1 60

2 430

3 4 900

No evolution of the strain was observed after storage for 24 h at 2degC ndash 8degC

Contamination level

The real contamination levels are provided in table 12

Table 12 ndash Real contamination level (CFUml)

Target inoculation

(CFU ml) Samples Ndeg

Real contamination levels

(CFUml)

0 1 - 8 0

50 5 - 6 45

500 3 - 7 440

5000 2 - 4 4 500

Sample temperature at receipt

Measured temperatures at receipt are listed in table 13

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2338 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Table 13 - Sample temperature on receipt

Laboratories Receipt

day

Hour

receipt

Measured temperature

at receipt (degC)

Measured temperature by the

temperature probe (degC)

A

B

C

D

E

F

G

H

I

J

K

L

M

30052006

10h45

10h15

11h15

10h00

09h30

8h30

11h50

10h00

08h30

14h45

08h30

09h45

08h45

82

55

45

63

60

62

81

38

48

-

14

67

76

03

03

-13

03

04

-14

05

04

01

05

-13

04

Conclusion

The temperatures during the transport were correct for all the labs except for

Lab J which didnrsquot provide the information

All the labs received their packages with several broken flasks

Three labs (B D and L) received some damaged samples but proceeded to

analyses

All the labs started the analyses at Day 1 except Lab J

2133 Results

Aerobic mesophilic flora

The count of aerobic mesophilic flora varied from 94 102 CFUml to

33 103 CFUml

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2438 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Expert lab results

The results obtained by the expert lab are the following

Table 14

Target inoculation level (CFUml)

Reference method ISO 11290-2

Alternative method Listeria Precis Enumeration

Rep 1 Rep 2 Rep 1 Rep 2

lt 10 lt 10 lt 10 lt 10 lt10

50 50 (Ne) 60 (Ne) 50 (Ne) 30 (Ne)

500 470 390 490 500

5 000 4 700 5 100 5 900 3 400

Ne estimated number

The obtained results are in agreement with those expected

Collaborator Labs results

A synthesis of the results obtained by the collaborator labs is provided

Table 15 (CFUml) and Table 16 (log CFUml)

Table 15 ndash Synthesis of results obtained by the alternative

and the reference methods (CFUml)

Labs Level 1 Level 2 Level 2 bis Level 3

Reference method

Alternative method

Reference method

Alternative method

Reference method

Alternative method

Reference method

Alternative method

A 40 40 70 40 480 410 210 340 1000 300 400 300 4900 4600 4900 5100

B 40 15 40 20 350 300 360 450 350 650 800 600 4100 4700 5100 3700

C 50 20 20 70 440 430 480 450 300 650 300 500 4600 5400 4600 5000

D 40 60 40 40 490 460 600 540 250 450 600 1000 4900 4900 4400 4600

E 50 40 50 20 490 520 530 560 750 550 400 200 5400 4500 4100 5400

F 60 20 60 50 370 250 300 130 300 350 600 300 4400 4700 4900 5900

G 70 35 120 50 440 390 570 370 450 550 700 700 5000 4500 4000 4400

H 110 40 100 90 520 440 450 540 150 650 300 300 4200 4700 5600 4800

L 35 60 20 40 390 390 170 380 650 350 800 200 5300 4600 5600 5900

M 45 45 30 40 450 490 550 410 400 550 800 180 4900 4900 6400 4800

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2538 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Table 16 ndash Synthesis of results obtained by the alternative

and the reference methods (log CFUml)

Labs Level 1 Level 2 Level 2 bis Level 3

Reference method

Alternative method

Reference method

Alternative method

Reference method

Alternative method

Reference method

Alternative method

A 1602 1602 1845 1602 2681 2613 2322 2531 3000 2477 2602 2477 3690 3663 3690 3708

B 1602 1176 1602 1301 2544 2477 2556 2653 2544 2813 2903 2778 3613 3672 3708 3568

C 1699 1301 1301 1845 2643 2633 2681 2653 2477 2813 2477 2699 3663 3732 3663 3699

D 1602 1778 1602 1602 2690 2663 2778 2732 2398 2653 2778 3000 3690 3690 3643 3663

E 1699 1602 1699 1301 2690 2716 2724 2748 2875 2740 2602 2301 3732 3653 3613 3732

F 1778 1301 1778 1699 2568 2398 2477 2114 2477 2544 2778 2477 3643 3672 3690 3771

G 1845 1544 2079 1699 2643 2591 2756 2568 2653 2740 2845 2845 3699 3653 3602 3643

H 2041 1602 2000 1954 2716 2643 2653 2732 2176 2813 2477 2477 3623 3672 3748 3681

L 1544 1778 1301 1602 2591 2591 2230 2580 2813 2544 2903 2301 3724 3663 3748 3771

M 1653 1653 1477 1602 2653 2690 2740 2613 2602 2740 2903 2255 3690 3690 3806 3681

Level 2 results obtained taking account the 3 plates inoculated with the mother-suspension

Level 2 bis results obtained taking account the plates inoculated with 01 ml of the mother-suspension

Scrutiny of the measurement results for consistency

In order to identify the measurement results or laboratories that could be

inconsistent two graphical consistency techniques were realised Mandelrsquos

h-ank-statistics Mandel indicators h and k at 5 significance highlighted

some possible inconsistent data (See tables and graphics in Appendix 3 and

Appendix 4)

Mandelrsquos values Number of values observed over the threshold

Reference method Alternative method

h gt 1 Lab F Level 2 Lab F Level 2

h gt 5 Lab F Level 2

Lab B Levels 1 and 2

Lab A Level 2

Lab D Level 3

Lab F Level 2

Lab L Level 2

kgt 1 Lab F Level 3

k gt 5 Lab F Level 2 Lab C Level 2

Lab F Level 3

Lab L Levels 1 and 3

Lab M Level 1

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2638 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

The emphasized results appear (i) coherent with the targeted contamination

levels (ii) concordant between both methods

All the results were thus kept for the statistical analysis

Comparison of the trueness and precision characteristics of

the reference method and alternative methods

The statistical values are summarised hereafter

Level p

Reference method Alternative method

Repeatability ratio

Reproducibility ratio Median

Repeatability standard deviation

Reproducibility standard deviation

Median Repeatability

standard deviation

Reproducibility standard deviation

1 10 16519 02415 02425 16021 02045 02433 0847 1007

2 10 26551 00543 00659 26719 00914 01325 1682 2011

2 bis 10 26581 02607 02607 25951 01900 01953 0729 0749

3 10 18671 00378 00378 18671 00478 00611 1264 1617

Bias of the alternative method

In order to estimate the bias of the alternative method with regard to the reference method for every

level Dij and t are calculated according to the following equations

RefAlt ijYijYDij

Diffp

Dijit

)2(

)(median

If the t value is superior to 2 the alternative method is significantly biased

with regard to the reference method

The values are given in table 17

Table 17 ndash t (d) values obtained by level

Level Bias t (d) Conclusion

1 0068 114 Non significant bias

2 0021 082 Non significant bias

2 bis - 0037 035 Non significant bias

3 - 0037 043 Non significant bias

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2738 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

The statistical test concludes to a non significant bias for all the inoculation

levels The bias varies from ndash 0037 to 0068 log CFUml

Comparison of the repeatability standard deviations

If the ratio Srj Alt Srj Ref of the repeatability standard deviations of the

alternative method and the reference method is larger than 2 the precision

under repeatability conditions of the alternative method is considered to be

lower than that of the reference method If this ratio is smaller than 05 the

precision under repeatability conditions of the alternative method is

considered to be greater than that of the reference method

The ratio values are given in table 18

Table 18

Level

Repeatability standard deviation

Reference method Alternative method Alternative method

reference method ratio

1 0242 0205 0847

2 0054 0091 1682

2 bis 0261 0190 0729

3 0038 0048 1264

Level Repeatability limit

Reference method Alternative method

1 0676 0573

2 0152 0256

2 bis 0730 0532

3 0106 0134

The ratios of the repeatability standard deviations are below 2 in all cases

The precision under repeatability conditions of the alternative method is

equivalent to that of the reference method

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2838 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Comparison of the reproducibility standard deviations

If the ratio Srj Alt Srj Ref of the reproducibility standard deviations of the alternative method and the

reference method is larger than 2 the precision under reproducibility conditions of the alternative

method is considered to be lower than that of the reference method If this ratio is smaller than 05 the

precision under reproducibility conditions of the alternative method is considered to be greater than

that of the reference method

The ratio values are given in table 19

Table 19

Level

Reproducibility standard deviation

Reference method Alternative method Alternative method

reference method ratio

1 0242 0243 1007

2 0066 0133 2011

2 bis 0261 0195 0749

3 0038 0061 1617

Level Reproducibility limit

Reference method Alternative method

1 0676 0681

2 0185 0371

2 bis 0730 0547

3 0106 0171

The ratios of the reproducibility standard deviations are below or close to 2

The precision under reproducibility conditions of the alternative method is

equivalent to that of the reference method

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2938 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

214 Conclusion

Method comparison study

The Listeria PrecisTM enumeration method shows satisfying linearity and

relative accuracy

The alternative method shows satisfying inclusivity and exclusivity results

Two Listeria monocytogenes strains gave negative results with the

alternative method These strains grew weakly on the reference method

media (OampA Palcam and Oxford)

The limits of repeatability of the alternative method are similar to those of the

reference method

The bias between both methods are low between - 0133 Log CFUg and

- 0008 Log CFUg

The Listeria PrecisTM enumeration method offers the saving for confirmatory

tests

Positive results are obtained with the alternative method in 2-3 days (with

OBIS Mono test) or in 4-7 days (by the classical tests described in the

standardized method) against 4-7 days for the reference method

Inter-laboratory study

The bias between the reference and the alternative methods shows satisfying

and correct values

- level 1 (100 - 1 000 CFUg) bias = + 007 log CFUg

- level 2 (1 000 - 10 000 CFUg) bias = 002 log CFUg

- level 2 bis (1 000 - 10 000 CFUg) bias = - 004 log CFUg

- level 3 (10 000 - 100 000 CFUg) bias = - 004 log CFUg

The repeatability and the reproducibility of the alternative method look similar

to those of the reference method

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3038 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

22 Extension study (realised by ASEPT 2007)

An extension study was performed in 2007 for a new confirmatory test OBIS

MONO

150 Listeria monocytogenes strains and 100 non-target strains were isolated

onto TSAYE and BrillianceTM Listeria Agar plates All the Listeria

monocytogenes and the non-target strains gave the expected results

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3138 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Appendix 1 ndash Flow diagram of the Listeria PrecisTM method

x g sample + 9 x ml buffered peptone water

Revivification step for 1 h plusmn 5 min at 20degC plusmn 1degC

Streaking 1 ml onto 3 Brilliance Listeria Agar plates (mother suspension)

or 01 ml onto 1 Brilliance Listeria Agar plates (mother suspension and decimal dilutions)

Incubation for 48 h plusmn 3 h at 37degC plusmn 1degC

Confirmatory tests on at least one typical colony

- by the tests described in the ISO method

- by applying the OBIS mono test on an isolated colony

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3238 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Appendix 2 ndash ISO 11290-2 (1998) amp ISO 11290-2A1 (2004) Horizontal

method for the detection and enumeration of Listeria monocytogenes -

Part 2 enumeration of Listeria monocytogenes in foods

10 g cheese + 90 ml buffered peptone water or Half Fraser Broth (without supplements) (110 dilution)

Stomach 1 min 30 seconds

Keep at 20degC plusmn 2degC for 1 h plusmn 5 min

Decimal dilutions in 9 ml peptone salt tubes

Dilution 110

Dilution 1100

Dilution 11000

1 ml on 3 Chromogenic Listeria medium (ISO) plates (x 2) (-1)

01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-2)

01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-3)

01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-4)

Incubation at 37degC plusmn 1degC

24 h - 48 h

Numerate typical colonies at 24 h

(bluegreen surrounded by an opaque halo)

Numerate at 48 h

Streak typical colonies on TSYEA

(5 coloniesplate 2 successive dilutions)

Confirmation tests

Catalase

Gram stain

For confirmation of L monocytogenes

Haemolysis

Carbohydrate (rhamnose and xylose) utilisation

CAMP Test

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3338 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Appendix 3 ndash Mandelrsquos graphics

Laboratoire h Ref

Niveau 1 h Ref

Niveau 2 h Ref

Niveau 3 h Alt

Niveau 1 h Alt

Niveau 2 h Alt

Niveau 3 h5 h1 h5 h1

A -050 -015 083 062 -212 -039 198 299 -198 -299

B -261 -270 021 -077 -058 173 198 299 -198 -299

C -151 -031 -014 -015 -004 -005 198 299 -198 -299

D 038 040 -137 000 072 207 198 299 -198 -299

E -001 090 155 -052 056 -101 198 299 -198 -299

F -112 -321 -153 070 -325 023 198 299 -198 -299

G 042 -071 040 147 -009 176 198 299 -198 -299

H 169 046 -169 192 018 -083 198 299 -198 -299

L 009 -120 021 -077 -231 005 198 299 -198 -299

M 001 031 014 -032 004 -011 198 299 -198 -299

-300

-200

-100

000

100

200

300

A B C D E F G H L M

Vale

urs

h d

e M

an

del(

rob

uste

s)

Laboratoires

Meacutethode de reacutefeacuterence analyse robuste

h Ref Niveau 1

h Ref Niveau 2

h Ref Niveau 3

h 5=198

h 1=299

-300

-200

-100

000

100

200

300

A B C D E F G H L M

Vale

urs

de h

de M

an

del(

rob

uste

s)

Laboratoires

Meacutethode alternative analyse robuste

h Alt Niveau 1

h Alt Niveau 2

h Alt Niveau 2

h 1=299

h 5=198

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3438 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Laboratoire k Ref

Niveau 1 k Ref

Niveau 2 k Ref

Niveau 3 k Alt

Niveau 1 k Alt

Niveau 2 k Alt

Niveau 3 k1 k5

A 000 089 142 084 162 046 259 181

B 125 087 073 104 075 046 259 181

C 117 013 091 188 022 083 259 181

D 052 036 069 000 035 083 259 181

E 028 034 037 138 019 112 259 181

F 140 222 018 027 281 112 259 181

G 088 068 024 131 145 000 259 181

H 129 094 173 016 061 000 259 181

L 069 000 073 104 270 224 259 181

M 000 048 038 043 099 241 259 181

000

050

100

150

200

250

300

350

400

A B C D E F G H L M

Vale

urs

k d

e M

an

del(

rob

uste

s)

Laboratoires

Meacutethode de reacutefeacuterence analyse robuste

k Ref Niveau 1k Ref Niveau 2k Ref Niveau 3

k 1=259

k 5=181

000

050

100

150

200

250

300

A B C D E F G H L M

Vale

urs

k d

e M

an

del

(ro

bu

ste

s)

Laboratoires

Meacutethode alternative analyse robuste

k Alt Niveau 3

k Alt Niveau 1

k Alt Niveau 2

k 1=259

k 5=181

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3538 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Appendix 4 ndash Statistical results

Level 1

Nombre de laboratoires (p) 10

A 1602 1602 1602 000 0000 0000 -0495 0000 A 1845 1602 1724 017 0122 -0122 0621 0840 0122

B 1602 1176 1389 030 0213 -0213 -2613 1247 B 1602 1301 1452 021 0151 -0151 -0769 1041 0062

C 1699 1301 1500 028 0199 -0199 -1510 1165 C 1301 1845 1573 038 -0272 0272 -0148 1881 0073

D 1602 1778 1690 012 -0088 0088 0380 0516 D 1602 1602 1602 000 0000 0000 0000 0000 -0088

E 1699 1602 1651 007 0048 -0048 -0013 0284 E 1699 1301 1500 028 0199 -0199 -0522 1376 -0151

F 1778 1301 1540 034 0239 -0239 -1116 1397 F 1778 1699 1739 006 0040 -0040 0698 0274 0199

G 1845 1544 1695 021 0151 -0151 0425 0881 G 2079 1699 1889 027 0190 -0190 1467 1315 0194

H 2041 1602 1822 031 0220 -0220 1689 1286 H 2000 1954 1977 003 0023 -0023 1917 0158 0155

L 1544 1778 1661 017 -0117 0117 0092 0685 L 1301 1602 1452 021 -0151 0151 -0769 1041 -0210

M 1653 1653 1653 000 0000 0000 0013 0000 M 1477 1602 1540 009 -0062 0062 -0319 0432 -0114

m 165186 m 160206 mediane D 00678

Cn(2p) 1867 Cn(2p) 1867 Cp 16101

Cn(p) 16101 Cn(p) 16101 Qn(D) 0092926

n=2p n 20 n=2p n 20 Qdiff 01496169

f 11 f 11 t 114

l 55 l 55

n=p n 10 n=p n 10

f 6 f 6

l 15 l 15

Qn(2p) 0091465 Qn(2p) 007745

Qn(p) 0062469 Qn(p) 0121519

Qintra 017078 Qintra 014461

Qinter 010058 Qinter 019565

Sr(eacutecart-type de reacutepeacutetabiliteacute) 02415 Sr 02045

RSDr(coefficient de variation de la reacutepeacutetabiliteacute)1462 RSDr 1277

r(repeatability limit)) 0676 r 0573

SL(eacutecart-type interlaboratoire) -001905 SLlt0 SL 001737

SR(eacutecart-type de reproductibiliteacute) 02415 SR 02433

RSDR(coefficient de variation de la reproductibiliteacute)0146 RSDR 0152

R(limite de reproductibiliteacute)) 06762 R(limite de reproductibiliteacute))068123

h k h MoyenneLaboratoires

Niveau 1

si LaboratoiresDeviationMoyenne Dk DeviationsiAlt

Niveau 1

Ref

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3638 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Level 2

Nombre de laboratoires (p) 10

A 2681 2613 2647 005 0034 -0034 -0151 0891 A 2322 2531 2427 015 -0105 0105 -2119 1620 -0220

B 2544 2477 2511 005 0033 -0033 -2699 0872 B 2556 2653 2605 007 -0048 0048 -0581 0750 0094

C 2643 2633 2638 001 0005 -0005 -0311 0130 C 2681 2653 2667 002 0014 -0014 -0040 0217 0029

D 2690 2663 2676 002 0014 -0014 0400 0357 D 2778 2732 2755 003 0023 -0023 0721 0354 0079

E 2690 2716 2703 002 -0013 0013 0897 0336 E 2724 2748 2736 002 -0012 0012 0556 0185 0033

F 2568 2398 2483 012 0085 -0085 -3214 2217 F 2477 2114 2296 026 0182 -0182 -3255 2811 -0188

G 2643 2591 2617 004 0026 -0026 -0707 0682 G 2756 2568 2662 013 0094 -0094 -0085 1453 0045

H 2716 2643 2680 005 0036 -0036 0460 0945 H 2653 2732 2693 006 -0040 0040 0181 0613 0013

L 2591 2591 2591 000 0000 0000 -1196 0000 L 2230 2580 2405 025 -0175 0175 -2307 2704 -0186

M 2653 2690 2672 003 -0018 0018 0311 0481 M 2740 2613 2677 009 0064 -0064 0040 0987 0005

m 2655082 m 26719001 mediane D 00209

Cn(2p) 1867 Cn(2p) 1867 Cp 16101

Cn(p) 16101 Cn(p) 16101 Qn(D) 0039911

n=2p n 20 n=2p n 20 Qdiff 00642591

f 11 f 11 t 082

l 55 l 55

n=p n 10 n=p n 10

f 6 f 6

l 15 l 15

Qn(2p) 0020570 Qn(2p) 00345985

Qn(p) 00332437 Qn(p) 00718158

Qintra 003841 Qintra 006460

Qinter 005352 Qinter 011563

Sr(eacutecart-type de reacutepeacutetabiliteacute) 00543 Sr 00914

RSDr(coefficient de variation de la reacutepeacutetabiliteacute)205 RSDr 342

r(repeatability limit)) 0152 r 0256

SL(eacutecart-type interlaboratoire) 000139 SL 000920

SR(eacutecart-type de reproductibiliteacute) 00659 SR 01325

RSDR(coefficient de variation de la reproductibiliteacute)0025 RSDR 0050

R(limite de reproductibiliteacute)) 018446 R(limite de reproductibiliteacute))03708618

Laboratoires

Niveau 2

Moyenne si Laboratoires

Niveau 2

DRef Alt Moyenne si Deviation h Deviation h k k

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3738 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Level 2 bis

Nombre de laboratoires (p) 10

A 3000 2477 2739 037 0261 -0261 0832 1418 A 2602 2477 2540 009 0062 -0062 -0391 0465 -0199

B 2544 2813 2678 019 -0134 0134 0211 0729 B 2903 2778 2841 009 0062 -0062 1732 0465 0162

C 2477 2813 2645 024 -0168 0168 -0135 0911 C 2477 2699 2588 016 -0111 0111 -0049 0826 -0057

D 2398 2653 2526 018 -0128 0128 -1370 0692 D 2778 3000 2889 016 -0111 0111 2074 0826 0363

E 2875 2740 2808 010 0067 -0067 1547 0365 E 2602 2301 2452 021 0151 -0151 -1012 1120 -0356

F 2477 2544 2511 005 -0033 0033 -1525 0182 F 2778 2477 2628 021 0151 -0151 0230 1120 0117

G 2653 2740 2697 006 -0044 0044 0400 0236 G 2845 2845 2845 000 0000 0000 1764 0000 0148

H 2176 2813 2495 045 -0318 0318 -1692 1727 H 2477 2477 2477 000 0000 0000 -0832 0000 -0017

L 2813 2544 2678 019 0134 -0134 0211 0729 L 2903 2301 2602 043 0301 -0301 0049 2241 -0076

M 2602 2740 2671 010 -0069 0069 0135 0375 M 2903 2255 2579 046 0324 -0324 -0112 2411 -0092

m 2658114 m 259505 mediane D -00372

Cn(2p) 1867 Cn(2p) 1867 Cp 16101

Cn(p) 16101 Cn(p) 16101 Qn(D) 01656915

n=2p n 20 n=2p n 20 Qdiff 02667753

f 11 f 11 t 035

l 55 l 55

n=p n 10 n=p n 10

f 6 f 6

l 15 l 15

Qn(2p) 0098745 Qn(2p) 007195

Qn(p) 006007 Qn(p) 008805

Qintra 018437 Qintra 013435

Qinter 009672 Qinter 014176

Sr(eacutecart-type de reacutepeacutetabiliteacute) 02607 Sr 01900

RSDr(coefficient de variation de la reacutepeacutetabiliteacute)981 RSDr 732

r(repeatability limit)) 0730 r 0532

SL(eacutecart-type interlaboratoire) -002464 SLlt0 SL 000205

SR(eacutecart-type de reproductibiliteacute) 02607 SR 01953

RSDR(coefficient de variation de la reproductibiliteacute)0098 RSDR 0075

R(limite de reproductibiliteacute)) 072996 R(limite de reproductibiliteacute))054686

Laboratoires

Niveau 2 bis

Moyenne si Laboratoires

Niveau 2 bis

DRef Alt Moyenne si Deviation h Deviation h k k

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3838 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Level 3

Nombre de laboratoires (p) 10

A 3690 3663 3676 002 0014 -0014 -0311 0513 A 3690 3708 3699 001 -0009 0009 0354 0257 0022

B 3613 3672 3642 004 -0030 0030 -1851 1109 B 3708 3568 3638 010 0070 -0070 -0844 2061 -0005

C 3663 3732 3698 005 -0035 0035 0645 1302 C 3663 3699 3681 003 -0018 0018 0000 0536 -0017

D 3690 3690 3690 000 0000 0000 0311 0000 D 3643 3663 3653 001 -0010 0010 -0545 0286 -0037

E 3732 3653 3693 006 0040 -0040 0429 1480 E 3613 3732 3673 008 -0060 0060 -0162 1769 -0020

F 3643 3672 3658 002 -0014 0014 -1157 0536 F 3690 3771 3731 006 -0040 0040 0975 1193 0073

G 3699 3653 3676 003 0023 -0023 -0328 0856 G 3602 3643 3623 003 -0021 0021 -1141 0612 -0053

H 3623 3672 3648 003 -0024 0024 -1615 0913 H 3748 3681 3715 005 0033 -0033 0664 0990 0067

L 3724 3663 3694 004 0031 -0031 0461 1150 L 3748 3771 3760 002 -0011 0011 1544 0335 0066

M 3690 3690 3690 000 0000 0000 0311 0000 M 3806 3681 3744 009 0062 -0062 1234 1848 0054

m 368334 m 368086 mediane D 00089

Cn(2p) 1867 Cn(2p) 1867 Cp 16101

Cn(p) 16101 Cn(p) 16101 Qn(D) 00325359

n=2p n 20 n=2p n 20 Qdiff 00523852

f 11 f 11 t 043

l 55 l 55

n=p n 10 n=p n 10

f 6 f 6

l 15 l 15

Qn(2p) 0014323 Qn(2p) 001811

Qn(p) 0013719 Qn(p) 003164

Qintra 002674 Qintra 003381

Qinter 002209 Qinter 005094

Sr(eacutecart-type de reacutepeacutetabiliteacute) 00378 Sr 00478

RSDr(coefficient de variation de la reacutepeacutetabiliteacute)103 RSDr 130

r(repeatability limit)) 0106 r 0134

SL(eacutecart-type interlaboratoire) -000023 SLlt0 SL 000145

SR(eacutecart-type de reproductibiliteacute) 00378 SR 00611

RSDR(coefficient de variation de la reproductibiliteacute)0010 RSDR 0017

R(limite de reproductibiliteacute)) 0105894 R(limite de reproductibiliteacute))017119

Ref Alt DLaboratoires

Niveau 3

Moyenne si Deviation h k Laboratoires

Niveau 3

Moyenne si Deviation h k

Page 4: ÞÀ¿¸7 ú Y5zñLjÜ< /u +æü t¬Á ... - NF Validation · NF VALIDATION Validation of alternative analytical methods Application in food microbiology ... 500 – 1 000 1 000 –

Thermo Fisher Scientific

ADRIA Deacuteveloppement 438 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

1 INTRODUCTION

11 Dates of the validation studies

The Listeria PrecisTM method for enumeration of Listeria monocytogenes was

validated on September 15 2006 (certificate number UNI 0305 ndash 0906) for

food products and environmental samples

In 2007 an extension study was obtained for a new confirmation test OBIS

MONO

The alternative method was renewed in 2010 and 2014

12 Protocol and principle of the alternative method

The Listeria PrecisTM method is based on a specific chromogenic media for

Listeria monocytogenes enumeration

The protocol is described in Appendix 1

13 Standard method

The reference method is the ISO 11290-2 (1998) amp ISO 11290-2A1 (2004)

Horizontal method for the detection and enumeration of Listeria

monocytogenes - Part 2 enumeration of Listeria monocytogenes in foods

(See Appendix 2)

Analysis performed according to the COFRAC accreditation

Thermo Fisher Scientific

ADRIA Deacuteveloppement 538 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

2 VALIDATION STUDY RESULTS

21 Initial validation (realised by ASEPT 2006)

211 Method Comparison Study

2111 Linearity

Linearity is the ability of the method when used with a given matrix to give results that are in proportion

to the amount of analyte present in the sample that is an increase in analyte corresponds to a linear or

proportional increase in results

Food matrices and protocols

Six matrixstrain pairs were analysed with five contamination levels and two

replicates At least 50 analyses were performed by the alternative and the

standard methods

The contamination levels the samples and the inoculated strains are

presented in the table below

Table 1

Samples Strains Contamination levels

(CFUg)

Rillettes Listeria monocytogenes 4e 10 ndash 50

50 ndash 100

100 ndash 500

500 ndash 1 000

1 000 ndash 10 000

Raw milk Listeria monocytogenes 12a

Lettuce Listeria monocytogenes 12a

Smoked salmon Listeria monocytogenes 12b

Raw egg product Listeria monocytogenes 12b

Process water Listeria monocytogenes 12a

The samples were individually inoculated and were analysed in duplicate by

the alternative and the reference methods

Results

The bi-dimensional graphs are shown figure 1

Thermo Fisher Scientific

ADRIA Deacuteveloppement 638 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Figure 1 ndash Bi-dimensional graphs

Rillettes Raw milk

Smoked salmon Raw egg product

Lettuce Process water

Thermo Fisher Scientific

ADRIA Deacuteveloppement 738 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Statistical interpretations

Statistical interpretation results are provided in Table 2

Table 2 ndash Statistical interpretations

Matrix R Selected

regression

RobF Critical

value

P Correlation

coefficient

Regression equation

Rillettes 180 GMFR 609 541 4 09960 log (Alt) = 09887 log (Ref) + 01141

Lettuce 108 GMFR 000 541 100 09992 Log (Alt) = 10081 log (Ref) - 00257

Raw milk 102 GMFR 000 541 100 09997 Log (Alt) = 10188 log (Ref) - 00737

Smoked salmon 1048 OLS1 1806 541 263 09984 Log (Alt) = 10406 log (Ref) - 01418

Raw egg product 130 GMFR 548 541 49 09958 Log (Alt) = 09775 log (Ref) - 00161

Process water 184 GMFR 0079 541 969 09985 Log (Alt) = 09680 log (Ref) + 01317

GMFR = orthogonal linear regression

OLS1 = ordinary least-square linear regression

x ndash axis = reference method

y ndash axis = alternative method

OLS1 = ordinary least-square linear regression

x axis = alternative method

y axis = reference method

Statistical Interpretation

P gt 5 not significant 1 lt P lt 5 significant

P lt 1 highly significant

The regressions straight lines are shown figure 2

Thermo Fisher Scientific

ADRIA Deacuteveloppement 838 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Figure 2 ndash Regression straight lines

Lineacuteariteacute - Œuf

Process water Process water (2) Process water

Smoked salmon (GMFR) Smoked salmon (OLS 1)

Raw milk Raw egg product

Rillettes Lettuce

Thermo Fisher Scientific

ADRIA Deacuteveloppement 938 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Discussion

The linearity is accepted in all cases with

P values 5 and correlation coefficients superior to 099

For the matrix ldquoRillettesldquo the linearity test is significant (P = 4) with a high

correlation coefficient value (0996)

The Listeria PrecisTM method shows satisfying linearity

2112 Relative accuracy

The accuracy is the closeness of agreement between a test result and the accepted reference value

The bias is the difference between the expectation of the test results and an accepted reference value

Number and nature of the samples

The repartition per category and types is provided in table 3

Table 3 ndash Number and nature of the samples

Categories Types Number of samples

Analysed Exploited (1)

Meat products

Raw meat products 6 4

Ready to cook products 4 2

Ready to eat delicatessen 10 4

Total 20 10

Dairy products

Raw milk cheeses 7 4

Raw milks 4 3

Other dairy products 5 5

Total 16 12

Fishery products

Raw fishes 5 4

Processed fishes 7 2

Ready-to-eat products 6 4

Total 18 10

Vegetables

Raw vegetables 5 4

Frozen vegetables 3 3

Lettuce raw vegetables 5 5

Total 13 12

Egg products

Raw egg 5 5

Pastries 3 3

Pasteurised egg products 4 3

Total 12 11

Environmental samples

Wipes swabs 8 4

Process water 3 3

Dusts 3 3

Total 14 10

TOTAL 93 65

(1) Some data cannot be exploited because enumeration was under the detection level for at least one of the

method

Thermo Fisher Scientific

ADRIA Deacuteveloppement 1038 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Artificial contamination of the samples

Artificial contaminations were realised by spiking after performing injury

protocols on pure cultures or by cross-contaminations

56 samples were artificially contaminated 53 gave exploitable results among

a total of 65 samples 8150 of the samples were artificially contaminated

The injury protocol and the strains inoculated are provided in Table 4

Table 4

Stress Ndeg Injury protocol Strain Origin

A 45 min at 50 degC

48

LM -H123

LM-H170

Smoked salmon

Lettuce

Lettuce

B 30 min at 50 degC

and 2 h at ndash20degC

18

43

LM-O1

Raw milk (environment)

Environment

Egg product

C 30 min at 55degC

18

48

LM-H123

Raw milk

Smoked salmon

Lettuce

D 30 min at 50degC

et 2 h at ndash80degC

34

LM-H171

LM-O1

Rillettes

Lettuce

Egg product

E 30 min at 60 degC LM-O1 Egg product

Confirmatory tests

For the reference and the alternative methods confirmations were realised

by the tests described in the ISO 12290-2A1 standard For the alternative

method only one colony was confirmed while 5 colonies were tested for the

reference method

Results

Samples were analysed in duplicate by the reference and the alternative

methods

The contamination range is provided in table 5 per tested category

Thermo Fisher Scientific

ADRIA Deacuteveloppement 1138 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Table 5 ndash Contamination range

Food category Contamination level (log CFUg)

Meat products 100 ndash 404

Dairy products 148 ndash 408

Fishery products 100 ndash 384

Vegetables 170 ndash 511

Egg products 130 ndash 499

Environmental samples 170 ndash 511

All categories 100 ndash 511

Bi-dimensional graphs are given figure 3

Figure 3 ndash Bi-dimensional graphs

Graphe bi-dimensionnel Produits carneacutes

Fishery products

Vegetables

Egg products

Meat products

Thermo Fisher Scientific

ADRIA Deacuteveloppement 1238 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Statistical interpretation according to EN ISO16140 method

The results of the statistical interpretation are given in Table 6

Table 6 ndash Statistical interpretation results

Category n R Regression

used a t(a) b t(b) Critical T

P

Ordinate

at 0

Slope

at 1

Meat products 10 158 GMFR -0455 3321 115 3123 2306 001 001

Dairy products 12 065 GMFR -0027 0385 1002 0108 2228 092 071

Fishery products 10 130 GMFR -0305 1024 1034 0312 2306 076 034

Vegetables 12 086 GMFR -0015 0128 0993 0191 2228 085 090

Egg products 11 158 GMFR -0038 0866 1001 0090 2262 093 041

Environmental

samples 10 176 GMFR -0139 265 1034 231 2306 005 003

All products 65 118 GMFR -0182 3042 1040 2145 2000 004 000

All products

(estimated number) 65 118 GMFR -0182 3042 1040 2145 2000 004 000

All products (non

estimated results) 20 115 GMFR -0367 151 1138 1141 2101 027 015

Repeatability limits obtained for the alternative and reference methods as

well as bias obtained are presented in Table 7

Dairy products Environmental samples

Log (Ref) Log (Ref)

All categories

Thermo Fisher Scientific

ADRIA Deacuteveloppement 1338 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Table 7

Categories Bias

Repeatability limits

Rob r

Ref Alt

Meat products 0008 0314 0343

Dairy products -0031 0136 0130

Fishery products -0133 0220 0214

Vegetables -0041 0180 0319

Egg products -0026 0219 0206

Environmental samples -0007 0198 0119

All products -0032 0196 0215

Regression straight lines (graph and equation representations) for each food

category and for all products are presented figure 4

Thermo Fisher Scientific

ADRIA Deacuteveloppement 1438 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Figure 4 ndash Regression straight lines

Meat products

Fishery products

Dairy products

Vegetables

Thermo Fisher Scientific

ADRIA Deacuteveloppement 1538 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Environmental samples

Egg products

All products (estimated number)

Thermo Fisher Scientific

ADRIA Deacuteveloppement 1638 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

The hypotheses that the ordinates are close to 0 and the slopes close to 1

are approved in all cases except for

- Meat products (slope P = 1 ordinate P = 1)

- Environmental samples (ordinate P = 3)

- All products (ordinate P = 0)

The bias between the two methods vary from ndash 0133 to 0008 log CFU For

all categories the bias is ndash 0032 log CFU The repeatability limits of the

alternative and the reference method are similar For all products the values

are respectively 0215 and 0196 log CFU For all products these

interpretations are provided taking into account data with low contamination

level or not (estimated colonies number)

For all food products the regression straight line is the following

log (Alt) = 1040 log Ref ndash 0182

The Listeria PrecisTM method shows satisfying relative accuracy

All products

All products (Level 2)

Thermo Fisher Scientific

ADRIA Deacuteveloppement 1738 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

2113 Detection limit (LOD) and quantification limit (LOQ)

The critical level is the defined as the smallest amount which can be detected (not null) but not

quantified as an exact value Below this value it cannot be sure that the true value is not null

The detection limit is defined as being higher than the critical level because it involves a power the

probability 1- which has to be well over 50 for example 95

The quantification limit is defined as the smallest amount of analyte (that is the lowest actual number of

organisms) which can be measured and quantified with defined precision and accuracy under the

experimental conditions by the method under validation

Protocol

The detection limit of the alternative method was realised with pure cultures

Three different inoculation levels were tested with six replicates per level ie

18 analyses were done by the alternative method

Listeria monocytogenes 18 was used for the detection limit of the alternative

method

Quantification limit was calculated for six independent blank samples

determinations

Results

These data are intrinsic to the method used and are presented in the

following tables

Table 8 ndash Inoculation 1 ml on 3 plates

Level (CFUml) Positive samples number Standard deviation Bias

0 06

05 26 1211 0

1 36 0816 05

5 66 2483 85

Thermo Fisher Scientific

ADRIA Deacuteveloppement 1838 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Table 9 ndash Inoculation 01 ml on 1 plate

Level (CFUml) Positive samples number Standard deviation Bias

0 06

5 36 0548 05

10 46 1414 2

50 66 2258 55

Table 10

Formulas Obtained values

LC 165 S0 + X0 18

LOD 33 S0 + X0 32

LOQ 10 S0 + X0 87

2114 Relative Sensitivity

The relative sensitivity is defined as the ability of the alternative method to detect two different amounts

of analyte measured by the reference method within a given matrix at a specified average value or

over the whole measurement range that is it is the minimal quantity variation (increase of the analyte

concentration x) which gives a significant variation of the measured signal (response y)

These data are intrinsic to the used method and are obtained from the results

of the linearity study

Sensitivity patterns obtained for tested (matrixstrain) pairs are presented in

figure 5

Thermo Fisher Scientific

ADRIA Deacuteveloppement 1938 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Figure 5 ndash Sensitivity patterns for the tested (matrixstrain) pairs

2115 SpecificitySelectivity

The specificity is defined as the degree to which a method is affected (or not) by the other components

present in a multi-component sample That is the ability of a method to measure exactly a given

analyte or its amount within the sample without interference from non-target components such as a

matrix effect or background noise

The selectivity is defined as a measure of the degree of non-interference in the presence of non-target

analytes A method is selective if it can be used to detect the analyte under examination and that a

guarantee can be provided that the detected signal can only be a product by that specific analyte

The specificity and selectivity study was performed for the Listeria PrecisTM

detection method no additional test was realised for the enumeration

method The results were the following

- Among the Listeria monocytogenes strains tested 2 strains (Listeria

monocytogenes CIP 7834 and Listeria monocytogenes CIP 105459) gave

negative results These 2 strains were tested twice by running 48 h

incubation time of the compared agars Positive results were then

observed by the alternative method Pale characteristic colonies were

observed on the ISO agar by streaking the Half Fraser broth only

- No cross reaction was observed with the non-target strains except with

Listeria ivanovii which gave characteristic colonies on Brilliancetrade Listeria

Agar but the confirmatory tests provide a clear discrimination between

Listeria monocytogenes and Listeria ivanovi species

Rillettes

Lettuce

Raw milk

Smoked salmon

Raw egg

Process water

x (CFUg)

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2038 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

212 Practicability

1 Packaging Ready to use Brilliance Listeria agar plates are available

(x 10 plates)

2 Reagent volume

3 Storage conditionsndash

Shelf life of products

not opened

The plates are stored at 6 - 12degC in darkness conditions The

expiration date is mentioned on the package and on the plates It

is around 3 months

4 Modalities of use

after first use

5 Required

equipments No specific equipment is required

6 Reagents Ready to use plates

7 Training 05 day

8 Real time handling

Reference method

9 min for one sample

55 min for 10 samples

Alternative method

7 min for one sample

35 minutes for 10 samples

9 Time to result

Negative results

Reference method Day 2

Alternative method Day 2

Negative results after confirmation or positive results

Reference method Day 4 ndash Day 7

Alternative method Day 4 ndash Day 7

Positive results

Reference method Day 4 ndash Day 7

Alternative method Day 2 ndash Day 3 (with OBIS Mono Test)

or Day 4 ndash Day 7 (by the classical tests described in the

standardised method)

10 Technician skill Identical to that required for the reference method

11 Steps common to

the reference method Mother suspension revivification step and decimal dilutions

12 Traceability

13 Maintenance

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2138 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

213 Inter-laboratory study

2131 Organisation

12 laboratories participated to this study Pasteurised milk was inoculated by

Listeria monocytogenes 12a isolated from raw milk The targeted

inoculation levels were

- 0 CFUml

- 50 CFU ml

- 500 CFU ml

- 5 000 CFU ml

Each laboratory received eight flasks of 10 ml sample ie two flasks per

inoculation level Furthermore one non-inoculated sample has been added

to the package for total viable count microflora (NF ISO 4833 method)

Detailed instructions were transmitted to the collaborators by the expert

laboratory

Collaborative study laboratories and the expert laboratory carried out the

analyses with the alternative and reference methods

2132 Verification of experimental parameters

Strain stability during transport

In order to evaluate the Listeria monocytogenes strain stability during

transport bacterial count of all inoculated flasks were checked at different

time ie inoculation time after 24 h of conservation at 2deg - 8degC Results are

reported in table 11

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2238 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Table 11 ndash Listeria monocytogenes count

by ISO 11290-2A1 method (log CFUml)

Day of the analyses Level contamination Reference method

Day 0

(29052006)

1 45

2 440

3 4 500

Day 1

(30052006)

1 60

2 430

3 4 900

No evolution of the strain was observed after storage for 24 h at 2degC ndash 8degC

Contamination level

The real contamination levels are provided in table 12

Table 12 ndash Real contamination level (CFUml)

Target inoculation

(CFU ml) Samples Ndeg

Real contamination levels

(CFUml)

0 1 - 8 0

50 5 - 6 45

500 3 - 7 440

5000 2 - 4 4 500

Sample temperature at receipt

Measured temperatures at receipt are listed in table 13

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2338 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Table 13 - Sample temperature on receipt

Laboratories Receipt

day

Hour

receipt

Measured temperature

at receipt (degC)

Measured temperature by the

temperature probe (degC)

A

B

C

D

E

F

G

H

I

J

K

L

M

30052006

10h45

10h15

11h15

10h00

09h30

8h30

11h50

10h00

08h30

14h45

08h30

09h45

08h45

82

55

45

63

60

62

81

38

48

-

14

67

76

03

03

-13

03

04

-14

05

04

01

05

-13

04

Conclusion

The temperatures during the transport were correct for all the labs except for

Lab J which didnrsquot provide the information

All the labs received their packages with several broken flasks

Three labs (B D and L) received some damaged samples but proceeded to

analyses

All the labs started the analyses at Day 1 except Lab J

2133 Results

Aerobic mesophilic flora

The count of aerobic mesophilic flora varied from 94 102 CFUml to

33 103 CFUml

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2438 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Expert lab results

The results obtained by the expert lab are the following

Table 14

Target inoculation level (CFUml)

Reference method ISO 11290-2

Alternative method Listeria Precis Enumeration

Rep 1 Rep 2 Rep 1 Rep 2

lt 10 lt 10 lt 10 lt 10 lt10

50 50 (Ne) 60 (Ne) 50 (Ne) 30 (Ne)

500 470 390 490 500

5 000 4 700 5 100 5 900 3 400

Ne estimated number

The obtained results are in agreement with those expected

Collaborator Labs results

A synthesis of the results obtained by the collaborator labs is provided

Table 15 (CFUml) and Table 16 (log CFUml)

Table 15 ndash Synthesis of results obtained by the alternative

and the reference methods (CFUml)

Labs Level 1 Level 2 Level 2 bis Level 3

Reference method

Alternative method

Reference method

Alternative method

Reference method

Alternative method

Reference method

Alternative method

A 40 40 70 40 480 410 210 340 1000 300 400 300 4900 4600 4900 5100

B 40 15 40 20 350 300 360 450 350 650 800 600 4100 4700 5100 3700

C 50 20 20 70 440 430 480 450 300 650 300 500 4600 5400 4600 5000

D 40 60 40 40 490 460 600 540 250 450 600 1000 4900 4900 4400 4600

E 50 40 50 20 490 520 530 560 750 550 400 200 5400 4500 4100 5400

F 60 20 60 50 370 250 300 130 300 350 600 300 4400 4700 4900 5900

G 70 35 120 50 440 390 570 370 450 550 700 700 5000 4500 4000 4400

H 110 40 100 90 520 440 450 540 150 650 300 300 4200 4700 5600 4800

L 35 60 20 40 390 390 170 380 650 350 800 200 5300 4600 5600 5900

M 45 45 30 40 450 490 550 410 400 550 800 180 4900 4900 6400 4800

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2538 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Table 16 ndash Synthesis of results obtained by the alternative

and the reference methods (log CFUml)

Labs Level 1 Level 2 Level 2 bis Level 3

Reference method

Alternative method

Reference method

Alternative method

Reference method

Alternative method

Reference method

Alternative method

A 1602 1602 1845 1602 2681 2613 2322 2531 3000 2477 2602 2477 3690 3663 3690 3708

B 1602 1176 1602 1301 2544 2477 2556 2653 2544 2813 2903 2778 3613 3672 3708 3568

C 1699 1301 1301 1845 2643 2633 2681 2653 2477 2813 2477 2699 3663 3732 3663 3699

D 1602 1778 1602 1602 2690 2663 2778 2732 2398 2653 2778 3000 3690 3690 3643 3663

E 1699 1602 1699 1301 2690 2716 2724 2748 2875 2740 2602 2301 3732 3653 3613 3732

F 1778 1301 1778 1699 2568 2398 2477 2114 2477 2544 2778 2477 3643 3672 3690 3771

G 1845 1544 2079 1699 2643 2591 2756 2568 2653 2740 2845 2845 3699 3653 3602 3643

H 2041 1602 2000 1954 2716 2643 2653 2732 2176 2813 2477 2477 3623 3672 3748 3681

L 1544 1778 1301 1602 2591 2591 2230 2580 2813 2544 2903 2301 3724 3663 3748 3771

M 1653 1653 1477 1602 2653 2690 2740 2613 2602 2740 2903 2255 3690 3690 3806 3681

Level 2 results obtained taking account the 3 plates inoculated with the mother-suspension

Level 2 bis results obtained taking account the plates inoculated with 01 ml of the mother-suspension

Scrutiny of the measurement results for consistency

In order to identify the measurement results or laboratories that could be

inconsistent two graphical consistency techniques were realised Mandelrsquos

h-ank-statistics Mandel indicators h and k at 5 significance highlighted

some possible inconsistent data (See tables and graphics in Appendix 3 and

Appendix 4)

Mandelrsquos values Number of values observed over the threshold

Reference method Alternative method

h gt 1 Lab F Level 2 Lab F Level 2

h gt 5 Lab F Level 2

Lab B Levels 1 and 2

Lab A Level 2

Lab D Level 3

Lab F Level 2

Lab L Level 2

kgt 1 Lab F Level 3

k gt 5 Lab F Level 2 Lab C Level 2

Lab F Level 3

Lab L Levels 1 and 3

Lab M Level 1

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2638 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

The emphasized results appear (i) coherent with the targeted contamination

levels (ii) concordant between both methods

All the results were thus kept for the statistical analysis

Comparison of the trueness and precision characteristics of

the reference method and alternative methods

The statistical values are summarised hereafter

Level p

Reference method Alternative method

Repeatability ratio

Reproducibility ratio Median

Repeatability standard deviation

Reproducibility standard deviation

Median Repeatability

standard deviation

Reproducibility standard deviation

1 10 16519 02415 02425 16021 02045 02433 0847 1007

2 10 26551 00543 00659 26719 00914 01325 1682 2011

2 bis 10 26581 02607 02607 25951 01900 01953 0729 0749

3 10 18671 00378 00378 18671 00478 00611 1264 1617

Bias of the alternative method

In order to estimate the bias of the alternative method with regard to the reference method for every

level Dij and t are calculated according to the following equations

RefAlt ijYijYDij

Diffp

Dijit

)2(

)(median

If the t value is superior to 2 the alternative method is significantly biased

with regard to the reference method

The values are given in table 17

Table 17 ndash t (d) values obtained by level

Level Bias t (d) Conclusion

1 0068 114 Non significant bias

2 0021 082 Non significant bias

2 bis - 0037 035 Non significant bias

3 - 0037 043 Non significant bias

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2738 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

The statistical test concludes to a non significant bias for all the inoculation

levels The bias varies from ndash 0037 to 0068 log CFUml

Comparison of the repeatability standard deviations

If the ratio Srj Alt Srj Ref of the repeatability standard deviations of the

alternative method and the reference method is larger than 2 the precision

under repeatability conditions of the alternative method is considered to be

lower than that of the reference method If this ratio is smaller than 05 the

precision under repeatability conditions of the alternative method is

considered to be greater than that of the reference method

The ratio values are given in table 18

Table 18

Level

Repeatability standard deviation

Reference method Alternative method Alternative method

reference method ratio

1 0242 0205 0847

2 0054 0091 1682

2 bis 0261 0190 0729

3 0038 0048 1264

Level Repeatability limit

Reference method Alternative method

1 0676 0573

2 0152 0256

2 bis 0730 0532

3 0106 0134

The ratios of the repeatability standard deviations are below 2 in all cases

The precision under repeatability conditions of the alternative method is

equivalent to that of the reference method

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2838 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Comparison of the reproducibility standard deviations

If the ratio Srj Alt Srj Ref of the reproducibility standard deviations of the alternative method and the

reference method is larger than 2 the precision under reproducibility conditions of the alternative

method is considered to be lower than that of the reference method If this ratio is smaller than 05 the

precision under reproducibility conditions of the alternative method is considered to be greater than

that of the reference method

The ratio values are given in table 19

Table 19

Level

Reproducibility standard deviation

Reference method Alternative method Alternative method

reference method ratio

1 0242 0243 1007

2 0066 0133 2011

2 bis 0261 0195 0749

3 0038 0061 1617

Level Reproducibility limit

Reference method Alternative method

1 0676 0681

2 0185 0371

2 bis 0730 0547

3 0106 0171

The ratios of the reproducibility standard deviations are below or close to 2

The precision under reproducibility conditions of the alternative method is

equivalent to that of the reference method

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2938 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

214 Conclusion

Method comparison study

The Listeria PrecisTM enumeration method shows satisfying linearity and

relative accuracy

The alternative method shows satisfying inclusivity and exclusivity results

Two Listeria monocytogenes strains gave negative results with the

alternative method These strains grew weakly on the reference method

media (OampA Palcam and Oxford)

The limits of repeatability of the alternative method are similar to those of the

reference method

The bias between both methods are low between - 0133 Log CFUg and

- 0008 Log CFUg

The Listeria PrecisTM enumeration method offers the saving for confirmatory

tests

Positive results are obtained with the alternative method in 2-3 days (with

OBIS Mono test) or in 4-7 days (by the classical tests described in the

standardized method) against 4-7 days for the reference method

Inter-laboratory study

The bias between the reference and the alternative methods shows satisfying

and correct values

- level 1 (100 - 1 000 CFUg) bias = + 007 log CFUg

- level 2 (1 000 - 10 000 CFUg) bias = 002 log CFUg

- level 2 bis (1 000 - 10 000 CFUg) bias = - 004 log CFUg

- level 3 (10 000 - 100 000 CFUg) bias = - 004 log CFUg

The repeatability and the reproducibility of the alternative method look similar

to those of the reference method

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3038 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

22 Extension study (realised by ASEPT 2007)

An extension study was performed in 2007 for a new confirmatory test OBIS

MONO

150 Listeria monocytogenes strains and 100 non-target strains were isolated

onto TSAYE and BrillianceTM Listeria Agar plates All the Listeria

monocytogenes and the non-target strains gave the expected results

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3138 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Appendix 1 ndash Flow diagram of the Listeria PrecisTM method

x g sample + 9 x ml buffered peptone water

Revivification step for 1 h plusmn 5 min at 20degC plusmn 1degC

Streaking 1 ml onto 3 Brilliance Listeria Agar plates (mother suspension)

or 01 ml onto 1 Brilliance Listeria Agar plates (mother suspension and decimal dilutions)

Incubation for 48 h plusmn 3 h at 37degC plusmn 1degC

Confirmatory tests on at least one typical colony

- by the tests described in the ISO method

- by applying the OBIS mono test on an isolated colony

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3238 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Appendix 2 ndash ISO 11290-2 (1998) amp ISO 11290-2A1 (2004) Horizontal

method for the detection and enumeration of Listeria monocytogenes -

Part 2 enumeration of Listeria monocytogenes in foods

10 g cheese + 90 ml buffered peptone water or Half Fraser Broth (without supplements) (110 dilution)

Stomach 1 min 30 seconds

Keep at 20degC plusmn 2degC for 1 h plusmn 5 min

Decimal dilutions in 9 ml peptone salt tubes

Dilution 110

Dilution 1100

Dilution 11000

1 ml on 3 Chromogenic Listeria medium (ISO) plates (x 2) (-1)

01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-2)

01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-3)

01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-4)

Incubation at 37degC plusmn 1degC

24 h - 48 h

Numerate typical colonies at 24 h

(bluegreen surrounded by an opaque halo)

Numerate at 48 h

Streak typical colonies on TSYEA

(5 coloniesplate 2 successive dilutions)

Confirmation tests

Catalase

Gram stain

For confirmation of L monocytogenes

Haemolysis

Carbohydrate (rhamnose and xylose) utilisation

CAMP Test

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3338 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Appendix 3 ndash Mandelrsquos graphics

Laboratoire h Ref

Niveau 1 h Ref

Niveau 2 h Ref

Niveau 3 h Alt

Niveau 1 h Alt

Niveau 2 h Alt

Niveau 3 h5 h1 h5 h1

A -050 -015 083 062 -212 -039 198 299 -198 -299

B -261 -270 021 -077 -058 173 198 299 -198 -299

C -151 -031 -014 -015 -004 -005 198 299 -198 -299

D 038 040 -137 000 072 207 198 299 -198 -299

E -001 090 155 -052 056 -101 198 299 -198 -299

F -112 -321 -153 070 -325 023 198 299 -198 -299

G 042 -071 040 147 -009 176 198 299 -198 -299

H 169 046 -169 192 018 -083 198 299 -198 -299

L 009 -120 021 -077 -231 005 198 299 -198 -299

M 001 031 014 -032 004 -011 198 299 -198 -299

-300

-200

-100

000

100

200

300

A B C D E F G H L M

Vale

urs

h d

e M

an

del(

rob

uste

s)

Laboratoires

Meacutethode de reacutefeacuterence analyse robuste

h Ref Niveau 1

h Ref Niveau 2

h Ref Niveau 3

h 5=198

h 1=299

-300

-200

-100

000

100

200

300

A B C D E F G H L M

Vale

urs

de h

de M

an

del(

rob

uste

s)

Laboratoires

Meacutethode alternative analyse robuste

h Alt Niveau 1

h Alt Niveau 2

h Alt Niveau 2

h 1=299

h 5=198

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3438 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Laboratoire k Ref

Niveau 1 k Ref

Niveau 2 k Ref

Niveau 3 k Alt

Niveau 1 k Alt

Niveau 2 k Alt

Niveau 3 k1 k5

A 000 089 142 084 162 046 259 181

B 125 087 073 104 075 046 259 181

C 117 013 091 188 022 083 259 181

D 052 036 069 000 035 083 259 181

E 028 034 037 138 019 112 259 181

F 140 222 018 027 281 112 259 181

G 088 068 024 131 145 000 259 181

H 129 094 173 016 061 000 259 181

L 069 000 073 104 270 224 259 181

M 000 048 038 043 099 241 259 181

000

050

100

150

200

250

300

350

400

A B C D E F G H L M

Vale

urs

k d

e M

an

del(

rob

uste

s)

Laboratoires

Meacutethode de reacutefeacuterence analyse robuste

k Ref Niveau 1k Ref Niveau 2k Ref Niveau 3

k 1=259

k 5=181

000

050

100

150

200

250

300

A B C D E F G H L M

Vale

urs

k d

e M

an

del

(ro

bu

ste

s)

Laboratoires

Meacutethode alternative analyse robuste

k Alt Niveau 3

k Alt Niveau 1

k Alt Niveau 2

k 1=259

k 5=181

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3538 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Appendix 4 ndash Statistical results

Level 1

Nombre de laboratoires (p) 10

A 1602 1602 1602 000 0000 0000 -0495 0000 A 1845 1602 1724 017 0122 -0122 0621 0840 0122

B 1602 1176 1389 030 0213 -0213 -2613 1247 B 1602 1301 1452 021 0151 -0151 -0769 1041 0062

C 1699 1301 1500 028 0199 -0199 -1510 1165 C 1301 1845 1573 038 -0272 0272 -0148 1881 0073

D 1602 1778 1690 012 -0088 0088 0380 0516 D 1602 1602 1602 000 0000 0000 0000 0000 -0088

E 1699 1602 1651 007 0048 -0048 -0013 0284 E 1699 1301 1500 028 0199 -0199 -0522 1376 -0151

F 1778 1301 1540 034 0239 -0239 -1116 1397 F 1778 1699 1739 006 0040 -0040 0698 0274 0199

G 1845 1544 1695 021 0151 -0151 0425 0881 G 2079 1699 1889 027 0190 -0190 1467 1315 0194

H 2041 1602 1822 031 0220 -0220 1689 1286 H 2000 1954 1977 003 0023 -0023 1917 0158 0155

L 1544 1778 1661 017 -0117 0117 0092 0685 L 1301 1602 1452 021 -0151 0151 -0769 1041 -0210

M 1653 1653 1653 000 0000 0000 0013 0000 M 1477 1602 1540 009 -0062 0062 -0319 0432 -0114

m 165186 m 160206 mediane D 00678

Cn(2p) 1867 Cn(2p) 1867 Cp 16101

Cn(p) 16101 Cn(p) 16101 Qn(D) 0092926

n=2p n 20 n=2p n 20 Qdiff 01496169

f 11 f 11 t 114

l 55 l 55

n=p n 10 n=p n 10

f 6 f 6

l 15 l 15

Qn(2p) 0091465 Qn(2p) 007745

Qn(p) 0062469 Qn(p) 0121519

Qintra 017078 Qintra 014461

Qinter 010058 Qinter 019565

Sr(eacutecart-type de reacutepeacutetabiliteacute) 02415 Sr 02045

RSDr(coefficient de variation de la reacutepeacutetabiliteacute)1462 RSDr 1277

r(repeatability limit)) 0676 r 0573

SL(eacutecart-type interlaboratoire) -001905 SLlt0 SL 001737

SR(eacutecart-type de reproductibiliteacute) 02415 SR 02433

RSDR(coefficient de variation de la reproductibiliteacute)0146 RSDR 0152

R(limite de reproductibiliteacute)) 06762 R(limite de reproductibiliteacute))068123

h k h MoyenneLaboratoires

Niveau 1

si LaboratoiresDeviationMoyenne Dk DeviationsiAlt

Niveau 1

Ref

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3638 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Level 2

Nombre de laboratoires (p) 10

A 2681 2613 2647 005 0034 -0034 -0151 0891 A 2322 2531 2427 015 -0105 0105 -2119 1620 -0220

B 2544 2477 2511 005 0033 -0033 -2699 0872 B 2556 2653 2605 007 -0048 0048 -0581 0750 0094

C 2643 2633 2638 001 0005 -0005 -0311 0130 C 2681 2653 2667 002 0014 -0014 -0040 0217 0029

D 2690 2663 2676 002 0014 -0014 0400 0357 D 2778 2732 2755 003 0023 -0023 0721 0354 0079

E 2690 2716 2703 002 -0013 0013 0897 0336 E 2724 2748 2736 002 -0012 0012 0556 0185 0033

F 2568 2398 2483 012 0085 -0085 -3214 2217 F 2477 2114 2296 026 0182 -0182 -3255 2811 -0188

G 2643 2591 2617 004 0026 -0026 -0707 0682 G 2756 2568 2662 013 0094 -0094 -0085 1453 0045

H 2716 2643 2680 005 0036 -0036 0460 0945 H 2653 2732 2693 006 -0040 0040 0181 0613 0013

L 2591 2591 2591 000 0000 0000 -1196 0000 L 2230 2580 2405 025 -0175 0175 -2307 2704 -0186

M 2653 2690 2672 003 -0018 0018 0311 0481 M 2740 2613 2677 009 0064 -0064 0040 0987 0005

m 2655082 m 26719001 mediane D 00209

Cn(2p) 1867 Cn(2p) 1867 Cp 16101

Cn(p) 16101 Cn(p) 16101 Qn(D) 0039911

n=2p n 20 n=2p n 20 Qdiff 00642591

f 11 f 11 t 082

l 55 l 55

n=p n 10 n=p n 10

f 6 f 6

l 15 l 15

Qn(2p) 0020570 Qn(2p) 00345985

Qn(p) 00332437 Qn(p) 00718158

Qintra 003841 Qintra 006460

Qinter 005352 Qinter 011563

Sr(eacutecart-type de reacutepeacutetabiliteacute) 00543 Sr 00914

RSDr(coefficient de variation de la reacutepeacutetabiliteacute)205 RSDr 342

r(repeatability limit)) 0152 r 0256

SL(eacutecart-type interlaboratoire) 000139 SL 000920

SR(eacutecart-type de reproductibiliteacute) 00659 SR 01325

RSDR(coefficient de variation de la reproductibiliteacute)0025 RSDR 0050

R(limite de reproductibiliteacute)) 018446 R(limite de reproductibiliteacute))03708618

Laboratoires

Niveau 2

Moyenne si Laboratoires

Niveau 2

DRef Alt Moyenne si Deviation h Deviation h k k

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3738 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Level 2 bis

Nombre de laboratoires (p) 10

A 3000 2477 2739 037 0261 -0261 0832 1418 A 2602 2477 2540 009 0062 -0062 -0391 0465 -0199

B 2544 2813 2678 019 -0134 0134 0211 0729 B 2903 2778 2841 009 0062 -0062 1732 0465 0162

C 2477 2813 2645 024 -0168 0168 -0135 0911 C 2477 2699 2588 016 -0111 0111 -0049 0826 -0057

D 2398 2653 2526 018 -0128 0128 -1370 0692 D 2778 3000 2889 016 -0111 0111 2074 0826 0363

E 2875 2740 2808 010 0067 -0067 1547 0365 E 2602 2301 2452 021 0151 -0151 -1012 1120 -0356

F 2477 2544 2511 005 -0033 0033 -1525 0182 F 2778 2477 2628 021 0151 -0151 0230 1120 0117

G 2653 2740 2697 006 -0044 0044 0400 0236 G 2845 2845 2845 000 0000 0000 1764 0000 0148

H 2176 2813 2495 045 -0318 0318 -1692 1727 H 2477 2477 2477 000 0000 0000 -0832 0000 -0017

L 2813 2544 2678 019 0134 -0134 0211 0729 L 2903 2301 2602 043 0301 -0301 0049 2241 -0076

M 2602 2740 2671 010 -0069 0069 0135 0375 M 2903 2255 2579 046 0324 -0324 -0112 2411 -0092

m 2658114 m 259505 mediane D -00372

Cn(2p) 1867 Cn(2p) 1867 Cp 16101

Cn(p) 16101 Cn(p) 16101 Qn(D) 01656915

n=2p n 20 n=2p n 20 Qdiff 02667753

f 11 f 11 t 035

l 55 l 55

n=p n 10 n=p n 10

f 6 f 6

l 15 l 15

Qn(2p) 0098745 Qn(2p) 007195

Qn(p) 006007 Qn(p) 008805

Qintra 018437 Qintra 013435

Qinter 009672 Qinter 014176

Sr(eacutecart-type de reacutepeacutetabiliteacute) 02607 Sr 01900

RSDr(coefficient de variation de la reacutepeacutetabiliteacute)981 RSDr 732

r(repeatability limit)) 0730 r 0532

SL(eacutecart-type interlaboratoire) -002464 SLlt0 SL 000205

SR(eacutecart-type de reproductibiliteacute) 02607 SR 01953

RSDR(coefficient de variation de la reproductibiliteacute)0098 RSDR 0075

R(limite de reproductibiliteacute)) 072996 R(limite de reproductibiliteacute))054686

Laboratoires

Niveau 2 bis

Moyenne si Laboratoires

Niveau 2 bis

DRef Alt Moyenne si Deviation h Deviation h k k

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3838 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Level 3

Nombre de laboratoires (p) 10

A 3690 3663 3676 002 0014 -0014 -0311 0513 A 3690 3708 3699 001 -0009 0009 0354 0257 0022

B 3613 3672 3642 004 -0030 0030 -1851 1109 B 3708 3568 3638 010 0070 -0070 -0844 2061 -0005

C 3663 3732 3698 005 -0035 0035 0645 1302 C 3663 3699 3681 003 -0018 0018 0000 0536 -0017

D 3690 3690 3690 000 0000 0000 0311 0000 D 3643 3663 3653 001 -0010 0010 -0545 0286 -0037

E 3732 3653 3693 006 0040 -0040 0429 1480 E 3613 3732 3673 008 -0060 0060 -0162 1769 -0020

F 3643 3672 3658 002 -0014 0014 -1157 0536 F 3690 3771 3731 006 -0040 0040 0975 1193 0073

G 3699 3653 3676 003 0023 -0023 -0328 0856 G 3602 3643 3623 003 -0021 0021 -1141 0612 -0053

H 3623 3672 3648 003 -0024 0024 -1615 0913 H 3748 3681 3715 005 0033 -0033 0664 0990 0067

L 3724 3663 3694 004 0031 -0031 0461 1150 L 3748 3771 3760 002 -0011 0011 1544 0335 0066

M 3690 3690 3690 000 0000 0000 0311 0000 M 3806 3681 3744 009 0062 -0062 1234 1848 0054

m 368334 m 368086 mediane D 00089

Cn(2p) 1867 Cn(2p) 1867 Cp 16101

Cn(p) 16101 Cn(p) 16101 Qn(D) 00325359

n=2p n 20 n=2p n 20 Qdiff 00523852

f 11 f 11 t 043

l 55 l 55

n=p n 10 n=p n 10

f 6 f 6

l 15 l 15

Qn(2p) 0014323 Qn(2p) 001811

Qn(p) 0013719 Qn(p) 003164

Qintra 002674 Qintra 003381

Qinter 002209 Qinter 005094

Sr(eacutecart-type de reacutepeacutetabiliteacute) 00378 Sr 00478

RSDr(coefficient de variation de la reacutepeacutetabiliteacute)103 RSDr 130

r(repeatability limit)) 0106 r 0134

SL(eacutecart-type interlaboratoire) -000023 SLlt0 SL 000145

SR(eacutecart-type de reproductibiliteacute) 00378 SR 00611

RSDR(coefficient de variation de la reproductibiliteacute)0010 RSDR 0017

R(limite de reproductibiliteacute)) 0105894 R(limite de reproductibiliteacute))017119

Ref Alt DLaboratoires

Niveau 3

Moyenne si Deviation h k Laboratoires

Niveau 3

Moyenne si Deviation h k

Page 5: ÞÀ¿¸7 ú Y5zñLjÜ< /u +æü t¬Á ... - NF Validation · NF VALIDATION Validation of alternative analytical methods Application in food microbiology ... 500 – 1 000 1 000 –

Thermo Fisher Scientific

ADRIA Deacuteveloppement 538 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

2 VALIDATION STUDY RESULTS

21 Initial validation (realised by ASEPT 2006)

211 Method Comparison Study

2111 Linearity

Linearity is the ability of the method when used with a given matrix to give results that are in proportion

to the amount of analyte present in the sample that is an increase in analyte corresponds to a linear or

proportional increase in results

Food matrices and protocols

Six matrixstrain pairs were analysed with five contamination levels and two

replicates At least 50 analyses were performed by the alternative and the

standard methods

The contamination levels the samples and the inoculated strains are

presented in the table below

Table 1

Samples Strains Contamination levels

(CFUg)

Rillettes Listeria monocytogenes 4e 10 ndash 50

50 ndash 100

100 ndash 500

500 ndash 1 000

1 000 ndash 10 000

Raw milk Listeria monocytogenes 12a

Lettuce Listeria monocytogenes 12a

Smoked salmon Listeria monocytogenes 12b

Raw egg product Listeria monocytogenes 12b

Process water Listeria monocytogenes 12a

The samples were individually inoculated and were analysed in duplicate by

the alternative and the reference methods

Results

The bi-dimensional graphs are shown figure 1

Thermo Fisher Scientific

ADRIA Deacuteveloppement 638 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Figure 1 ndash Bi-dimensional graphs

Rillettes Raw milk

Smoked salmon Raw egg product

Lettuce Process water

Thermo Fisher Scientific

ADRIA Deacuteveloppement 738 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Statistical interpretations

Statistical interpretation results are provided in Table 2

Table 2 ndash Statistical interpretations

Matrix R Selected

regression

RobF Critical

value

P Correlation

coefficient

Regression equation

Rillettes 180 GMFR 609 541 4 09960 log (Alt) = 09887 log (Ref) + 01141

Lettuce 108 GMFR 000 541 100 09992 Log (Alt) = 10081 log (Ref) - 00257

Raw milk 102 GMFR 000 541 100 09997 Log (Alt) = 10188 log (Ref) - 00737

Smoked salmon 1048 OLS1 1806 541 263 09984 Log (Alt) = 10406 log (Ref) - 01418

Raw egg product 130 GMFR 548 541 49 09958 Log (Alt) = 09775 log (Ref) - 00161

Process water 184 GMFR 0079 541 969 09985 Log (Alt) = 09680 log (Ref) + 01317

GMFR = orthogonal linear regression

OLS1 = ordinary least-square linear regression

x ndash axis = reference method

y ndash axis = alternative method

OLS1 = ordinary least-square linear regression

x axis = alternative method

y axis = reference method

Statistical Interpretation

P gt 5 not significant 1 lt P lt 5 significant

P lt 1 highly significant

The regressions straight lines are shown figure 2

Thermo Fisher Scientific

ADRIA Deacuteveloppement 838 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Figure 2 ndash Regression straight lines

Lineacuteariteacute - Œuf

Process water Process water (2) Process water

Smoked salmon (GMFR) Smoked salmon (OLS 1)

Raw milk Raw egg product

Rillettes Lettuce

Thermo Fisher Scientific

ADRIA Deacuteveloppement 938 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Discussion

The linearity is accepted in all cases with

P values 5 and correlation coefficients superior to 099

For the matrix ldquoRillettesldquo the linearity test is significant (P = 4) with a high

correlation coefficient value (0996)

The Listeria PrecisTM method shows satisfying linearity

2112 Relative accuracy

The accuracy is the closeness of agreement between a test result and the accepted reference value

The bias is the difference between the expectation of the test results and an accepted reference value

Number and nature of the samples

The repartition per category and types is provided in table 3

Table 3 ndash Number and nature of the samples

Categories Types Number of samples

Analysed Exploited (1)

Meat products

Raw meat products 6 4

Ready to cook products 4 2

Ready to eat delicatessen 10 4

Total 20 10

Dairy products

Raw milk cheeses 7 4

Raw milks 4 3

Other dairy products 5 5

Total 16 12

Fishery products

Raw fishes 5 4

Processed fishes 7 2

Ready-to-eat products 6 4

Total 18 10

Vegetables

Raw vegetables 5 4

Frozen vegetables 3 3

Lettuce raw vegetables 5 5

Total 13 12

Egg products

Raw egg 5 5

Pastries 3 3

Pasteurised egg products 4 3

Total 12 11

Environmental samples

Wipes swabs 8 4

Process water 3 3

Dusts 3 3

Total 14 10

TOTAL 93 65

(1) Some data cannot be exploited because enumeration was under the detection level for at least one of the

method

Thermo Fisher Scientific

ADRIA Deacuteveloppement 1038 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Artificial contamination of the samples

Artificial contaminations were realised by spiking after performing injury

protocols on pure cultures or by cross-contaminations

56 samples were artificially contaminated 53 gave exploitable results among

a total of 65 samples 8150 of the samples were artificially contaminated

The injury protocol and the strains inoculated are provided in Table 4

Table 4

Stress Ndeg Injury protocol Strain Origin

A 45 min at 50 degC

48

LM -H123

LM-H170

Smoked salmon

Lettuce

Lettuce

B 30 min at 50 degC

and 2 h at ndash20degC

18

43

LM-O1

Raw milk (environment)

Environment

Egg product

C 30 min at 55degC

18

48

LM-H123

Raw milk

Smoked salmon

Lettuce

D 30 min at 50degC

et 2 h at ndash80degC

34

LM-H171

LM-O1

Rillettes

Lettuce

Egg product

E 30 min at 60 degC LM-O1 Egg product

Confirmatory tests

For the reference and the alternative methods confirmations were realised

by the tests described in the ISO 12290-2A1 standard For the alternative

method only one colony was confirmed while 5 colonies were tested for the

reference method

Results

Samples were analysed in duplicate by the reference and the alternative

methods

The contamination range is provided in table 5 per tested category

Thermo Fisher Scientific

ADRIA Deacuteveloppement 1138 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Table 5 ndash Contamination range

Food category Contamination level (log CFUg)

Meat products 100 ndash 404

Dairy products 148 ndash 408

Fishery products 100 ndash 384

Vegetables 170 ndash 511

Egg products 130 ndash 499

Environmental samples 170 ndash 511

All categories 100 ndash 511

Bi-dimensional graphs are given figure 3

Figure 3 ndash Bi-dimensional graphs

Graphe bi-dimensionnel Produits carneacutes

Fishery products

Vegetables

Egg products

Meat products

Thermo Fisher Scientific

ADRIA Deacuteveloppement 1238 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Statistical interpretation according to EN ISO16140 method

The results of the statistical interpretation are given in Table 6

Table 6 ndash Statistical interpretation results

Category n R Regression

used a t(a) b t(b) Critical T

P

Ordinate

at 0

Slope

at 1

Meat products 10 158 GMFR -0455 3321 115 3123 2306 001 001

Dairy products 12 065 GMFR -0027 0385 1002 0108 2228 092 071

Fishery products 10 130 GMFR -0305 1024 1034 0312 2306 076 034

Vegetables 12 086 GMFR -0015 0128 0993 0191 2228 085 090

Egg products 11 158 GMFR -0038 0866 1001 0090 2262 093 041

Environmental

samples 10 176 GMFR -0139 265 1034 231 2306 005 003

All products 65 118 GMFR -0182 3042 1040 2145 2000 004 000

All products

(estimated number) 65 118 GMFR -0182 3042 1040 2145 2000 004 000

All products (non

estimated results) 20 115 GMFR -0367 151 1138 1141 2101 027 015

Repeatability limits obtained for the alternative and reference methods as

well as bias obtained are presented in Table 7

Dairy products Environmental samples

Log (Ref) Log (Ref)

All categories

Thermo Fisher Scientific

ADRIA Deacuteveloppement 1338 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Table 7

Categories Bias

Repeatability limits

Rob r

Ref Alt

Meat products 0008 0314 0343

Dairy products -0031 0136 0130

Fishery products -0133 0220 0214

Vegetables -0041 0180 0319

Egg products -0026 0219 0206

Environmental samples -0007 0198 0119

All products -0032 0196 0215

Regression straight lines (graph and equation representations) for each food

category and for all products are presented figure 4

Thermo Fisher Scientific

ADRIA Deacuteveloppement 1438 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Figure 4 ndash Regression straight lines

Meat products

Fishery products

Dairy products

Vegetables

Thermo Fisher Scientific

ADRIA Deacuteveloppement 1538 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Environmental samples

Egg products

All products (estimated number)

Thermo Fisher Scientific

ADRIA Deacuteveloppement 1638 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

The hypotheses that the ordinates are close to 0 and the slopes close to 1

are approved in all cases except for

- Meat products (slope P = 1 ordinate P = 1)

- Environmental samples (ordinate P = 3)

- All products (ordinate P = 0)

The bias between the two methods vary from ndash 0133 to 0008 log CFU For

all categories the bias is ndash 0032 log CFU The repeatability limits of the

alternative and the reference method are similar For all products the values

are respectively 0215 and 0196 log CFU For all products these

interpretations are provided taking into account data with low contamination

level or not (estimated colonies number)

For all food products the regression straight line is the following

log (Alt) = 1040 log Ref ndash 0182

The Listeria PrecisTM method shows satisfying relative accuracy

All products

All products (Level 2)

Thermo Fisher Scientific

ADRIA Deacuteveloppement 1738 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

2113 Detection limit (LOD) and quantification limit (LOQ)

The critical level is the defined as the smallest amount which can be detected (not null) but not

quantified as an exact value Below this value it cannot be sure that the true value is not null

The detection limit is defined as being higher than the critical level because it involves a power the

probability 1- which has to be well over 50 for example 95

The quantification limit is defined as the smallest amount of analyte (that is the lowest actual number of

organisms) which can be measured and quantified with defined precision and accuracy under the

experimental conditions by the method under validation

Protocol

The detection limit of the alternative method was realised with pure cultures

Three different inoculation levels were tested with six replicates per level ie

18 analyses were done by the alternative method

Listeria monocytogenes 18 was used for the detection limit of the alternative

method

Quantification limit was calculated for six independent blank samples

determinations

Results

These data are intrinsic to the method used and are presented in the

following tables

Table 8 ndash Inoculation 1 ml on 3 plates

Level (CFUml) Positive samples number Standard deviation Bias

0 06

05 26 1211 0

1 36 0816 05

5 66 2483 85

Thermo Fisher Scientific

ADRIA Deacuteveloppement 1838 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Table 9 ndash Inoculation 01 ml on 1 plate

Level (CFUml) Positive samples number Standard deviation Bias

0 06

5 36 0548 05

10 46 1414 2

50 66 2258 55

Table 10

Formulas Obtained values

LC 165 S0 + X0 18

LOD 33 S0 + X0 32

LOQ 10 S0 + X0 87

2114 Relative Sensitivity

The relative sensitivity is defined as the ability of the alternative method to detect two different amounts

of analyte measured by the reference method within a given matrix at a specified average value or

over the whole measurement range that is it is the minimal quantity variation (increase of the analyte

concentration x) which gives a significant variation of the measured signal (response y)

These data are intrinsic to the used method and are obtained from the results

of the linearity study

Sensitivity patterns obtained for tested (matrixstrain) pairs are presented in

figure 5

Thermo Fisher Scientific

ADRIA Deacuteveloppement 1938 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Figure 5 ndash Sensitivity patterns for the tested (matrixstrain) pairs

2115 SpecificitySelectivity

The specificity is defined as the degree to which a method is affected (or not) by the other components

present in a multi-component sample That is the ability of a method to measure exactly a given

analyte or its amount within the sample without interference from non-target components such as a

matrix effect or background noise

The selectivity is defined as a measure of the degree of non-interference in the presence of non-target

analytes A method is selective if it can be used to detect the analyte under examination and that a

guarantee can be provided that the detected signal can only be a product by that specific analyte

The specificity and selectivity study was performed for the Listeria PrecisTM

detection method no additional test was realised for the enumeration

method The results were the following

- Among the Listeria monocytogenes strains tested 2 strains (Listeria

monocytogenes CIP 7834 and Listeria monocytogenes CIP 105459) gave

negative results These 2 strains were tested twice by running 48 h

incubation time of the compared agars Positive results were then

observed by the alternative method Pale characteristic colonies were

observed on the ISO agar by streaking the Half Fraser broth only

- No cross reaction was observed with the non-target strains except with

Listeria ivanovii which gave characteristic colonies on Brilliancetrade Listeria

Agar but the confirmatory tests provide a clear discrimination between

Listeria monocytogenes and Listeria ivanovi species

Rillettes

Lettuce

Raw milk

Smoked salmon

Raw egg

Process water

x (CFUg)

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2038 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

212 Practicability

1 Packaging Ready to use Brilliance Listeria agar plates are available

(x 10 plates)

2 Reagent volume

3 Storage conditionsndash

Shelf life of products

not opened

The plates are stored at 6 - 12degC in darkness conditions The

expiration date is mentioned on the package and on the plates It

is around 3 months

4 Modalities of use

after first use

5 Required

equipments No specific equipment is required

6 Reagents Ready to use plates

7 Training 05 day

8 Real time handling

Reference method

9 min for one sample

55 min for 10 samples

Alternative method

7 min for one sample

35 minutes for 10 samples

9 Time to result

Negative results

Reference method Day 2

Alternative method Day 2

Negative results after confirmation or positive results

Reference method Day 4 ndash Day 7

Alternative method Day 4 ndash Day 7

Positive results

Reference method Day 4 ndash Day 7

Alternative method Day 2 ndash Day 3 (with OBIS Mono Test)

or Day 4 ndash Day 7 (by the classical tests described in the

standardised method)

10 Technician skill Identical to that required for the reference method

11 Steps common to

the reference method Mother suspension revivification step and decimal dilutions

12 Traceability

13 Maintenance

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2138 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

213 Inter-laboratory study

2131 Organisation

12 laboratories participated to this study Pasteurised milk was inoculated by

Listeria monocytogenes 12a isolated from raw milk The targeted

inoculation levels were

- 0 CFUml

- 50 CFU ml

- 500 CFU ml

- 5 000 CFU ml

Each laboratory received eight flasks of 10 ml sample ie two flasks per

inoculation level Furthermore one non-inoculated sample has been added

to the package for total viable count microflora (NF ISO 4833 method)

Detailed instructions were transmitted to the collaborators by the expert

laboratory

Collaborative study laboratories and the expert laboratory carried out the

analyses with the alternative and reference methods

2132 Verification of experimental parameters

Strain stability during transport

In order to evaluate the Listeria monocytogenes strain stability during

transport bacterial count of all inoculated flasks were checked at different

time ie inoculation time after 24 h of conservation at 2deg - 8degC Results are

reported in table 11

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2238 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Table 11 ndash Listeria monocytogenes count

by ISO 11290-2A1 method (log CFUml)

Day of the analyses Level contamination Reference method

Day 0

(29052006)

1 45

2 440

3 4 500

Day 1

(30052006)

1 60

2 430

3 4 900

No evolution of the strain was observed after storage for 24 h at 2degC ndash 8degC

Contamination level

The real contamination levels are provided in table 12

Table 12 ndash Real contamination level (CFUml)

Target inoculation

(CFU ml) Samples Ndeg

Real contamination levels

(CFUml)

0 1 - 8 0

50 5 - 6 45

500 3 - 7 440

5000 2 - 4 4 500

Sample temperature at receipt

Measured temperatures at receipt are listed in table 13

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2338 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Table 13 - Sample temperature on receipt

Laboratories Receipt

day

Hour

receipt

Measured temperature

at receipt (degC)

Measured temperature by the

temperature probe (degC)

A

B

C

D

E

F

G

H

I

J

K

L

M

30052006

10h45

10h15

11h15

10h00

09h30

8h30

11h50

10h00

08h30

14h45

08h30

09h45

08h45

82

55

45

63

60

62

81

38

48

-

14

67

76

03

03

-13

03

04

-14

05

04

01

05

-13

04

Conclusion

The temperatures during the transport were correct for all the labs except for

Lab J which didnrsquot provide the information

All the labs received their packages with several broken flasks

Three labs (B D and L) received some damaged samples but proceeded to

analyses

All the labs started the analyses at Day 1 except Lab J

2133 Results

Aerobic mesophilic flora

The count of aerobic mesophilic flora varied from 94 102 CFUml to

33 103 CFUml

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2438 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Expert lab results

The results obtained by the expert lab are the following

Table 14

Target inoculation level (CFUml)

Reference method ISO 11290-2

Alternative method Listeria Precis Enumeration

Rep 1 Rep 2 Rep 1 Rep 2

lt 10 lt 10 lt 10 lt 10 lt10

50 50 (Ne) 60 (Ne) 50 (Ne) 30 (Ne)

500 470 390 490 500

5 000 4 700 5 100 5 900 3 400

Ne estimated number

The obtained results are in agreement with those expected

Collaborator Labs results

A synthesis of the results obtained by the collaborator labs is provided

Table 15 (CFUml) and Table 16 (log CFUml)

Table 15 ndash Synthesis of results obtained by the alternative

and the reference methods (CFUml)

Labs Level 1 Level 2 Level 2 bis Level 3

Reference method

Alternative method

Reference method

Alternative method

Reference method

Alternative method

Reference method

Alternative method

A 40 40 70 40 480 410 210 340 1000 300 400 300 4900 4600 4900 5100

B 40 15 40 20 350 300 360 450 350 650 800 600 4100 4700 5100 3700

C 50 20 20 70 440 430 480 450 300 650 300 500 4600 5400 4600 5000

D 40 60 40 40 490 460 600 540 250 450 600 1000 4900 4900 4400 4600

E 50 40 50 20 490 520 530 560 750 550 400 200 5400 4500 4100 5400

F 60 20 60 50 370 250 300 130 300 350 600 300 4400 4700 4900 5900

G 70 35 120 50 440 390 570 370 450 550 700 700 5000 4500 4000 4400

H 110 40 100 90 520 440 450 540 150 650 300 300 4200 4700 5600 4800

L 35 60 20 40 390 390 170 380 650 350 800 200 5300 4600 5600 5900

M 45 45 30 40 450 490 550 410 400 550 800 180 4900 4900 6400 4800

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2538 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Table 16 ndash Synthesis of results obtained by the alternative

and the reference methods (log CFUml)

Labs Level 1 Level 2 Level 2 bis Level 3

Reference method

Alternative method

Reference method

Alternative method

Reference method

Alternative method

Reference method

Alternative method

A 1602 1602 1845 1602 2681 2613 2322 2531 3000 2477 2602 2477 3690 3663 3690 3708

B 1602 1176 1602 1301 2544 2477 2556 2653 2544 2813 2903 2778 3613 3672 3708 3568

C 1699 1301 1301 1845 2643 2633 2681 2653 2477 2813 2477 2699 3663 3732 3663 3699

D 1602 1778 1602 1602 2690 2663 2778 2732 2398 2653 2778 3000 3690 3690 3643 3663

E 1699 1602 1699 1301 2690 2716 2724 2748 2875 2740 2602 2301 3732 3653 3613 3732

F 1778 1301 1778 1699 2568 2398 2477 2114 2477 2544 2778 2477 3643 3672 3690 3771

G 1845 1544 2079 1699 2643 2591 2756 2568 2653 2740 2845 2845 3699 3653 3602 3643

H 2041 1602 2000 1954 2716 2643 2653 2732 2176 2813 2477 2477 3623 3672 3748 3681

L 1544 1778 1301 1602 2591 2591 2230 2580 2813 2544 2903 2301 3724 3663 3748 3771

M 1653 1653 1477 1602 2653 2690 2740 2613 2602 2740 2903 2255 3690 3690 3806 3681

Level 2 results obtained taking account the 3 plates inoculated with the mother-suspension

Level 2 bis results obtained taking account the plates inoculated with 01 ml of the mother-suspension

Scrutiny of the measurement results for consistency

In order to identify the measurement results or laboratories that could be

inconsistent two graphical consistency techniques were realised Mandelrsquos

h-ank-statistics Mandel indicators h and k at 5 significance highlighted

some possible inconsistent data (See tables and graphics in Appendix 3 and

Appendix 4)

Mandelrsquos values Number of values observed over the threshold

Reference method Alternative method

h gt 1 Lab F Level 2 Lab F Level 2

h gt 5 Lab F Level 2

Lab B Levels 1 and 2

Lab A Level 2

Lab D Level 3

Lab F Level 2

Lab L Level 2

kgt 1 Lab F Level 3

k gt 5 Lab F Level 2 Lab C Level 2

Lab F Level 3

Lab L Levels 1 and 3

Lab M Level 1

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2638 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

The emphasized results appear (i) coherent with the targeted contamination

levels (ii) concordant between both methods

All the results were thus kept for the statistical analysis

Comparison of the trueness and precision characteristics of

the reference method and alternative methods

The statistical values are summarised hereafter

Level p

Reference method Alternative method

Repeatability ratio

Reproducibility ratio Median

Repeatability standard deviation

Reproducibility standard deviation

Median Repeatability

standard deviation

Reproducibility standard deviation

1 10 16519 02415 02425 16021 02045 02433 0847 1007

2 10 26551 00543 00659 26719 00914 01325 1682 2011

2 bis 10 26581 02607 02607 25951 01900 01953 0729 0749

3 10 18671 00378 00378 18671 00478 00611 1264 1617

Bias of the alternative method

In order to estimate the bias of the alternative method with regard to the reference method for every

level Dij and t are calculated according to the following equations

RefAlt ijYijYDij

Diffp

Dijit

)2(

)(median

If the t value is superior to 2 the alternative method is significantly biased

with regard to the reference method

The values are given in table 17

Table 17 ndash t (d) values obtained by level

Level Bias t (d) Conclusion

1 0068 114 Non significant bias

2 0021 082 Non significant bias

2 bis - 0037 035 Non significant bias

3 - 0037 043 Non significant bias

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2738 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

The statistical test concludes to a non significant bias for all the inoculation

levels The bias varies from ndash 0037 to 0068 log CFUml

Comparison of the repeatability standard deviations

If the ratio Srj Alt Srj Ref of the repeatability standard deviations of the

alternative method and the reference method is larger than 2 the precision

under repeatability conditions of the alternative method is considered to be

lower than that of the reference method If this ratio is smaller than 05 the

precision under repeatability conditions of the alternative method is

considered to be greater than that of the reference method

The ratio values are given in table 18

Table 18

Level

Repeatability standard deviation

Reference method Alternative method Alternative method

reference method ratio

1 0242 0205 0847

2 0054 0091 1682

2 bis 0261 0190 0729

3 0038 0048 1264

Level Repeatability limit

Reference method Alternative method

1 0676 0573

2 0152 0256

2 bis 0730 0532

3 0106 0134

The ratios of the repeatability standard deviations are below 2 in all cases

The precision under repeatability conditions of the alternative method is

equivalent to that of the reference method

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2838 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Comparison of the reproducibility standard deviations

If the ratio Srj Alt Srj Ref of the reproducibility standard deviations of the alternative method and the

reference method is larger than 2 the precision under reproducibility conditions of the alternative

method is considered to be lower than that of the reference method If this ratio is smaller than 05 the

precision under reproducibility conditions of the alternative method is considered to be greater than

that of the reference method

The ratio values are given in table 19

Table 19

Level

Reproducibility standard deviation

Reference method Alternative method Alternative method

reference method ratio

1 0242 0243 1007

2 0066 0133 2011

2 bis 0261 0195 0749

3 0038 0061 1617

Level Reproducibility limit

Reference method Alternative method

1 0676 0681

2 0185 0371

2 bis 0730 0547

3 0106 0171

The ratios of the reproducibility standard deviations are below or close to 2

The precision under reproducibility conditions of the alternative method is

equivalent to that of the reference method

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2938 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

214 Conclusion

Method comparison study

The Listeria PrecisTM enumeration method shows satisfying linearity and

relative accuracy

The alternative method shows satisfying inclusivity and exclusivity results

Two Listeria monocytogenes strains gave negative results with the

alternative method These strains grew weakly on the reference method

media (OampA Palcam and Oxford)

The limits of repeatability of the alternative method are similar to those of the

reference method

The bias between both methods are low between - 0133 Log CFUg and

- 0008 Log CFUg

The Listeria PrecisTM enumeration method offers the saving for confirmatory

tests

Positive results are obtained with the alternative method in 2-3 days (with

OBIS Mono test) or in 4-7 days (by the classical tests described in the

standardized method) against 4-7 days for the reference method

Inter-laboratory study

The bias between the reference and the alternative methods shows satisfying

and correct values

- level 1 (100 - 1 000 CFUg) bias = + 007 log CFUg

- level 2 (1 000 - 10 000 CFUg) bias = 002 log CFUg

- level 2 bis (1 000 - 10 000 CFUg) bias = - 004 log CFUg

- level 3 (10 000 - 100 000 CFUg) bias = - 004 log CFUg

The repeatability and the reproducibility of the alternative method look similar

to those of the reference method

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3038 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

22 Extension study (realised by ASEPT 2007)

An extension study was performed in 2007 for a new confirmatory test OBIS

MONO

150 Listeria monocytogenes strains and 100 non-target strains were isolated

onto TSAYE and BrillianceTM Listeria Agar plates All the Listeria

monocytogenes and the non-target strains gave the expected results

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3138 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Appendix 1 ndash Flow diagram of the Listeria PrecisTM method

x g sample + 9 x ml buffered peptone water

Revivification step for 1 h plusmn 5 min at 20degC plusmn 1degC

Streaking 1 ml onto 3 Brilliance Listeria Agar plates (mother suspension)

or 01 ml onto 1 Brilliance Listeria Agar plates (mother suspension and decimal dilutions)

Incubation for 48 h plusmn 3 h at 37degC plusmn 1degC

Confirmatory tests on at least one typical colony

- by the tests described in the ISO method

- by applying the OBIS mono test on an isolated colony

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3238 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Appendix 2 ndash ISO 11290-2 (1998) amp ISO 11290-2A1 (2004) Horizontal

method for the detection and enumeration of Listeria monocytogenes -

Part 2 enumeration of Listeria monocytogenes in foods

10 g cheese + 90 ml buffered peptone water or Half Fraser Broth (without supplements) (110 dilution)

Stomach 1 min 30 seconds

Keep at 20degC plusmn 2degC for 1 h plusmn 5 min

Decimal dilutions in 9 ml peptone salt tubes

Dilution 110

Dilution 1100

Dilution 11000

1 ml on 3 Chromogenic Listeria medium (ISO) plates (x 2) (-1)

01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-2)

01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-3)

01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-4)

Incubation at 37degC plusmn 1degC

24 h - 48 h

Numerate typical colonies at 24 h

(bluegreen surrounded by an opaque halo)

Numerate at 48 h

Streak typical colonies on TSYEA

(5 coloniesplate 2 successive dilutions)

Confirmation tests

Catalase

Gram stain

For confirmation of L monocytogenes

Haemolysis

Carbohydrate (rhamnose and xylose) utilisation

CAMP Test

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3338 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Appendix 3 ndash Mandelrsquos graphics

Laboratoire h Ref

Niveau 1 h Ref

Niveau 2 h Ref

Niveau 3 h Alt

Niveau 1 h Alt

Niveau 2 h Alt

Niveau 3 h5 h1 h5 h1

A -050 -015 083 062 -212 -039 198 299 -198 -299

B -261 -270 021 -077 -058 173 198 299 -198 -299

C -151 -031 -014 -015 -004 -005 198 299 -198 -299

D 038 040 -137 000 072 207 198 299 -198 -299

E -001 090 155 -052 056 -101 198 299 -198 -299

F -112 -321 -153 070 -325 023 198 299 -198 -299

G 042 -071 040 147 -009 176 198 299 -198 -299

H 169 046 -169 192 018 -083 198 299 -198 -299

L 009 -120 021 -077 -231 005 198 299 -198 -299

M 001 031 014 -032 004 -011 198 299 -198 -299

-300

-200

-100

000

100

200

300

A B C D E F G H L M

Vale

urs

h d

e M

an

del(

rob

uste

s)

Laboratoires

Meacutethode de reacutefeacuterence analyse robuste

h Ref Niveau 1

h Ref Niveau 2

h Ref Niveau 3

h 5=198

h 1=299

-300

-200

-100

000

100

200

300

A B C D E F G H L M

Vale

urs

de h

de M

an

del(

rob

uste

s)

Laboratoires

Meacutethode alternative analyse robuste

h Alt Niveau 1

h Alt Niveau 2

h Alt Niveau 2

h 1=299

h 5=198

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3438 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Laboratoire k Ref

Niveau 1 k Ref

Niveau 2 k Ref

Niveau 3 k Alt

Niveau 1 k Alt

Niveau 2 k Alt

Niveau 3 k1 k5

A 000 089 142 084 162 046 259 181

B 125 087 073 104 075 046 259 181

C 117 013 091 188 022 083 259 181

D 052 036 069 000 035 083 259 181

E 028 034 037 138 019 112 259 181

F 140 222 018 027 281 112 259 181

G 088 068 024 131 145 000 259 181

H 129 094 173 016 061 000 259 181

L 069 000 073 104 270 224 259 181

M 000 048 038 043 099 241 259 181

000

050

100

150

200

250

300

350

400

A B C D E F G H L M

Vale

urs

k d

e M

an

del(

rob

uste

s)

Laboratoires

Meacutethode de reacutefeacuterence analyse robuste

k Ref Niveau 1k Ref Niveau 2k Ref Niveau 3

k 1=259

k 5=181

000

050

100

150

200

250

300

A B C D E F G H L M

Vale

urs

k d

e M

an

del

(ro

bu

ste

s)

Laboratoires

Meacutethode alternative analyse robuste

k Alt Niveau 3

k Alt Niveau 1

k Alt Niveau 2

k 1=259

k 5=181

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3538 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Appendix 4 ndash Statistical results

Level 1

Nombre de laboratoires (p) 10

A 1602 1602 1602 000 0000 0000 -0495 0000 A 1845 1602 1724 017 0122 -0122 0621 0840 0122

B 1602 1176 1389 030 0213 -0213 -2613 1247 B 1602 1301 1452 021 0151 -0151 -0769 1041 0062

C 1699 1301 1500 028 0199 -0199 -1510 1165 C 1301 1845 1573 038 -0272 0272 -0148 1881 0073

D 1602 1778 1690 012 -0088 0088 0380 0516 D 1602 1602 1602 000 0000 0000 0000 0000 -0088

E 1699 1602 1651 007 0048 -0048 -0013 0284 E 1699 1301 1500 028 0199 -0199 -0522 1376 -0151

F 1778 1301 1540 034 0239 -0239 -1116 1397 F 1778 1699 1739 006 0040 -0040 0698 0274 0199

G 1845 1544 1695 021 0151 -0151 0425 0881 G 2079 1699 1889 027 0190 -0190 1467 1315 0194

H 2041 1602 1822 031 0220 -0220 1689 1286 H 2000 1954 1977 003 0023 -0023 1917 0158 0155

L 1544 1778 1661 017 -0117 0117 0092 0685 L 1301 1602 1452 021 -0151 0151 -0769 1041 -0210

M 1653 1653 1653 000 0000 0000 0013 0000 M 1477 1602 1540 009 -0062 0062 -0319 0432 -0114

m 165186 m 160206 mediane D 00678

Cn(2p) 1867 Cn(2p) 1867 Cp 16101

Cn(p) 16101 Cn(p) 16101 Qn(D) 0092926

n=2p n 20 n=2p n 20 Qdiff 01496169

f 11 f 11 t 114

l 55 l 55

n=p n 10 n=p n 10

f 6 f 6

l 15 l 15

Qn(2p) 0091465 Qn(2p) 007745

Qn(p) 0062469 Qn(p) 0121519

Qintra 017078 Qintra 014461

Qinter 010058 Qinter 019565

Sr(eacutecart-type de reacutepeacutetabiliteacute) 02415 Sr 02045

RSDr(coefficient de variation de la reacutepeacutetabiliteacute)1462 RSDr 1277

r(repeatability limit)) 0676 r 0573

SL(eacutecart-type interlaboratoire) -001905 SLlt0 SL 001737

SR(eacutecart-type de reproductibiliteacute) 02415 SR 02433

RSDR(coefficient de variation de la reproductibiliteacute)0146 RSDR 0152

R(limite de reproductibiliteacute)) 06762 R(limite de reproductibiliteacute))068123

h k h MoyenneLaboratoires

Niveau 1

si LaboratoiresDeviationMoyenne Dk DeviationsiAlt

Niveau 1

Ref

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3638 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Level 2

Nombre de laboratoires (p) 10

A 2681 2613 2647 005 0034 -0034 -0151 0891 A 2322 2531 2427 015 -0105 0105 -2119 1620 -0220

B 2544 2477 2511 005 0033 -0033 -2699 0872 B 2556 2653 2605 007 -0048 0048 -0581 0750 0094

C 2643 2633 2638 001 0005 -0005 -0311 0130 C 2681 2653 2667 002 0014 -0014 -0040 0217 0029

D 2690 2663 2676 002 0014 -0014 0400 0357 D 2778 2732 2755 003 0023 -0023 0721 0354 0079

E 2690 2716 2703 002 -0013 0013 0897 0336 E 2724 2748 2736 002 -0012 0012 0556 0185 0033

F 2568 2398 2483 012 0085 -0085 -3214 2217 F 2477 2114 2296 026 0182 -0182 -3255 2811 -0188

G 2643 2591 2617 004 0026 -0026 -0707 0682 G 2756 2568 2662 013 0094 -0094 -0085 1453 0045

H 2716 2643 2680 005 0036 -0036 0460 0945 H 2653 2732 2693 006 -0040 0040 0181 0613 0013

L 2591 2591 2591 000 0000 0000 -1196 0000 L 2230 2580 2405 025 -0175 0175 -2307 2704 -0186

M 2653 2690 2672 003 -0018 0018 0311 0481 M 2740 2613 2677 009 0064 -0064 0040 0987 0005

m 2655082 m 26719001 mediane D 00209

Cn(2p) 1867 Cn(2p) 1867 Cp 16101

Cn(p) 16101 Cn(p) 16101 Qn(D) 0039911

n=2p n 20 n=2p n 20 Qdiff 00642591

f 11 f 11 t 082

l 55 l 55

n=p n 10 n=p n 10

f 6 f 6

l 15 l 15

Qn(2p) 0020570 Qn(2p) 00345985

Qn(p) 00332437 Qn(p) 00718158

Qintra 003841 Qintra 006460

Qinter 005352 Qinter 011563

Sr(eacutecart-type de reacutepeacutetabiliteacute) 00543 Sr 00914

RSDr(coefficient de variation de la reacutepeacutetabiliteacute)205 RSDr 342

r(repeatability limit)) 0152 r 0256

SL(eacutecart-type interlaboratoire) 000139 SL 000920

SR(eacutecart-type de reproductibiliteacute) 00659 SR 01325

RSDR(coefficient de variation de la reproductibiliteacute)0025 RSDR 0050

R(limite de reproductibiliteacute)) 018446 R(limite de reproductibiliteacute))03708618

Laboratoires

Niveau 2

Moyenne si Laboratoires

Niveau 2

DRef Alt Moyenne si Deviation h Deviation h k k

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3738 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Level 2 bis

Nombre de laboratoires (p) 10

A 3000 2477 2739 037 0261 -0261 0832 1418 A 2602 2477 2540 009 0062 -0062 -0391 0465 -0199

B 2544 2813 2678 019 -0134 0134 0211 0729 B 2903 2778 2841 009 0062 -0062 1732 0465 0162

C 2477 2813 2645 024 -0168 0168 -0135 0911 C 2477 2699 2588 016 -0111 0111 -0049 0826 -0057

D 2398 2653 2526 018 -0128 0128 -1370 0692 D 2778 3000 2889 016 -0111 0111 2074 0826 0363

E 2875 2740 2808 010 0067 -0067 1547 0365 E 2602 2301 2452 021 0151 -0151 -1012 1120 -0356

F 2477 2544 2511 005 -0033 0033 -1525 0182 F 2778 2477 2628 021 0151 -0151 0230 1120 0117

G 2653 2740 2697 006 -0044 0044 0400 0236 G 2845 2845 2845 000 0000 0000 1764 0000 0148

H 2176 2813 2495 045 -0318 0318 -1692 1727 H 2477 2477 2477 000 0000 0000 -0832 0000 -0017

L 2813 2544 2678 019 0134 -0134 0211 0729 L 2903 2301 2602 043 0301 -0301 0049 2241 -0076

M 2602 2740 2671 010 -0069 0069 0135 0375 M 2903 2255 2579 046 0324 -0324 -0112 2411 -0092

m 2658114 m 259505 mediane D -00372

Cn(2p) 1867 Cn(2p) 1867 Cp 16101

Cn(p) 16101 Cn(p) 16101 Qn(D) 01656915

n=2p n 20 n=2p n 20 Qdiff 02667753

f 11 f 11 t 035

l 55 l 55

n=p n 10 n=p n 10

f 6 f 6

l 15 l 15

Qn(2p) 0098745 Qn(2p) 007195

Qn(p) 006007 Qn(p) 008805

Qintra 018437 Qintra 013435

Qinter 009672 Qinter 014176

Sr(eacutecart-type de reacutepeacutetabiliteacute) 02607 Sr 01900

RSDr(coefficient de variation de la reacutepeacutetabiliteacute)981 RSDr 732

r(repeatability limit)) 0730 r 0532

SL(eacutecart-type interlaboratoire) -002464 SLlt0 SL 000205

SR(eacutecart-type de reproductibiliteacute) 02607 SR 01953

RSDR(coefficient de variation de la reproductibiliteacute)0098 RSDR 0075

R(limite de reproductibiliteacute)) 072996 R(limite de reproductibiliteacute))054686

Laboratoires

Niveau 2 bis

Moyenne si Laboratoires

Niveau 2 bis

DRef Alt Moyenne si Deviation h Deviation h k k

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3838 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Level 3

Nombre de laboratoires (p) 10

A 3690 3663 3676 002 0014 -0014 -0311 0513 A 3690 3708 3699 001 -0009 0009 0354 0257 0022

B 3613 3672 3642 004 -0030 0030 -1851 1109 B 3708 3568 3638 010 0070 -0070 -0844 2061 -0005

C 3663 3732 3698 005 -0035 0035 0645 1302 C 3663 3699 3681 003 -0018 0018 0000 0536 -0017

D 3690 3690 3690 000 0000 0000 0311 0000 D 3643 3663 3653 001 -0010 0010 -0545 0286 -0037

E 3732 3653 3693 006 0040 -0040 0429 1480 E 3613 3732 3673 008 -0060 0060 -0162 1769 -0020

F 3643 3672 3658 002 -0014 0014 -1157 0536 F 3690 3771 3731 006 -0040 0040 0975 1193 0073

G 3699 3653 3676 003 0023 -0023 -0328 0856 G 3602 3643 3623 003 -0021 0021 -1141 0612 -0053

H 3623 3672 3648 003 -0024 0024 -1615 0913 H 3748 3681 3715 005 0033 -0033 0664 0990 0067

L 3724 3663 3694 004 0031 -0031 0461 1150 L 3748 3771 3760 002 -0011 0011 1544 0335 0066

M 3690 3690 3690 000 0000 0000 0311 0000 M 3806 3681 3744 009 0062 -0062 1234 1848 0054

m 368334 m 368086 mediane D 00089

Cn(2p) 1867 Cn(2p) 1867 Cp 16101

Cn(p) 16101 Cn(p) 16101 Qn(D) 00325359

n=2p n 20 n=2p n 20 Qdiff 00523852

f 11 f 11 t 043

l 55 l 55

n=p n 10 n=p n 10

f 6 f 6

l 15 l 15

Qn(2p) 0014323 Qn(2p) 001811

Qn(p) 0013719 Qn(p) 003164

Qintra 002674 Qintra 003381

Qinter 002209 Qinter 005094

Sr(eacutecart-type de reacutepeacutetabiliteacute) 00378 Sr 00478

RSDr(coefficient de variation de la reacutepeacutetabiliteacute)103 RSDr 130

r(repeatability limit)) 0106 r 0134

SL(eacutecart-type interlaboratoire) -000023 SLlt0 SL 000145

SR(eacutecart-type de reproductibiliteacute) 00378 SR 00611

RSDR(coefficient de variation de la reproductibiliteacute)0010 RSDR 0017

R(limite de reproductibiliteacute)) 0105894 R(limite de reproductibiliteacute))017119

Ref Alt DLaboratoires

Niveau 3

Moyenne si Deviation h k Laboratoires

Niveau 3

Moyenne si Deviation h k

Page 6: ÞÀ¿¸7 ú Y5zñLjÜ< /u +æü t¬Á ... - NF Validation · NF VALIDATION Validation of alternative analytical methods Application in food microbiology ... 500 – 1 000 1 000 –

Thermo Fisher Scientific

ADRIA Deacuteveloppement 638 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Figure 1 ndash Bi-dimensional graphs

Rillettes Raw milk

Smoked salmon Raw egg product

Lettuce Process water

Thermo Fisher Scientific

ADRIA Deacuteveloppement 738 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Statistical interpretations

Statistical interpretation results are provided in Table 2

Table 2 ndash Statistical interpretations

Matrix R Selected

regression

RobF Critical

value

P Correlation

coefficient

Regression equation

Rillettes 180 GMFR 609 541 4 09960 log (Alt) = 09887 log (Ref) + 01141

Lettuce 108 GMFR 000 541 100 09992 Log (Alt) = 10081 log (Ref) - 00257

Raw milk 102 GMFR 000 541 100 09997 Log (Alt) = 10188 log (Ref) - 00737

Smoked salmon 1048 OLS1 1806 541 263 09984 Log (Alt) = 10406 log (Ref) - 01418

Raw egg product 130 GMFR 548 541 49 09958 Log (Alt) = 09775 log (Ref) - 00161

Process water 184 GMFR 0079 541 969 09985 Log (Alt) = 09680 log (Ref) + 01317

GMFR = orthogonal linear regression

OLS1 = ordinary least-square linear regression

x ndash axis = reference method

y ndash axis = alternative method

OLS1 = ordinary least-square linear regression

x axis = alternative method

y axis = reference method

Statistical Interpretation

P gt 5 not significant 1 lt P lt 5 significant

P lt 1 highly significant

The regressions straight lines are shown figure 2

Thermo Fisher Scientific

ADRIA Deacuteveloppement 838 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Figure 2 ndash Regression straight lines

Lineacuteariteacute - Œuf

Process water Process water (2) Process water

Smoked salmon (GMFR) Smoked salmon (OLS 1)

Raw milk Raw egg product

Rillettes Lettuce

Thermo Fisher Scientific

ADRIA Deacuteveloppement 938 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Discussion

The linearity is accepted in all cases with

P values 5 and correlation coefficients superior to 099

For the matrix ldquoRillettesldquo the linearity test is significant (P = 4) with a high

correlation coefficient value (0996)

The Listeria PrecisTM method shows satisfying linearity

2112 Relative accuracy

The accuracy is the closeness of agreement between a test result and the accepted reference value

The bias is the difference between the expectation of the test results and an accepted reference value

Number and nature of the samples

The repartition per category and types is provided in table 3

Table 3 ndash Number and nature of the samples

Categories Types Number of samples

Analysed Exploited (1)

Meat products

Raw meat products 6 4

Ready to cook products 4 2

Ready to eat delicatessen 10 4

Total 20 10

Dairy products

Raw milk cheeses 7 4

Raw milks 4 3

Other dairy products 5 5

Total 16 12

Fishery products

Raw fishes 5 4

Processed fishes 7 2

Ready-to-eat products 6 4

Total 18 10

Vegetables

Raw vegetables 5 4

Frozen vegetables 3 3

Lettuce raw vegetables 5 5

Total 13 12

Egg products

Raw egg 5 5

Pastries 3 3

Pasteurised egg products 4 3

Total 12 11

Environmental samples

Wipes swabs 8 4

Process water 3 3

Dusts 3 3

Total 14 10

TOTAL 93 65

(1) Some data cannot be exploited because enumeration was under the detection level for at least one of the

method

Thermo Fisher Scientific

ADRIA Deacuteveloppement 1038 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Artificial contamination of the samples

Artificial contaminations were realised by spiking after performing injury

protocols on pure cultures or by cross-contaminations

56 samples were artificially contaminated 53 gave exploitable results among

a total of 65 samples 8150 of the samples were artificially contaminated

The injury protocol and the strains inoculated are provided in Table 4

Table 4

Stress Ndeg Injury protocol Strain Origin

A 45 min at 50 degC

48

LM -H123

LM-H170

Smoked salmon

Lettuce

Lettuce

B 30 min at 50 degC

and 2 h at ndash20degC

18

43

LM-O1

Raw milk (environment)

Environment

Egg product

C 30 min at 55degC

18

48

LM-H123

Raw milk

Smoked salmon

Lettuce

D 30 min at 50degC

et 2 h at ndash80degC

34

LM-H171

LM-O1

Rillettes

Lettuce

Egg product

E 30 min at 60 degC LM-O1 Egg product

Confirmatory tests

For the reference and the alternative methods confirmations were realised

by the tests described in the ISO 12290-2A1 standard For the alternative

method only one colony was confirmed while 5 colonies were tested for the

reference method

Results

Samples were analysed in duplicate by the reference and the alternative

methods

The contamination range is provided in table 5 per tested category

Thermo Fisher Scientific

ADRIA Deacuteveloppement 1138 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Table 5 ndash Contamination range

Food category Contamination level (log CFUg)

Meat products 100 ndash 404

Dairy products 148 ndash 408

Fishery products 100 ndash 384

Vegetables 170 ndash 511

Egg products 130 ndash 499

Environmental samples 170 ndash 511

All categories 100 ndash 511

Bi-dimensional graphs are given figure 3

Figure 3 ndash Bi-dimensional graphs

Graphe bi-dimensionnel Produits carneacutes

Fishery products

Vegetables

Egg products

Meat products

Thermo Fisher Scientific

ADRIA Deacuteveloppement 1238 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Statistical interpretation according to EN ISO16140 method

The results of the statistical interpretation are given in Table 6

Table 6 ndash Statistical interpretation results

Category n R Regression

used a t(a) b t(b) Critical T

P

Ordinate

at 0

Slope

at 1

Meat products 10 158 GMFR -0455 3321 115 3123 2306 001 001

Dairy products 12 065 GMFR -0027 0385 1002 0108 2228 092 071

Fishery products 10 130 GMFR -0305 1024 1034 0312 2306 076 034

Vegetables 12 086 GMFR -0015 0128 0993 0191 2228 085 090

Egg products 11 158 GMFR -0038 0866 1001 0090 2262 093 041

Environmental

samples 10 176 GMFR -0139 265 1034 231 2306 005 003

All products 65 118 GMFR -0182 3042 1040 2145 2000 004 000

All products

(estimated number) 65 118 GMFR -0182 3042 1040 2145 2000 004 000

All products (non

estimated results) 20 115 GMFR -0367 151 1138 1141 2101 027 015

Repeatability limits obtained for the alternative and reference methods as

well as bias obtained are presented in Table 7

Dairy products Environmental samples

Log (Ref) Log (Ref)

All categories

Thermo Fisher Scientific

ADRIA Deacuteveloppement 1338 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Table 7

Categories Bias

Repeatability limits

Rob r

Ref Alt

Meat products 0008 0314 0343

Dairy products -0031 0136 0130

Fishery products -0133 0220 0214

Vegetables -0041 0180 0319

Egg products -0026 0219 0206

Environmental samples -0007 0198 0119

All products -0032 0196 0215

Regression straight lines (graph and equation representations) for each food

category and for all products are presented figure 4

Thermo Fisher Scientific

ADRIA Deacuteveloppement 1438 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Figure 4 ndash Regression straight lines

Meat products

Fishery products

Dairy products

Vegetables

Thermo Fisher Scientific

ADRIA Deacuteveloppement 1538 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Environmental samples

Egg products

All products (estimated number)

Thermo Fisher Scientific

ADRIA Deacuteveloppement 1638 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

The hypotheses that the ordinates are close to 0 and the slopes close to 1

are approved in all cases except for

- Meat products (slope P = 1 ordinate P = 1)

- Environmental samples (ordinate P = 3)

- All products (ordinate P = 0)

The bias between the two methods vary from ndash 0133 to 0008 log CFU For

all categories the bias is ndash 0032 log CFU The repeatability limits of the

alternative and the reference method are similar For all products the values

are respectively 0215 and 0196 log CFU For all products these

interpretations are provided taking into account data with low contamination

level or not (estimated colonies number)

For all food products the regression straight line is the following

log (Alt) = 1040 log Ref ndash 0182

The Listeria PrecisTM method shows satisfying relative accuracy

All products

All products (Level 2)

Thermo Fisher Scientific

ADRIA Deacuteveloppement 1738 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

2113 Detection limit (LOD) and quantification limit (LOQ)

The critical level is the defined as the smallest amount which can be detected (not null) but not

quantified as an exact value Below this value it cannot be sure that the true value is not null

The detection limit is defined as being higher than the critical level because it involves a power the

probability 1- which has to be well over 50 for example 95

The quantification limit is defined as the smallest amount of analyte (that is the lowest actual number of

organisms) which can be measured and quantified with defined precision and accuracy under the

experimental conditions by the method under validation

Protocol

The detection limit of the alternative method was realised with pure cultures

Three different inoculation levels were tested with six replicates per level ie

18 analyses were done by the alternative method

Listeria monocytogenes 18 was used for the detection limit of the alternative

method

Quantification limit was calculated for six independent blank samples

determinations

Results

These data are intrinsic to the method used and are presented in the

following tables

Table 8 ndash Inoculation 1 ml on 3 plates

Level (CFUml) Positive samples number Standard deviation Bias

0 06

05 26 1211 0

1 36 0816 05

5 66 2483 85

Thermo Fisher Scientific

ADRIA Deacuteveloppement 1838 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Table 9 ndash Inoculation 01 ml on 1 plate

Level (CFUml) Positive samples number Standard deviation Bias

0 06

5 36 0548 05

10 46 1414 2

50 66 2258 55

Table 10

Formulas Obtained values

LC 165 S0 + X0 18

LOD 33 S0 + X0 32

LOQ 10 S0 + X0 87

2114 Relative Sensitivity

The relative sensitivity is defined as the ability of the alternative method to detect two different amounts

of analyte measured by the reference method within a given matrix at a specified average value or

over the whole measurement range that is it is the minimal quantity variation (increase of the analyte

concentration x) which gives a significant variation of the measured signal (response y)

These data are intrinsic to the used method and are obtained from the results

of the linearity study

Sensitivity patterns obtained for tested (matrixstrain) pairs are presented in

figure 5

Thermo Fisher Scientific

ADRIA Deacuteveloppement 1938 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Figure 5 ndash Sensitivity patterns for the tested (matrixstrain) pairs

2115 SpecificitySelectivity

The specificity is defined as the degree to which a method is affected (or not) by the other components

present in a multi-component sample That is the ability of a method to measure exactly a given

analyte or its amount within the sample without interference from non-target components such as a

matrix effect or background noise

The selectivity is defined as a measure of the degree of non-interference in the presence of non-target

analytes A method is selective if it can be used to detect the analyte under examination and that a

guarantee can be provided that the detected signal can only be a product by that specific analyte

The specificity and selectivity study was performed for the Listeria PrecisTM

detection method no additional test was realised for the enumeration

method The results were the following

- Among the Listeria monocytogenes strains tested 2 strains (Listeria

monocytogenes CIP 7834 and Listeria monocytogenes CIP 105459) gave

negative results These 2 strains were tested twice by running 48 h

incubation time of the compared agars Positive results were then

observed by the alternative method Pale characteristic colonies were

observed on the ISO agar by streaking the Half Fraser broth only

- No cross reaction was observed with the non-target strains except with

Listeria ivanovii which gave characteristic colonies on Brilliancetrade Listeria

Agar but the confirmatory tests provide a clear discrimination between

Listeria monocytogenes and Listeria ivanovi species

Rillettes

Lettuce

Raw milk

Smoked salmon

Raw egg

Process water

x (CFUg)

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2038 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

212 Practicability

1 Packaging Ready to use Brilliance Listeria agar plates are available

(x 10 plates)

2 Reagent volume

3 Storage conditionsndash

Shelf life of products

not opened

The plates are stored at 6 - 12degC in darkness conditions The

expiration date is mentioned on the package and on the plates It

is around 3 months

4 Modalities of use

after first use

5 Required

equipments No specific equipment is required

6 Reagents Ready to use plates

7 Training 05 day

8 Real time handling

Reference method

9 min for one sample

55 min for 10 samples

Alternative method

7 min for one sample

35 minutes for 10 samples

9 Time to result

Negative results

Reference method Day 2

Alternative method Day 2

Negative results after confirmation or positive results

Reference method Day 4 ndash Day 7

Alternative method Day 4 ndash Day 7

Positive results

Reference method Day 4 ndash Day 7

Alternative method Day 2 ndash Day 3 (with OBIS Mono Test)

or Day 4 ndash Day 7 (by the classical tests described in the

standardised method)

10 Technician skill Identical to that required for the reference method

11 Steps common to

the reference method Mother suspension revivification step and decimal dilutions

12 Traceability

13 Maintenance

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2138 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

213 Inter-laboratory study

2131 Organisation

12 laboratories participated to this study Pasteurised milk was inoculated by

Listeria monocytogenes 12a isolated from raw milk The targeted

inoculation levels were

- 0 CFUml

- 50 CFU ml

- 500 CFU ml

- 5 000 CFU ml

Each laboratory received eight flasks of 10 ml sample ie two flasks per

inoculation level Furthermore one non-inoculated sample has been added

to the package for total viable count microflora (NF ISO 4833 method)

Detailed instructions were transmitted to the collaborators by the expert

laboratory

Collaborative study laboratories and the expert laboratory carried out the

analyses with the alternative and reference methods

2132 Verification of experimental parameters

Strain stability during transport

In order to evaluate the Listeria monocytogenes strain stability during

transport bacterial count of all inoculated flasks were checked at different

time ie inoculation time after 24 h of conservation at 2deg - 8degC Results are

reported in table 11

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2238 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Table 11 ndash Listeria monocytogenes count

by ISO 11290-2A1 method (log CFUml)

Day of the analyses Level contamination Reference method

Day 0

(29052006)

1 45

2 440

3 4 500

Day 1

(30052006)

1 60

2 430

3 4 900

No evolution of the strain was observed after storage for 24 h at 2degC ndash 8degC

Contamination level

The real contamination levels are provided in table 12

Table 12 ndash Real contamination level (CFUml)

Target inoculation

(CFU ml) Samples Ndeg

Real contamination levels

(CFUml)

0 1 - 8 0

50 5 - 6 45

500 3 - 7 440

5000 2 - 4 4 500

Sample temperature at receipt

Measured temperatures at receipt are listed in table 13

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2338 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Table 13 - Sample temperature on receipt

Laboratories Receipt

day

Hour

receipt

Measured temperature

at receipt (degC)

Measured temperature by the

temperature probe (degC)

A

B

C

D

E

F

G

H

I

J

K

L

M

30052006

10h45

10h15

11h15

10h00

09h30

8h30

11h50

10h00

08h30

14h45

08h30

09h45

08h45

82

55

45

63

60

62

81

38

48

-

14

67

76

03

03

-13

03

04

-14

05

04

01

05

-13

04

Conclusion

The temperatures during the transport were correct for all the labs except for

Lab J which didnrsquot provide the information

All the labs received their packages with several broken flasks

Three labs (B D and L) received some damaged samples but proceeded to

analyses

All the labs started the analyses at Day 1 except Lab J

2133 Results

Aerobic mesophilic flora

The count of aerobic mesophilic flora varied from 94 102 CFUml to

33 103 CFUml

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2438 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Expert lab results

The results obtained by the expert lab are the following

Table 14

Target inoculation level (CFUml)

Reference method ISO 11290-2

Alternative method Listeria Precis Enumeration

Rep 1 Rep 2 Rep 1 Rep 2

lt 10 lt 10 lt 10 lt 10 lt10

50 50 (Ne) 60 (Ne) 50 (Ne) 30 (Ne)

500 470 390 490 500

5 000 4 700 5 100 5 900 3 400

Ne estimated number

The obtained results are in agreement with those expected

Collaborator Labs results

A synthesis of the results obtained by the collaborator labs is provided

Table 15 (CFUml) and Table 16 (log CFUml)

Table 15 ndash Synthesis of results obtained by the alternative

and the reference methods (CFUml)

Labs Level 1 Level 2 Level 2 bis Level 3

Reference method

Alternative method

Reference method

Alternative method

Reference method

Alternative method

Reference method

Alternative method

A 40 40 70 40 480 410 210 340 1000 300 400 300 4900 4600 4900 5100

B 40 15 40 20 350 300 360 450 350 650 800 600 4100 4700 5100 3700

C 50 20 20 70 440 430 480 450 300 650 300 500 4600 5400 4600 5000

D 40 60 40 40 490 460 600 540 250 450 600 1000 4900 4900 4400 4600

E 50 40 50 20 490 520 530 560 750 550 400 200 5400 4500 4100 5400

F 60 20 60 50 370 250 300 130 300 350 600 300 4400 4700 4900 5900

G 70 35 120 50 440 390 570 370 450 550 700 700 5000 4500 4000 4400

H 110 40 100 90 520 440 450 540 150 650 300 300 4200 4700 5600 4800

L 35 60 20 40 390 390 170 380 650 350 800 200 5300 4600 5600 5900

M 45 45 30 40 450 490 550 410 400 550 800 180 4900 4900 6400 4800

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2538 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Table 16 ndash Synthesis of results obtained by the alternative

and the reference methods (log CFUml)

Labs Level 1 Level 2 Level 2 bis Level 3

Reference method

Alternative method

Reference method

Alternative method

Reference method

Alternative method

Reference method

Alternative method

A 1602 1602 1845 1602 2681 2613 2322 2531 3000 2477 2602 2477 3690 3663 3690 3708

B 1602 1176 1602 1301 2544 2477 2556 2653 2544 2813 2903 2778 3613 3672 3708 3568

C 1699 1301 1301 1845 2643 2633 2681 2653 2477 2813 2477 2699 3663 3732 3663 3699

D 1602 1778 1602 1602 2690 2663 2778 2732 2398 2653 2778 3000 3690 3690 3643 3663

E 1699 1602 1699 1301 2690 2716 2724 2748 2875 2740 2602 2301 3732 3653 3613 3732

F 1778 1301 1778 1699 2568 2398 2477 2114 2477 2544 2778 2477 3643 3672 3690 3771

G 1845 1544 2079 1699 2643 2591 2756 2568 2653 2740 2845 2845 3699 3653 3602 3643

H 2041 1602 2000 1954 2716 2643 2653 2732 2176 2813 2477 2477 3623 3672 3748 3681

L 1544 1778 1301 1602 2591 2591 2230 2580 2813 2544 2903 2301 3724 3663 3748 3771

M 1653 1653 1477 1602 2653 2690 2740 2613 2602 2740 2903 2255 3690 3690 3806 3681

Level 2 results obtained taking account the 3 plates inoculated with the mother-suspension

Level 2 bis results obtained taking account the plates inoculated with 01 ml of the mother-suspension

Scrutiny of the measurement results for consistency

In order to identify the measurement results or laboratories that could be

inconsistent two graphical consistency techniques were realised Mandelrsquos

h-ank-statistics Mandel indicators h and k at 5 significance highlighted

some possible inconsistent data (See tables and graphics in Appendix 3 and

Appendix 4)

Mandelrsquos values Number of values observed over the threshold

Reference method Alternative method

h gt 1 Lab F Level 2 Lab F Level 2

h gt 5 Lab F Level 2

Lab B Levels 1 and 2

Lab A Level 2

Lab D Level 3

Lab F Level 2

Lab L Level 2

kgt 1 Lab F Level 3

k gt 5 Lab F Level 2 Lab C Level 2

Lab F Level 3

Lab L Levels 1 and 3

Lab M Level 1

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2638 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

The emphasized results appear (i) coherent with the targeted contamination

levels (ii) concordant between both methods

All the results were thus kept for the statistical analysis

Comparison of the trueness and precision characteristics of

the reference method and alternative methods

The statistical values are summarised hereafter

Level p

Reference method Alternative method

Repeatability ratio

Reproducibility ratio Median

Repeatability standard deviation

Reproducibility standard deviation

Median Repeatability

standard deviation

Reproducibility standard deviation

1 10 16519 02415 02425 16021 02045 02433 0847 1007

2 10 26551 00543 00659 26719 00914 01325 1682 2011

2 bis 10 26581 02607 02607 25951 01900 01953 0729 0749

3 10 18671 00378 00378 18671 00478 00611 1264 1617

Bias of the alternative method

In order to estimate the bias of the alternative method with regard to the reference method for every

level Dij and t are calculated according to the following equations

RefAlt ijYijYDij

Diffp

Dijit

)2(

)(median

If the t value is superior to 2 the alternative method is significantly biased

with regard to the reference method

The values are given in table 17

Table 17 ndash t (d) values obtained by level

Level Bias t (d) Conclusion

1 0068 114 Non significant bias

2 0021 082 Non significant bias

2 bis - 0037 035 Non significant bias

3 - 0037 043 Non significant bias

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2738 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

The statistical test concludes to a non significant bias for all the inoculation

levels The bias varies from ndash 0037 to 0068 log CFUml

Comparison of the repeatability standard deviations

If the ratio Srj Alt Srj Ref of the repeatability standard deviations of the

alternative method and the reference method is larger than 2 the precision

under repeatability conditions of the alternative method is considered to be

lower than that of the reference method If this ratio is smaller than 05 the

precision under repeatability conditions of the alternative method is

considered to be greater than that of the reference method

The ratio values are given in table 18

Table 18

Level

Repeatability standard deviation

Reference method Alternative method Alternative method

reference method ratio

1 0242 0205 0847

2 0054 0091 1682

2 bis 0261 0190 0729

3 0038 0048 1264

Level Repeatability limit

Reference method Alternative method

1 0676 0573

2 0152 0256

2 bis 0730 0532

3 0106 0134

The ratios of the repeatability standard deviations are below 2 in all cases

The precision under repeatability conditions of the alternative method is

equivalent to that of the reference method

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2838 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Comparison of the reproducibility standard deviations

If the ratio Srj Alt Srj Ref of the reproducibility standard deviations of the alternative method and the

reference method is larger than 2 the precision under reproducibility conditions of the alternative

method is considered to be lower than that of the reference method If this ratio is smaller than 05 the

precision under reproducibility conditions of the alternative method is considered to be greater than

that of the reference method

The ratio values are given in table 19

Table 19

Level

Reproducibility standard deviation

Reference method Alternative method Alternative method

reference method ratio

1 0242 0243 1007

2 0066 0133 2011

2 bis 0261 0195 0749

3 0038 0061 1617

Level Reproducibility limit

Reference method Alternative method

1 0676 0681

2 0185 0371

2 bis 0730 0547

3 0106 0171

The ratios of the reproducibility standard deviations are below or close to 2

The precision under reproducibility conditions of the alternative method is

equivalent to that of the reference method

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2938 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

214 Conclusion

Method comparison study

The Listeria PrecisTM enumeration method shows satisfying linearity and

relative accuracy

The alternative method shows satisfying inclusivity and exclusivity results

Two Listeria monocytogenes strains gave negative results with the

alternative method These strains grew weakly on the reference method

media (OampA Palcam and Oxford)

The limits of repeatability of the alternative method are similar to those of the

reference method

The bias between both methods are low between - 0133 Log CFUg and

- 0008 Log CFUg

The Listeria PrecisTM enumeration method offers the saving for confirmatory

tests

Positive results are obtained with the alternative method in 2-3 days (with

OBIS Mono test) or in 4-7 days (by the classical tests described in the

standardized method) against 4-7 days for the reference method

Inter-laboratory study

The bias between the reference and the alternative methods shows satisfying

and correct values

- level 1 (100 - 1 000 CFUg) bias = + 007 log CFUg

- level 2 (1 000 - 10 000 CFUg) bias = 002 log CFUg

- level 2 bis (1 000 - 10 000 CFUg) bias = - 004 log CFUg

- level 3 (10 000 - 100 000 CFUg) bias = - 004 log CFUg

The repeatability and the reproducibility of the alternative method look similar

to those of the reference method

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3038 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

22 Extension study (realised by ASEPT 2007)

An extension study was performed in 2007 for a new confirmatory test OBIS

MONO

150 Listeria monocytogenes strains and 100 non-target strains were isolated

onto TSAYE and BrillianceTM Listeria Agar plates All the Listeria

monocytogenes and the non-target strains gave the expected results

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3138 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Appendix 1 ndash Flow diagram of the Listeria PrecisTM method

x g sample + 9 x ml buffered peptone water

Revivification step for 1 h plusmn 5 min at 20degC plusmn 1degC

Streaking 1 ml onto 3 Brilliance Listeria Agar plates (mother suspension)

or 01 ml onto 1 Brilliance Listeria Agar plates (mother suspension and decimal dilutions)

Incubation for 48 h plusmn 3 h at 37degC plusmn 1degC

Confirmatory tests on at least one typical colony

- by the tests described in the ISO method

- by applying the OBIS mono test on an isolated colony

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3238 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Appendix 2 ndash ISO 11290-2 (1998) amp ISO 11290-2A1 (2004) Horizontal

method for the detection and enumeration of Listeria monocytogenes -

Part 2 enumeration of Listeria monocytogenes in foods

10 g cheese + 90 ml buffered peptone water or Half Fraser Broth (without supplements) (110 dilution)

Stomach 1 min 30 seconds

Keep at 20degC plusmn 2degC for 1 h plusmn 5 min

Decimal dilutions in 9 ml peptone salt tubes

Dilution 110

Dilution 1100

Dilution 11000

1 ml on 3 Chromogenic Listeria medium (ISO) plates (x 2) (-1)

01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-2)

01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-3)

01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-4)

Incubation at 37degC plusmn 1degC

24 h - 48 h

Numerate typical colonies at 24 h

(bluegreen surrounded by an opaque halo)

Numerate at 48 h

Streak typical colonies on TSYEA

(5 coloniesplate 2 successive dilutions)

Confirmation tests

Catalase

Gram stain

For confirmation of L monocytogenes

Haemolysis

Carbohydrate (rhamnose and xylose) utilisation

CAMP Test

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3338 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Appendix 3 ndash Mandelrsquos graphics

Laboratoire h Ref

Niveau 1 h Ref

Niveau 2 h Ref

Niveau 3 h Alt

Niveau 1 h Alt

Niveau 2 h Alt

Niveau 3 h5 h1 h5 h1

A -050 -015 083 062 -212 -039 198 299 -198 -299

B -261 -270 021 -077 -058 173 198 299 -198 -299

C -151 -031 -014 -015 -004 -005 198 299 -198 -299

D 038 040 -137 000 072 207 198 299 -198 -299

E -001 090 155 -052 056 -101 198 299 -198 -299

F -112 -321 -153 070 -325 023 198 299 -198 -299

G 042 -071 040 147 -009 176 198 299 -198 -299

H 169 046 -169 192 018 -083 198 299 -198 -299

L 009 -120 021 -077 -231 005 198 299 -198 -299

M 001 031 014 -032 004 -011 198 299 -198 -299

-300

-200

-100

000

100

200

300

A B C D E F G H L M

Vale

urs

h d

e M

an

del(

rob

uste

s)

Laboratoires

Meacutethode de reacutefeacuterence analyse robuste

h Ref Niveau 1

h Ref Niveau 2

h Ref Niveau 3

h 5=198

h 1=299

-300

-200

-100

000

100

200

300

A B C D E F G H L M

Vale

urs

de h

de M

an

del(

rob

uste

s)

Laboratoires

Meacutethode alternative analyse robuste

h Alt Niveau 1

h Alt Niveau 2

h Alt Niveau 2

h 1=299

h 5=198

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3438 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Laboratoire k Ref

Niveau 1 k Ref

Niveau 2 k Ref

Niveau 3 k Alt

Niveau 1 k Alt

Niveau 2 k Alt

Niveau 3 k1 k5

A 000 089 142 084 162 046 259 181

B 125 087 073 104 075 046 259 181

C 117 013 091 188 022 083 259 181

D 052 036 069 000 035 083 259 181

E 028 034 037 138 019 112 259 181

F 140 222 018 027 281 112 259 181

G 088 068 024 131 145 000 259 181

H 129 094 173 016 061 000 259 181

L 069 000 073 104 270 224 259 181

M 000 048 038 043 099 241 259 181

000

050

100

150

200

250

300

350

400

A B C D E F G H L M

Vale

urs

k d

e M

an

del(

rob

uste

s)

Laboratoires

Meacutethode de reacutefeacuterence analyse robuste

k Ref Niveau 1k Ref Niveau 2k Ref Niveau 3

k 1=259

k 5=181

000

050

100

150

200

250

300

A B C D E F G H L M

Vale

urs

k d

e M

an

del

(ro

bu

ste

s)

Laboratoires

Meacutethode alternative analyse robuste

k Alt Niveau 3

k Alt Niveau 1

k Alt Niveau 2

k 1=259

k 5=181

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3538 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Appendix 4 ndash Statistical results

Level 1

Nombre de laboratoires (p) 10

A 1602 1602 1602 000 0000 0000 -0495 0000 A 1845 1602 1724 017 0122 -0122 0621 0840 0122

B 1602 1176 1389 030 0213 -0213 -2613 1247 B 1602 1301 1452 021 0151 -0151 -0769 1041 0062

C 1699 1301 1500 028 0199 -0199 -1510 1165 C 1301 1845 1573 038 -0272 0272 -0148 1881 0073

D 1602 1778 1690 012 -0088 0088 0380 0516 D 1602 1602 1602 000 0000 0000 0000 0000 -0088

E 1699 1602 1651 007 0048 -0048 -0013 0284 E 1699 1301 1500 028 0199 -0199 -0522 1376 -0151

F 1778 1301 1540 034 0239 -0239 -1116 1397 F 1778 1699 1739 006 0040 -0040 0698 0274 0199

G 1845 1544 1695 021 0151 -0151 0425 0881 G 2079 1699 1889 027 0190 -0190 1467 1315 0194

H 2041 1602 1822 031 0220 -0220 1689 1286 H 2000 1954 1977 003 0023 -0023 1917 0158 0155

L 1544 1778 1661 017 -0117 0117 0092 0685 L 1301 1602 1452 021 -0151 0151 -0769 1041 -0210

M 1653 1653 1653 000 0000 0000 0013 0000 M 1477 1602 1540 009 -0062 0062 -0319 0432 -0114

m 165186 m 160206 mediane D 00678

Cn(2p) 1867 Cn(2p) 1867 Cp 16101

Cn(p) 16101 Cn(p) 16101 Qn(D) 0092926

n=2p n 20 n=2p n 20 Qdiff 01496169

f 11 f 11 t 114

l 55 l 55

n=p n 10 n=p n 10

f 6 f 6

l 15 l 15

Qn(2p) 0091465 Qn(2p) 007745

Qn(p) 0062469 Qn(p) 0121519

Qintra 017078 Qintra 014461

Qinter 010058 Qinter 019565

Sr(eacutecart-type de reacutepeacutetabiliteacute) 02415 Sr 02045

RSDr(coefficient de variation de la reacutepeacutetabiliteacute)1462 RSDr 1277

r(repeatability limit)) 0676 r 0573

SL(eacutecart-type interlaboratoire) -001905 SLlt0 SL 001737

SR(eacutecart-type de reproductibiliteacute) 02415 SR 02433

RSDR(coefficient de variation de la reproductibiliteacute)0146 RSDR 0152

R(limite de reproductibiliteacute)) 06762 R(limite de reproductibiliteacute))068123

h k h MoyenneLaboratoires

Niveau 1

si LaboratoiresDeviationMoyenne Dk DeviationsiAlt

Niveau 1

Ref

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3638 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Level 2

Nombre de laboratoires (p) 10

A 2681 2613 2647 005 0034 -0034 -0151 0891 A 2322 2531 2427 015 -0105 0105 -2119 1620 -0220

B 2544 2477 2511 005 0033 -0033 -2699 0872 B 2556 2653 2605 007 -0048 0048 -0581 0750 0094

C 2643 2633 2638 001 0005 -0005 -0311 0130 C 2681 2653 2667 002 0014 -0014 -0040 0217 0029

D 2690 2663 2676 002 0014 -0014 0400 0357 D 2778 2732 2755 003 0023 -0023 0721 0354 0079

E 2690 2716 2703 002 -0013 0013 0897 0336 E 2724 2748 2736 002 -0012 0012 0556 0185 0033

F 2568 2398 2483 012 0085 -0085 -3214 2217 F 2477 2114 2296 026 0182 -0182 -3255 2811 -0188

G 2643 2591 2617 004 0026 -0026 -0707 0682 G 2756 2568 2662 013 0094 -0094 -0085 1453 0045

H 2716 2643 2680 005 0036 -0036 0460 0945 H 2653 2732 2693 006 -0040 0040 0181 0613 0013

L 2591 2591 2591 000 0000 0000 -1196 0000 L 2230 2580 2405 025 -0175 0175 -2307 2704 -0186

M 2653 2690 2672 003 -0018 0018 0311 0481 M 2740 2613 2677 009 0064 -0064 0040 0987 0005

m 2655082 m 26719001 mediane D 00209

Cn(2p) 1867 Cn(2p) 1867 Cp 16101

Cn(p) 16101 Cn(p) 16101 Qn(D) 0039911

n=2p n 20 n=2p n 20 Qdiff 00642591

f 11 f 11 t 082

l 55 l 55

n=p n 10 n=p n 10

f 6 f 6

l 15 l 15

Qn(2p) 0020570 Qn(2p) 00345985

Qn(p) 00332437 Qn(p) 00718158

Qintra 003841 Qintra 006460

Qinter 005352 Qinter 011563

Sr(eacutecart-type de reacutepeacutetabiliteacute) 00543 Sr 00914

RSDr(coefficient de variation de la reacutepeacutetabiliteacute)205 RSDr 342

r(repeatability limit)) 0152 r 0256

SL(eacutecart-type interlaboratoire) 000139 SL 000920

SR(eacutecart-type de reproductibiliteacute) 00659 SR 01325

RSDR(coefficient de variation de la reproductibiliteacute)0025 RSDR 0050

R(limite de reproductibiliteacute)) 018446 R(limite de reproductibiliteacute))03708618

Laboratoires

Niveau 2

Moyenne si Laboratoires

Niveau 2

DRef Alt Moyenne si Deviation h Deviation h k k

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3738 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Level 2 bis

Nombre de laboratoires (p) 10

A 3000 2477 2739 037 0261 -0261 0832 1418 A 2602 2477 2540 009 0062 -0062 -0391 0465 -0199

B 2544 2813 2678 019 -0134 0134 0211 0729 B 2903 2778 2841 009 0062 -0062 1732 0465 0162

C 2477 2813 2645 024 -0168 0168 -0135 0911 C 2477 2699 2588 016 -0111 0111 -0049 0826 -0057

D 2398 2653 2526 018 -0128 0128 -1370 0692 D 2778 3000 2889 016 -0111 0111 2074 0826 0363

E 2875 2740 2808 010 0067 -0067 1547 0365 E 2602 2301 2452 021 0151 -0151 -1012 1120 -0356

F 2477 2544 2511 005 -0033 0033 -1525 0182 F 2778 2477 2628 021 0151 -0151 0230 1120 0117

G 2653 2740 2697 006 -0044 0044 0400 0236 G 2845 2845 2845 000 0000 0000 1764 0000 0148

H 2176 2813 2495 045 -0318 0318 -1692 1727 H 2477 2477 2477 000 0000 0000 -0832 0000 -0017

L 2813 2544 2678 019 0134 -0134 0211 0729 L 2903 2301 2602 043 0301 -0301 0049 2241 -0076

M 2602 2740 2671 010 -0069 0069 0135 0375 M 2903 2255 2579 046 0324 -0324 -0112 2411 -0092

m 2658114 m 259505 mediane D -00372

Cn(2p) 1867 Cn(2p) 1867 Cp 16101

Cn(p) 16101 Cn(p) 16101 Qn(D) 01656915

n=2p n 20 n=2p n 20 Qdiff 02667753

f 11 f 11 t 035

l 55 l 55

n=p n 10 n=p n 10

f 6 f 6

l 15 l 15

Qn(2p) 0098745 Qn(2p) 007195

Qn(p) 006007 Qn(p) 008805

Qintra 018437 Qintra 013435

Qinter 009672 Qinter 014176

Sr(eacutecart-type de reacutepeacutetabiliteacute) 02607 Sr 01900

RSDr(coefficient de variation de la reacutepeacutetabiliteacute)981 RSDr 732

r(repeatability limit)) 0730 r 0532

SL(eacutecart-type interlaboratoire) -002464 SLlt0 SL 000205

SR(eacutecart-type de reproductibiliteacute) 02607 SR 01953

RSDR(coefficient de variation de la reproductibiliteacute)0098 RSDR 0075

R(limite de reproductibiliteacute)) 072996 R(limite de reproductibiliteacute))054686

Laboratoires

Niveau 2 bis

Moyenne si Laboratoires

Niveau 2 bis

DRef Alt Moyenne si Deviation h Deviation h k k

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3838 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Level 3

Nombre de laboratoires (p) 10

A 3690 3663 3676 002 0014 -0014 -0311 0513 A 3690 3708 3699 001 -0009 0009 0354 0257 0022

B 3613 3672 3642 004 -0030 0030 -1851 1109 B 3708 3568 3638 010 0070 -0070 -0844 2061 -0005

C 3663 3732 3698 005 -0035 0035 0645 1302 C 3663 3699 3681 003 -0018 0018 0000 0536 -0017

D 3690 3690 3690 000 0000 0000 0311 0000 D 3643 3663 3653 001 -0010 0010 -0545 0286 -0037

E 3732 3653 3693 006 0040 -0040 0429 1480 E 3613 3732 3673 008 -0060 0060 -0162 1769 -0020

F 3643 3672 3658 002 -0014 0014 -1157 0536 F 3690 3771 3731 006 -0040 0040 0975 1193 0073

G 3699 3653 3676 003 0023 -0023 -0328 0856 G 3602 3643 3623 003 -0021 0021 -1141 0612 -0053

H 3623 3672 3648 003 -0024 0024 -1615 0913 H 3748 3681 3715 005 0033 -0033 0664 0990 0067

L 3724 3663 3694 004 0031 -0031 0461 1150 L 3748 3771 3760 002 -0011 0011 1544 0335 0066

M 3690 3690 3690 000 0000 0000 0311 0000 M 3806 3681 3744 009 0062 -0062 1234 1848 0054

m 368334 m 368086 mediane D 00089

Cn(2p) 1867 Cn(2p) 1867 Cp 16101

Cn(p) 16101 Cn(p) 16101 Qn(D) 00325359

n=2p n 20 n=2p n 20 Qdiff 00523852

f 11 f 11 t 043

l 55 l 55

n=p n 10 n=p n 10

f 6 f 6

l 15 l 15

Qn(2p) 0014323 Qn(2p) 001811

Qn(p) 0013719 Qn(p) 003164

Qintra 002674 Qintra 003381

Qinter 002209 Qinter 005094

Sr(eacutecart-type de reacutepeacutetabiliteacute) 00378 Sr 00478

RSDr(coefficient de variation de la reacutepeacutetabiliteacute)103 RSDr 130

r(repeatability limit)) 0106 r 0134

SL(eacutecart-type interlaboratoire) -000023 SLlt0 SL 000145

SR(eacutecart-type de reproductibiliteacute) 00378 SR 00611

RSDR(coefficient de variation de la reproductibiliteacute)0010 RSDR 0017

R(limite de reproductibiliteacute)) 0105894 R(limite de reproductibiliteacute))017119

Ref Alt DLaboratoires

Niveau 3

Moyenne si Deviation h k Laboratoires

Niveau 3

Moyenne si Deviation h k

Page 7: ÞÀ¿¸7 ú Y5zñLjÜ< /u +æü t¬Á ... - NF Validation · NF VALIDATION Validation of alternative analytical methods Application in food microbiology ... 500 – 1 000 1 000 –

Thermo Fisher Scientific

ADRIA Deacuteveloppement 738 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Statistical interpretations

Statistical interpretation results are provided in Table 2

Table 2 ndash Statistical interpretations

Matrix R Selected

regression

RobF Critical

value

P Correlation

coefficient

Regression equation

Rillettes 180 GMFR 609 541 4 09960 log (Alt) = 09887 log (Ref) + 01141

Lettuce 108 GMFR 000 541 100 09992 Log (Alt) = 10081 log (Ref) - 00257

Raw milk 102 GMFR 000 541 100 09997 Log (Alt) = 10188 log (Ref) - 00737

Smoked salmon 1048 OLS1 1806 541 263 09984 Log (Alt) = 10406 log (Ref) - 01418

Raw egg product 130 GMFR 548 541 49 09958 Log (Alt) = 09775 log (Ref) - 00161

Process water 184 GMFR 0079 541 969 09985 Log (Alt) = 09680 log (Ref) + 01317

GMFR = orthogonal linear regression

OLS1 = ordinary least-square linear regression

x ndash axis = reference method

y ndash axis = alternative method

OLS1 = ordinary least-square linear regression

x axis = alternative method

y axis = reference method

Statistical Interpretation

P gt 5 not significant 1 lt P lt 5 significant

P lt 1 highly significant

The regressions straight lines are shown figure 2

Thermo Fisher Scientific

ADRIA Deacuteveloppement 838 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Figure 2 ndash Regression straight lines

Lineacuteariteacute - Œuf

Process water Process water (2) Process water

Smoked salmon (GMFR) Smoked salmon (OLS 1)

Raw milk Raw egg product

Rillettes Lettuce

Thermo Fisher Scientific

ADRIA Deacuteveloppement 938 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Discussion

The linearity is accepted in all cases with

P values 5 and correlation coefficients superior to 099

For the matrix ldquoRillettesldquo the linearity test is significant (P = 4) with a high

correlation coefficient value (0996)

The Listeria PrecisTM method shows satisfying linearity

2112 Relative accuracy

The accuracy is the closeness of agreement between a test result and the accepted reference value

The bias is the difference between the expectation of the test results and an accepted reference value

Number and nature of the samples

The repartition per category and types is provided in table 3

Table 3 ndash Number and nature of the samples

Categories Types Number of samples

Analysed Exploited (1)

Meat products

Raw meat products 6 4

Ready to cook products 4 2

Ready to eat delicatessen 10 4

Total 20 10

Dairy products

Raw milk cheeses 7 4

Raw milks 4 3

Other dairy products 5 5

Total 16 12

Fishery products

Raw fishes 5 4

Processed fishes 7 2

Ready-to-eat products 6 4

Total 18 10

Vegetables

Raw vegetables 5 4

Frozen vegetables 3 3

Lettuce raw vegetables 5 5

Total 13 12

Egg products

Raw egg 5 5

Pastries 3 3

Pasteurised egg products 4 3

Total 12 11

Environmental samples

Wipes swabs 8 4

Process water 3 3

Dusts 3 3

Total 14 10

TOTAL 93 65

(1) Some data cannot be exploited because enumeration was under the detection level for at least one of the

method

Thermo Fisher Scientific

ADRIA Deacuteveloppement 1038 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Artificial contamination of the samples

Artificial contaminations were realised by spiking after performing injury

protocols on pure cultures or by cross-contaminations

56 samples were artificially contaminated 53 gave exploitable results among

a total of 65 samples 8150 of the samples were artificially contaminated

The injury protocol and the strains inoculated are provided in Table 4

Table 4

Stress Ndeg Injury protocol Strain Origin

A 45 min at 50 degC

48

LM -H123

LM-H170

Smoked salmon

Lettuce

Lettuce

B 30 min at 50 degC

and 2 h at ndash20degC

18

43

LM-O1

Raw milk (environment)

Environment

Egg product

C 30 min at 55degC

18

48

LM-H123

Raw milk

Smoked salmon

Lettuce

D 30 min at 50degC

et 2 h at ndash80degC

34

LM-H171

LM-O1

Rillettes

Lettuce

Egg product

E 30 min at 60 degC LM-O1 Egg product

Confirmatory tests

For the reference and the alternative methods confirmations were realised

by the tests described in the ISO 12290-2A1 standard For the alternative

method only one colony was confirmed while 5 colonies were tested for the

reference method

Results

Samples were analysed in duplicate by the reference and the alternative

methods

The contamination range is provided in table 5 per tested category

Thermo Fisher Scientific

ADRIA Deacuteveloppement 1138 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Table 5 ndash Contamination range

Food category Contamination level (log CFUg)

Meat products 100 ndash 404

Dairy products 148 ndash 408

Fishery products 100 ndash 384

Vegetables 170 ndash 511

Egg products 130 ndash 499

Environmental samples 170 ndash 511

All categories 100 ndash 511

Bi-dimensional graphs are given figure 3

Figure 3 ndash Bi-dimensional graphs

Graphe bi-dimensionnel Produits carneacutes

Fishery products

Vegetables

Egg products

Meat products

Thermo Fisher Scientific

ADRIA Deacuteveloppement 1238 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Statistical interpretation according to EN ISO16140 method

The results of the statistical interpretation are given in Table 6

Table 6 ndash Statistical interpretation results

Category n R Regression

used a t(a) b t(b) Critical T

P

Ordinate

at 0

Slope

at 1

Meat products 10 158 GMFR -0455 3321 115 3123 2306 001 001

Dairy products 12 065 GMFR -0027 0385 1002 0108 2228 092 071

Fishery products 10 130 GMFR -0305 1024 1034 0312 2306 076 034

Vegetables 12 086 GMFR -0015 0128 0993 0191 2228 085 090

Egg products 11 158 GMFR -0038 0866 1001 0090 2262 093 041

Environmental

samples 10 176 GMFR -0139 265 1034 231 2306 005 003

All products 65 118 GMFR -0182 3042 1040 2145 2000 004 000

All products

(estimated number) 65 118 GMFR -0182 3042 1040 2145 2000 004 000

All products (non

estimated results) 20 115 GMFR -0367 151 1138 1141 2101 027 015

Repeatability limits obtained for the alternative and reference methods as

well as bias obtained are presented in Table 7

Dairy products Environmental samples

Log (Ref) Log (Ref)

All categories

Thermo Fisher Scientific

ADRIA Deacuteveloppement 1338 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Table 7

Categories Bias

Repeatability limits

Rob r

Ref Alt

Meat products 0008 0314 0343

Dairy products -0031 0136 0130

Fishery products -0133 0220 0214

Vegetables -0041 0180 0319

Egg products -0026 0219 0206

Environmental samples -0007 0198 0119

All products -0032 0196 0215

Regression straight lines (graph and equation representations) for each food

category and for all products are presented figure 4

Thermo Fisher Scientific

ADRIA Deacuteveloppement 1438 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Figure 4 ndash Regression straight lines

Meat products

Fishery products

Dairy products

Vegetables

Thermo Fisher Scientific

ADRIA Deacuteveloppement 1538 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Environmental samples

Egg products

All products (estimated number)

Thermo Fisher Scientific

ADRIA Deacuteveloppement 1638 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

The hypotheses that the ordinates are close to 0 and the slopes close to 1

are approved in all cases except for

- Meat products (slope P = 1 ordinate P = 1)

- Environmental samples (ordinate P = 3)

- All products (ordinate P = 0)

The bias between the two methods vary from ndash 0133 to 0008 log CFU For

all categories the bias is ndash 0032 log CFU The repeatability limits of the

alternative and the reference method are similar For all products the values

are respectively 0215 and 0196 log CFU For all products these

interpretations are provided taking into account data with low contamination

level or not (estimated colonies number)

For all food products the regression straight line is the following

log (Alt) = 1040 log Ref ndash 0182

The Listeria PrecisTM method shows satisfying relative accuracy

All products

All products (Level 2)

Thermo Fisher Scientific

ADRIA Deacuteveloppement 1738 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

2113 Detection limit (LOD) and quantification limit (LOQ)

The critical level is the defined as the smallest amount which can be detected (not null) but not

quantified as an exact value Below this value it cannot be sure that the true value is not null

The detection limit is defined as being higher than the critical level because it involves a power the

probability 1- which has to be well over 50 for example 95

The quantification limit is defined as the smallest amount of analyte (that is the lowest actual number of

organisms) which can be measured and quantified with defined precision and accuracy under the

experimental conditions by the method under validation

Protocol

The detection limit of the alternative method was realised with pure cultures

Three different inoculation levels were tested with six replicates per level ie

18 analyses were done by the alternative method

Listeria monocytogenes 18 was used for the detection limit of the alternative

method

Quantification limit was calculated for six independent blank samples

determinations

Results

These data are intrinsic to the method used and are presented in the

following tables

Table 8 ndash Inoculation 1 ml on 3 plates

Level (CFUml) Positive samples number Standard deviation Bias

0 06

05 26 1211 0

1 36 0816 05

5 66 2483 85

Thermo Fisher Scientific

ADRIA Deacuteveloppement 1838 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Table 9 ndash Inoculation 01 ml on 1 plate

Level (CFUml) Positive samples number Standard deviation Bias

0 06

5 36 0548 05

10 46 1414 2

50 66 2258 55

Table 10

Formulas Obtained values

LC 165 S0 + X0 18

LOD 33 S0 + X0 32

LOQ 10 S0 + X0 87

2114 Relative Sensitivity

The relative sensitivity is defined as the ability of the alternative method to detect two different amounts

of analyte measured by the reference method within a given matrix at a specified average value or

over the whole measurement range that is it is the minimal quantity variation (increase of the analyte

concentration x) which gives a significant variation of the measured signal (response y)

These data are intrinsic to the used method and are obtained from the results

of the linearity study

Sensitivity patterns obtained for tested (matrixstrain) pairs are presented in

figure 5

Thermo Fisher Scientific

ADRIA Deacuteveloppement 1938 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Figure 5 ndash Sensitivity patterns for the tested (matrixstrain) pairs

2115 SpecificitySelectivity

The specificity is defined as the degree to which a method is affected (or not) by the other components

present in a multi-component sample That is the ability of a method to measure exactly a given

analyte or its amount within the sample without interference from non-target components such as a

matrix effect or background noise

The selectivity is defined as a measure of the degree of non-interference in the presence of non-target

analytes A method is selective if it can be used to detect the analyte under examination and that a

guarantee can be provided that the detected signal can only be a product by that specific analyte

The specificity and selectivity study was performed for the Listeria PrecisTM

detection method no additional test was realised for the enumeration

method The results were the following

- Among the Listeria monocytogenes strains tested 2 strains (Listeria

monocytogenes CIP 7834 and Listeria monocytogenes CIP 105459) gave

negative results These 2 strains were tested twice by running 48 h

incubation time of the compared agars Positive results were then

observed by the alternative method Pale characteristic colonies were

observed on the ISO agar by streaking the Half Fraser broth only

- No cross reaction was observed with the non-target strains except with

Listeria ivanovii which gave characteristic colonies on Brilliancetrade Listeria

Agar but the confirmatory tests provide a clear discrimination between

Listeria monocytogenes and Listeria ivanovi species

Rillettes

Lettuce

Raw milk

Smoked salmon

Raw egg

Process water

x (CFUg)

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2038 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

212 Practicability

1 Packaging Ready to use Brilliance Listeria agar plates are available

(x 10 plates)

2 Reagent volume

3 Storage conditionsndash

Shelf life of products

not opened

The plates are stored at 6 - 12degC in darkness conditions The

expiration date is mentioned on the package and on the plates It

is around 3 months

4 Modalities of use

after first use

5 Required

equipments No specific equipment is required

6 Reagents Ready to use plates

7 Training 05 day

8 Real time handling

Reference method

9 min for one sample

55 min for 10 samples

Alternative method

7 min for one sample

35 minutes for 10 samples

9 Time to result

Negative results

Reference method Day 2

Alternative method Day 2

Negative results after confirmation or positive results

Reference method Day 4 ndash Day 7

Alternative method Day 4 ndash Day 7

Positive results

Reference method Day 4 ndash Day 7

Alternative method Day 2 ndash Day 3 (with OBIS Mono Test)

or Day 4 ndash Day 7 (by the classical tests described in the

standardised method)

10 Technician skill Identical to that required for the reference method

11 Steps common to

the reference method Mother suspension revivification step and decimal dilutions

12 Traceability

13 Maintenance

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2138 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

213 Inter-laboratory study

2131 Organisation

12 laboratories participated to this study Pasteurised milk was inoculated by

Listeria monocytogenes 12a isolated from raw milk The targeted

inoculation levels were

- 0 CFUml

- 50 CFU ml

- 500 CFU ml

- 5 000 CFU ml

Each laboratory received eight flasks of 10 ml sample ie two flasks per

inoculation level Furthermore one non-inoculated sample has been added

to the package for total viable count microflora (NF ISO 4833 method)

Detailed instructions were transmitted to the collaborators by the expert

laboratory

Collaborative study laboratories and the expert laboratory carried out the

analyses with the alternative and reference methods

2132 Verification of experimental parameters

Strain stability during transport

In order to evaluate the Listeria monocytogenes strain stability during

transport bacterial count of all inoculated flasks were checked at different

time ie inoculation time after 24 h of conservation at 2deg - 8degC Results are

reported in table 11

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2238 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Table 11 ndash Listeria monocytogenes count

by ISO 11290-2A1 method (log CFUml)

Day of the analyses Level contamination Reference method

Day 0

(29052006)

1 45

2 440

3 4 500

Day 1

(30052006)

1 60

2 430

3 4 900

No evolution of the strain was observed after storage for 24 h at 2degC ndash 8degC

Contamination level

The real contamination levels are provided in table 12

Table 12 ndash Real contamination level (CFUml)

Target inoculation

(CFU ml) Samples Ndeg

Real contamination levels

(CFUml)

0 1 - 8 0

50 5 - 6 45

500 3 - 7 440

5000 2 - 4 4 500

Sample temperature at receipt

Measured temperatures at receipt are listed in table 13

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2338 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Table 13 - Sample temperature on receipt

Laboratories Receipt

day

Hour

receipt

Measured temperature

at receipt (degC)

Measured temperature by the

temperature probe (degC)

A

B

C

D

E

F

G

H

I

J

K

L

M

30052006

10h45

10h15

11h15

10h00

09h30

8h30

11h50

10h00

08h30

14h45

08h30

09h45

08h45

82

55

45

63

60

62

81

38

48

-

14

67

76

03

03

-13

03

04

-14

05

04

01

05

-13

04

Conclusion

The temperatures during the transport were correct for all the labs except for

Lab J which didnrsquot provide the information

All the labs received their packages with several broken flasks

Three labs (B D and L) received some damaged samples but proceeded to

analyses

All the labs started the analyses at Day 1 except Lab J

2133 Results

Aerobic mesophilic flora

The count of aerobic mesophilic flora varied from 94 102 CFUml to

33 103 CFUml

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2438 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Expert lab results

The results obtained by the expert lab are the following

Table 14

Target inoculation level (CFUml)

Reference method ISO 11290-2

Alternative method Listeria Precis Enumeration

Rep 1 Rep 2 Rep 1 Rep 2

lt 10 lt 10 lt 10 lt 10 lt10

50 50 (Ne) 60 (Ne) 50 (Ne) 30 (Ne)

500 470 390 490 500

5 000 4 700 5 100 5 900 3 400

Ne estimated number

The obtained results are in agreement with those expected

Collaborator Labs results

A synthesis of the results obtained by the collaborator labs is provided

Table 15 (CFUml) and Table 16 (log CFUml)

Table 15 ndash Synthesis of results obtained by the alternative

and the reference methods (CFUml)

Labs Level 1 Level 2 Level 2 bis Level 3

Reference method

Alternative method

Reference method

Alternative method

Reference method

Alternative method

Reference method

Alternative method

A 40 40 70 40 480 410 210 340 1000 300 400 300 4900 4600 4900 5100

B 40 15 40 20 350 300 360 450 350 650 800 600 4100 4700 5100 3700

C 50 20 20 70 440 430 480 450 300 650 300 500 4600 5400 4600 5000

D 40 60 40 40 490 460 600 540 250 450 600 1000 4900 4900 4400 4600

E 50 40 50 20 490 520 530 560 750 550 400 200 5400 4500 4100 5400

F 60 20 60 50 370 250 300 130 300 350 600 300 4400 4700 4900 5900

G 70 35 120 50 440 390 570 370 450 550 700 700 5000 4500 4000 4400

H 110 40 100 90 520 440 450 540 150 650 300 300 4200 4700 5600 4800

L 35 60 20 40 390 390 170 380 650 350 800 200 5300 4600 5600 5900

M 45 45 30 40 450 490 550 410 400 550 800 180 4900 4900 6400 4800

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2538 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Table 16 ndash Synthesis of results obtained by the alternative

and the reference methods (log CFUml)

Labs Level 1 Level 2 Level 2 bis Level 3

Reference method

Alternative method

Reference method

Alternative method

Reference method

Alternative method

Reference method

Alternative method

A 1602 1602 1845 1602 2681 2613 2322 2531 3000 2477 2602 2477 3690 3663 3690 3708

B 1602 1176 1602 1301 2544 2477 2556 2653 2544 2813 2903 2778 3613 3672 3708 3568

C 1699 1301 1301 1845 2643 2633 2681 2653 2477 2813 2477 2699 3663 3732 3663 3699

D 1602 1778 1602 1602 2690 2663 2778 2732 2398 2653 2778 3000 3690 3690 3643 3663

E 1699 1602 1699 1301 2690 2716 2724 2748 2875 2740 2602 2301 3732 3653 3613 3732

F 1778 1301 1778 1699 2568 2398 2477 2114 2477 2544 2778 2477 3643 3672 3690 3771

G 1845 1544 2079 1699 2643 2591 2756 2568 2653 2740 2845 2845 3699 3653 3602 3643

H 2041 1602 2000 1954 2716 2643 2653 2732 2176 2813 2477 2477 3623 3672 3748 3681

L 1544 1778 1301 1602 2591 2591 2230 2580 2813 2544 2903 2301 3724 3663 3748 3771

M 1653 1653 1477 1602 2653 2690 2740 2613 2602 2740 2903 2255 3690 3690 3806 3681

Level 2 results obtained taking account the 3 plates inoculated with the mother-suspension

Level 2 bis results obtained taking account the plates inoculated with 01 ml of the mother-suspension

Scrutiny of the measurement results for consistency

In order to identify the measurement results or laboratories that could be

inconsistent two graphical consistency techniques were realised Mandelrsquos

h-ank-statistics Mandel indicators h and k at 5 significance highlighted

some possible inconsistent data (See tables and graphics in Appendix 3 and

Appendix 4)

Mandelrsquos values Number of values observed over the threshold

Reference method Alternative method

h gt 1 Lab F Level 2 Lab F Level 2

h gt 5 Lab F Level 2

Lab B Levels 1 and 2

Lab A Level 2

Lab D Level 3

Lab F Level 2

Lab L Level 2

kgt 1 Lab F Level 3

k gt 5 Lab F Level 2 Lab C Level 2

Lab F Level 3

Lab L Levels 1 and 3

Lab M Level 1

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2638 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

The emphasized results appear (i) coherent with the targeted contamination

levels (ii) concordant between both methods

All the results were thus kept for the statistical analysis

Comparison of the trueness and precision characteristics of

the reference method and alternative methods

The statistical values are summarised hereafter

Level p

Reference method Alternative method

Repeatability ratio

Reproducibility ratio Median

Repeatability standard deviation

Reproducibility standard deviation

Median Repeatability

standard deviation

Reproducibility standard deviation

1 10 16519 02415 02425 16021 02045 02433 0847 1007

2 10 26551 00543 00659 26719 00914 01325 1682 2011

2 bis 10 26581 02607 02607 25951 01900 01953 0729 0749

3 10 18671 00378 00378 18671 00478 00611 1264 1617

Bias of the alternative method

In order to estimate the bias of the alternative method with regard to the reference method for every

level Dij and t are calculated according to the following equations

RefAlt ijYijYDij

Diffp

Dijit

)2(

)(median

If the t value is superior to 2 the alternative method is significantly biased

with regard to the reference method

The values are given in table 17

Table 17 ndash t (d) values obtained by level

Level Bias t (d) Conclusion

1 0068 114 Non significant bias

2 0021 082 Non significant bias

2 bis - 0037 035 Non significant bias

3 - 0037 043 Non significant bias

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2738 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

The statistical test concludes to a non significant bias for all the inoculation

levels The bias varies from ndash 0037 to 0068 log CFUml

Comparison of the repeatability standard deviations

If the ratio Srj Alt Srj Ref of the repeatability standard deviations of the

alternative method and the reference method is larger than 2 the precision

under repeatability conditions of the alternative method is considered to be

lower than that of the reference method If this ratio is smaller than 05 the

precision under repeatability conditions of the alternative method is

considered to be greater than that of the reference method

The ratio values are given in table 18

Table 18

Level

Repeatability standard deviation

Reference method Alternative method Alternative method

reference method ratio

1 0242 0205 0847

2 0054 0091 1682

2 bis 0261 0190 0729

3 0038 0048 1264

Level Repeatability limit

Reference method Alternative method

1 0676 0573

2 0152 0256

2 bis 0730 0532

3 0106 0134

The ratios of the repeatability standard deviations are below 2 in all cases

The precision under repeatability conditions of the alternative method is

equivalent to that of the reference method

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2838 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Comparison of the reproducibility standard deviations

If the ratio Srj Alt Srj Ref of the reproducibility standard deviations of the alternative method and the

reference method is larger than 2 the precision under reproducibility conditions of the alternative

method is considered to be lower than that of the reference method If this ratio is smaller than 05 the

precision under reproducibility conditions of the alternative method is considered to be greater than

that of the reference method

The ratio values are given in table 19

Table 19

Level

Reproducibility standard deviation

Reference method Alternative method Alternative method

reference method ratio

1 0242 0243 1007

2 0066 0133 2011

2 bis 0261 0195 0749

3 0038 0061 1617

Level Reproducibility limit

Reference method Alternative method

1 0676 0681

2 0185 0371

2 bis 0730 0547

3 0106 0171

The ratios of the reproducibility standard deviations are below or close to 2

The precision under reproducibility conditions of the alternative method is

equivalent to that of the reference method

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2938 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

214 Conclusion

Method comparison study

The Listeria PrecisTM enumeration method shows satisfying linearity and

relative accuracy

The alternative method shows satisfying inclusivity and exclusivity results

Two Listeria monocytogenes strains gave negative results with the

alternative method These strains grew weakly on the reference method

media (OampA Palcam and Oxford)

The limits of repeatability of the alternative method are similar to those of the

reference method

The bias between both methods are low between - 0133 Log CFUg and

- 0008 Log CFUg

The Listeria PrecisTM enumeration method offers the saving for confirmatory

tests

Positive results are obtained with the alternative method in 2-3 days (with

OBIS Mono test) or in 4-7 days (by the classical tests described in the

standardized method) against 4-7 days for the reference method

Inter-laboratory study

The bias between the reference and the alternative methods shows satisfying

and correct values

- level 1 (100 - 1 000 CFUg) bias = + 007 log CFUg

- level 2 (1 000 - 10 000 CFUg) bias = 002 log CFUg

- level 2 bis (1 000 - 10 000 CFUg) bias = - 004 log CFUg

- level 3 (10 000 - 100 000 CFUg) bias = - 004 log CFUg

The repeatability and the reproducibility of the alternative method look similar

to those of the reference method

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3038 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

22 Extension study (realised by ASEPT 2007)

An extension study was performed in 2007 for a new confirmatory test OBIS

MONO

150 Listeria monocytogenes strains and 100 non-target strains were isolated

onto TSAYE and BrillianceTM Listeria Agar plates All the Listeria

monocytogenes and the non-target strains gave the expected results

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3138 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Appendix 1 ndash Flow diagram of the Listeria PrecisTM method

x g sample + 9 x ml buffered peptone water

Revivification step for 1 h plusmn 5 min at 20degC plusmn 1degC

Streaking 1 ml onto 3 Brilliance Listeria Agar plates (mother suspension)

or 01 ml onto 1 Brilliance Listeria Agar plates (mother suspension and decimal dilutions)

Incubation for 48 h plusmn 3 h at 37degC plusmn 1degC

Confirmatory tests on at least one typical colony

- by the tests described in the ISO method

- by applying the OBIS mono test on an isolated colony

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3238 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Appendix 2 ndash ISO 11290-2 (1998) amp ISO 11290-2A1 (2004) Horizontal

method for the detection and enumeration of Listeria monocytogenes -

Part 2 enumeration of Listeria monocytogenes in foods

10 g cheese + 90 ml buffered peptone water or Half Fraser Broth (without supplements) (110 dilution)

Stomach 1 min 30 seconds

Keep at 20degC plusmn 2degC for 1 h plusmn 5 min

Decimal dilutions in 9 ml peptone salt tubes

Dilution 110

Dilution 1100

Dilution 11000

1 ml on 3 Chromogenic Listeria medium (ISO) plates (x 2) (-1)

01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-2)

01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-3)

01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-4)

Incubation at 37degC plusmn 1degC

24 h - 48 h

Numerate typical colonies at 24 h

(bluegreen surrounded by an opaque halo)

Numerate at 48 h

Streak typical colonies on TSYEA

(5 coloniesplate 2 successive dilutions)

Confirmation tests

Catalase

Gram stain

For confirmation of L monocytogenes

Haemolysis

Carbohydrate (rhamnose and xylose) utilisation

CAMP Test

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3338 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Appendix 3 ndash Mandelrsquos graphics

Laboratoire h Ref

Niveau 1 h Ref

Niveau 2 h Ref

Niveau 3 h Alt

Niveau 1 h Alt

Niveau 2 h Alt

Niveau 3 h5 h1 h5 h1

A -050 -015 083 062 -212 -039 198 299 -198 -299

B -261 -270 021 -077 -058 173 198 299 -198 -299

C -151 -031 -014 -015 -004 -005 198 299 -198 -299

D 038 040 -137 000 072 207 198 299 -198 -299

E -001 090 155 -052 056 -101 198 299 -198 -299

F -112 -321 -153 070 -325 023 198 299 -198 -299

G 042 -071 040 147 -009 176 198 299 -198 -299

H 169 046 -169 192 018 -083 198 299 -198 -299

L 009 -120 021 -077 -231 005 198 299 -198 -299

M 001 031 014 -032 004 -011 198 299 -198 -299

-300

-200

-100

000

100

200

300

A B C D E F G H L M

Vale

urs

h d

e M

an

del(

rob

uste

s)

Laboratoires

Meacutethode de reacutefeacuterence analyse robuste

h Ref Niveau 1

h Ref Niveau 2

h Ref Niveau 3

h 5=198

h 1=299

-300

-200

-100

000

100

200

300

A B C D E F G H L M

Vale

urs

de h

de M

an

del(

rob

uste

s)

Laboratoires

Meacutethode alternative analyse robuste

h Alt Niveau 1

h Alt Niveau 2

h Alt Niveau 2

h 1=299

h 5=198

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3438 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Laboratoire k Ref

Niveau 1 k Ref

Niveau 2 k Ref

Niveau 3 k Alt

Niveau 1 k Alt

Niveau 2 k Alt

Niveau 3 k1 k5

A 000 089 142 084 162 046 259 181

B 125 087 073 104 075 046 259 181

C 117 013 091 188 022 083 259 181

D 052 036 069 000 035 083 259 181

E 028 034 037 138 019 112 259 181

F 140 222 018 027 281 112 259 181

G 088 068 024 131 145 000 259 181

H 129 094 173 016 061 000 259 181

L 069 000 073 104 270 224 259 181

M 000 048 038 043 099 241 259 181

000

050

100

150

200

250

300

350

400

A B C D E F G H L M

Vale

urs

k d

e M

an

del(

rob

uste

s)

Laboratoires

Meacutethode de reacutefeacuterence analyse robuste

k Ref Niveau 1k Ref Niveau 2k Ref Niveau 3

k 1=259

k 5=181

000

050

100

150

200

250

300

A B C D E F G H L M

Vale

urs

k d

e M

an

del

(ro

bu

ste

s)

Laboratoires

Meacutethode alternative analyse robuste

k Alt Niveau 3

k Alt Niveau 1

k Alt Niveau 2

k 1=259

k 5=181

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3538 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Appendix 4 ndash Statistical results

Level 1

Nombre de laboratoires (p) 10

A 1602 1602 1602 000 0000 0000 -0495 0000 A 1845 1602 1724 017 0122 -0122 0621 0840 0122

B 1602 1176 1389 030 0213 -0213 -2613 1247 B 1602 1301 1452 021 0151 -0151 -0769 1041 0062

C 1699 1301 1500 028 0199 -0199 -1510 1165 C 1301 1845 1573 038 -0272 0272 -0148 1881 0073

D 1602 1778 1690 012 -0088 0088 0380 0516 D 1602 1602 1602 000 0000 0000 0000 0000 -0088

E 1699 1602 1651 007 0048 -0048 -0013 0284 E 1699 1301 1500 028 0199 -0199 -0522 1376 -0151

F 1778 1301 1540 034 0239 -0239 -1116 1397 F 1778 1699 1739 006 0040 -0040 0698 0274 0199

G 1845 1544 1695 021 0151 -0151 0425 0881 G 2079 1699 1889 027 0190 -0190 1467 1315 0194

H 2041 1602 1822 031 0220 -0220 1689 1286 H 2000 1954 1977 003 0023 -0023 1917 0158 0155

L 1544 1778 1661 017 -0117 0117 0092 0685 L 1301 1602 1452 021 -0151 0151 -0769 1041 -0210

M 1653 1653 1653 000 0000 0000 0013 0000 M 1477 1602 1540 009 -0062 0062 -0319 0432 -0114

m 165186 m 160206 mediane D 00678

Cn(2p) 1867 Cn(2p) 1867 Cp 16101

Cn(p) 16101 Cn(p) 16101 Qn(D) 0092926

n=2p n 20 n=2p n 20 Qdiff 01496169

f 11 f 11 t 114

l 55 l 55

n=p n 10 n=p n 10

f 6 f 6

l 15 l 15

Qn(2p) 0091465 Qn(2p) 007745

Qn(p) 0062469 Qn(p) 0121519

Qintra 017078 Qintra 014461

Qinter 010058 Qinter 019565

Sr(eacutecart-type de reacutepeacutetabiliteacute) 02415 Sr 02045

RSDr(coefficient de variation de la reacutepeacutetabiliteacute)1462 RSDr 1277

r(repeatability limit)) 0676 r 0573

SL(eacutecart-type interlaboratoire) -001905 SLlt0 SL 001737

SR(eacutecart-type de reproductibiliteacute) 02415 SR 02433

RSDR(coefficient de variation de la reproductibiliteacute)0146 RSDR 0152

R(limite de reproductibiliteacute)) 06762 R(limite de reproductibiliteacute))068123

h k h MoyenneLaboratoires

Niveau 1

si LaboratoiresDeviationMoyenne Dk DeviationsiAlt

Niveau 1

Ref

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3638 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Level 2

Nombre de laboratoires (p) 10

A 2681 2613 2647 005 0034 -0034 -0151 0891 A 2322 2531 2427 015 -0105 0105 -2119 1620 -0220

B 2544 2477 2511 005 0033 -0033 -2699 0872 B 2556 2653 2605 007 -0048 0048 -0581 0750 0094

C 2643 2633 2638 001 0005 -0005 -0311 0130 C 2681 2653 2667 002 0014 -0014 -0040 0217 0029

D 2690 2663 2676 002 0014 -0014 0400 0357 D 2778 2732 2755 003 0023 -0023 0721 0354 0079

E 2690 2716 2703 002 -0013 0013 0897 0336 E 2724 2748 2736 002 -0012 0012 0556 0185 0033

F 2568 2398 2483 012 0085 -0085 -3214 2217 F 2477 2114 2296 026 0182 -0182 -3255 2811 -0188

G 2643 2591 2617 004 0026 -0026 -0707 0682 G 2756 2568 2662 013 0094 -0094 -0085 1453 0045

H 2716 2643 2680 005 0036 -0036 0460 0945 H 2653 2732 2693 006 -0040 0040 0181 0613 0013

L 2591 2591 2591 000 0000 0000 -1196 0000 L 2230 2580 2405 025 -0175 0175 -2307 2704 -0186

M 2653 2690 2672 003 -0018 0018 0311 0481 M 2740 2613 2677 009 0064 -0064 0040 0987 0005

m 2655082 m 26719001 mediane D 00209

Cn(2p) 1867 Cn(2p) 1867 Cp 16101

Cn(p) 16101 Cn(p) 16101 Qn(D) 0039911

n=2p n 20 n=2p n 20 Qdiff 00642591

f 11 f 11 t 082

l 55 l 55

n=p n 10 n=p n 10

f 6 f 6

l 15 l 15

Qn(2p) 0020570 Qn(2p) 00345985

Qn(p) 00332437 Qn(p) 00718158

Qintra 003841 Qintra 006460

Qinter 005352 Qinter 011563

Sr(eacutecart-type de reacutepeacutetabiliteacute) 00543 Sr 00914

RSDr(coefficient de variation de la reacutepeacutetabiliteacute)205 RSDr 342

r(repeatability limit)) 0152 r 0256

SL(eacutecart-type interlaboratoire) 000139 SL 000920

SR(eacutecart-type de reproductibiliteacute) 00659 SR 01325

RSDR(coefficient de variation de la reproductibiliteacute)0025 RSDR 0050

R(limite de reproductibiliteacute)) 018446 R(limite de reproductibiliteacute))03708618

Laboratoires

Niveau 2

Moyenne si Laboratoires

Niveau 2

DRef Alt Moyenne si Deviation h Deviation h k k

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3738 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Level 2 bis

Nombre de laboratoires (p) 10

A 3000 2477 2739 037 0261 -0261 0832 1418 A 2602 2477 2540 009 0062 -0062 -0391 0465 -0199

B 2544 2813 2678 019 -0134 0134 0211 0729 B 2903 2778 2841 009 0062 -0062 1732 0465 0162

C 2477 2813 2645 024 -0168 0168 -0135 0911 C 2477 2699 2588 016 -0111 0111 -0049 0826 -0057

D 2398 2653 2526 018 -0128 0128 -1370 0692 D 2778 3000 2889 016 -0111 0111 2074 0826 0363

E 2875 2740 2808 010 0067 -0067 1547 0365 E 2602 2301 2452 021 0151 -0151 -1012 1120 -0356

F 2477 2544 2511 005 -0033 0033 -1525 0182 F 2778 2477 2628 021 0151 -0151 0230 1120 0117

G 2653 2740 2697 006 -0044 0044 0400 0236 G 2845 2845 2845 000 0000 0000 1764 0000 0148

H 2176 2813 2495 045 -0318 0318 -1692 1727 H 2477 2477 2477 000 0000 0000 -0832 0000 -0017

L 2813 2544 2678 019 0134 -0134 0211 0729 L 2903 2301 2602 043 0301 -0301 0049 2241 -0076

M 2602 2740 2671 010 -0069 0069 0135 0375 M 2903 2255 2579 046 0324 -0324 -0112 2411 -0092

m 2658114 m 259505 mediane D -00372

Cn(2p) 1867 Cn(2p) 1867 Cp 16101

Cn(p) 16101 Cn(p) 16101 Qn(D) 01656915

n=2p n 20 n=2p n 20 Qdiff 02667753

f 11 f 11 t 035

l 55 l 55

n=p n 10 n=p n 10

f 6 f 6

l 15 l 15

Qn(2p) 0098745 Qn(2p) 007195

Qn(p) 006007 Qn(p) 008805

Qintra 018437 Qintra 013435

Qinter 009672 Qinter 014176

Sr(eacutecart-type de reacutepeacutetabiliteacute) 02607 Sr 01900

RSDr(coefficient de variation de la reacutepeacutetabiliteacute)981 RSDr 732

r(repeatability limit)) 0730 r 0532

SL(eacutecart-type interlaboratoire) -002464 SLlt0 SL 000205

SR(eacutecart-type de reproductibiliteacute) 02607 SR 01953

RSDR(coefficient de variation de la reproductibiliteacute)0098 RSDR 0075

R(limite de reproductibiliteacute)) 072996 R(limite de reproductibiliteacute))054686

Laboratoires

Niveau 2 bis

Moyenne si Laboratoires

Niveau 2 bis

DRef Alt Moyenne si Deviation h Deviation h k k

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3838 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Level 3

Nombre de laboratoires (p) 10

A 3690 3663 3676 002 0014 -0014 -0311 0513 A 3690 3708 3699 001 -0009 0009 0354 0257 0022

B 3613 3672 3642 004 -0030 0030 -1851 1109 B 3708 3568 3638 010 0070 -0070 -0844 2061 -0005

C 3663 3732 3698 005 -0035 0035 0645 1302 C 3663 3699 3681 003 -0018 0018 0000 0536 -0017

D 3690 3690 3690 000 0000 0000 0311 0000 D 3643 3663 3653 001 -0010 0010 -0545 0286 -0037

E 3732 3653 3693 006 0040 -0040 0429 1480 E 3613 3732 3673 008 -0060 0060 -0162 1769 -0020

F 3643 3672 3658 002 -0014 0014 -1157 0536 F 3690 3771 3731 006 -0040 0040 0975 1193 0073

G 3699 3653 3676 003 0023 -0023 -0328 0856 G 3602 3643 3623 003 -0021 0021 -1141 0612 -0053

H 3623 3672 3648 003 -0024 0024 -1615 0913 H 3748 3681 3715 005 0033 -0033 0664 0990 0067

L 3724 3663 3694 004 0031 -0031 0461 1150 L 3748 3771 3760 002 -0011 0011 1544 0335 0066

M 3690 3690 3690 000 0000 0000 0311 0000 M 3806 3681 3744 009 0062 -0062 1234 1848 0054

m 368334 m 368086 mediane D 00089

Cn(2p) 1867 Cn(2p) 1867 Cp 16101

Cn(p) 16101 Cn(p) 16101 Qn(D) 00325359

n=2p n 20 n=2p n 20 Qdiff 00523852

f 11 f 11 t 043

l 55 l 55

n=p n 10 n=p n 10

f 6 f 6

l 15 l 15

Qn(2p) 0014323 Qn(2p) 001811

Qn(p) 0013719 Qn(p) 003164

Qintra 002674 Qintra 003381

Qinter 002209 Qinter 005094

Sr(eacutecart-type de reacutepeacutetabiliteacute) 00378 Sr 00478

RSDr(coefficient de variation de la reacutepeacutetabiliteacute)103 RSDr 130

r(repeatability limit)) 0106 r 0134

SL(eacutecart-type interlaboratoire) -000023 SLlt0 SL 000145

SR(eacutecart-type de reproductibiliteacute) 00378 SR 00611

RSDR(coefficient de variation de la reproductibiliteacute)0010 RSDR 0017

R(limite de reproductibiliteacute)) 0105894 R(limite de reproductibiliteacute))017119

Ref Alt DLaboratoires

Niveau 3

Moyenne si Deviation h k Laboratoires

Niveau 3

Moyenne si Deviation h k

Page 8: ÞÀ¿¸7 ú Y5zñLjÜ< /u +æü t¬Á ... - NF Validation · NF VALIDATION Validation of alternative analytical methods Application in food microbiology ... 500 – 1 000 1 000 –

Thermo Fisher Scientific

ADRIA Deacuteveloppement 838 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Figure 2 ndash Regression straight lines

Lineacuteariteacute - Œuf

Process water Process water (2) Process water

Smoked salmon (GMFR) Smoked salmon (OLS 1)

Raw milk Raw egg product

Rillettes Lettuce

Thermo Fisher Scientific

ADRIA Deacuteveloppement 938 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Discussion

The linearity is accepted in all cases with

P values 5 and correlation coefficients superior to 099

For the matrix ldquoRillettesldquo the linearity test is significant (P = 4) with a high

correlation coefficient value (0996)

The Listeria PrecisTM method shows satisfying linearity

2112 Relative accuracy

The accuracy is the closeness of agreement between a test result and the accepted reference value

The bias is the difference between the expectation of the test results and an accepted reference value

Number and nature of the samples

The repartition per category and types is provided in table 3

Table 3 ndash Number and nature of the samples

Categories Types Number of samples

Analysed Exploited (1)

Meat products

Raw meat products 6 4

Ready to cook products 4 2

Ready to eat delicatessen 10 4

Total 20 10

Dairy products

Raw milk cheeses 7 4

Raw milks 4 3

Other dairy products 5 5

Total 16 12

Fishery products

Raw fishes 5 4

Processed fishes 7 2

Ready-to-eat products 6 4

Total 18 10

Vegetables

Raw vegetables 5 4

Frozen vegetables 3 3

Lettuce raw vegetables 5 5

Total 13 12

Egg products

Raw egg 5 5

Pastries 3 3

Pasteurised egg products 4 3

Total 12 11

Environmental samples

Wipes swabs 8 4

Process water 3 3

Dusts 3 3

Total 14 10

TOTAL 93 65

(1) Some data cannot be exploited because enumeration was under the detection level for at least one of the

method

Thermo Fisher Scientific

ADRIA Deacuteveloppement 1038 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Artificial contamination of the samples

Artificial contaminations were realised by spiking after performing injury

protocols on pure cultures or by cross-contaminations

56 samples were artificially contaminated 53 gave exploitable results among

a total of 65 samples 8150 of the samples were artificially contaminated

The injury protocol and the strains inoculated are provided in Table 4

Table 4

Stress Ndeg Injury protocol Strain Origin

A 45 min at 50 degC

48

LM -H123

LM-H170

Smoked salmon

Lettuce

Lettuce

B 30 min at 50 degC

and 2 h at ndash20degC

18

43

LM-O1

Raw milk (environment)

Environment

Egg product

C 30 min at 55degC

18

48

LM-H123

Raw milk

Smoked salmon

Lettuce

D 30 min at 50degC

et 2 h at ndash80degC

34

LM-H171

LM-O1

Rillettes

Lettuce

Egg product

E 30 min at 60 degC LM-O1 Egg product

Confirmatory tests

For the reference and the alternative methods confirmations were realised

by the tests described in the ISO 12290-2A1 standard For the alternative

method only one colony was confirmed while 5 colonies were tested for the

reference method

Results

Samples were analysed in duplicate by the reference and the alternative

methods

The contamination range is provided in table 5 per tested category

Thermo Fisher Scientific

ADRIA Deacuteveloppement 1138 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Table 5 ndash Contamination range

Food category Contamination level (log CFUg)

Meat products 100 ndash 404

Dairy products 148 ndash 408

Fishery products 100 ndash 384

Vegetables 170 ndash 511

Egg products 130 ndash 499

Environmental samples 170 ndash 511

All categories 100 ndash 511

Bi-dimensional graphs are given figure 3

Figure 3 ndash Bi-dimensional graphs

Graphe bi-dimensionnel Produits carneacutes

Fishery products

Vegetables

Egg products

Meat products

Thermo Fisher Scientific

ADRIA Deacuteveloppement 1238 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Statistical interpretation according to EN ISO16140 method

The results of the statistical interpretation are given in Table 6

Table 6 ndash Statistical interpretation results

Category n R Regression

used a t(a) b t(b) Critical T

P

Ordinate

at 0

Slope

at 1

Meat products 10 158 GMFR -0455 3321 115 3123 2306 001 001

Dairy products 12 065 GMFR -0027 0385 1002 0108 2228 092 071

Fishery products 10 130 GMFR -0305 1024 1034 0312 2306 076 034

Vegetables 12 086 GMFR -0015 0128 0993 0191 2228 085 090

Egg products 11 158 GMFR -0038 0866 1001 0090 2262 093 041

Environmental

samples 10 176 GMFR -0139 265 1034 231 2306 005 003

All products 65 118 GMFR -0182 3042 1040 2145 2000 004 000

All products

(estimated number) 65 118 GMFR -0182 3042 1040 2145 2000 004 000

All products (non

estimated results) 20 115 GMFR -0367 151 1138 1141 2101 027 015

Repeatability limits obtained for the alternative and reference methods as

well as bias obtained are presented in Table 7

Dairy products Environmental samples

Log (Ref) Log (Ref)

All categories

Thermo Fisher Scientific

ADRIA Deacuteveloppement 1338 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Table 7

Categories Bias

Repeatability limits

Rob r

Ref Alt

Meat products 0008 0314 0343

Dairy products -0031 0136 0130

Fishery products -0133 0220 0214

Vegetables -0041 0180 0319

Egg products -0026 0219 0206

Environmental samples -0007 0198 0119

All products -0032 0196 0215

Regression straight lines (graph and equation representations) for each food

category and for all products are presented figure 4

Thermo Fisher Scientific

ADRIA Deacuteveloppement 1438 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Figure 4 ndash Regression straight lines

Meat products

Fishery products

Dairy products

Vegetables

Thermo Fisher Scientific

ADRIA Deacuteveloppement 1538 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Environmental samples

Egg products

All products (estimated number)

Thermo Fisher Scientific

ADRIA Deacuteveloppement 1638 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

The hypotheses that the ordinates are close to 0 and the slopes close to 1

are approved in all cases except for

- Meat products (slope P = 1 ordinate P = 1)

- Environmental samples (ordinate P = 3)

- All products (ordinate P = 0)

The bias between the two methods vary from ndash 0133 to 0008 log CFU For

all categories the bias is ndash 0032 log CFU The repeatability limits of the

alternative and the reference method are similar For all products the values

are respectively 0215 and 0196 log CFU For all products these

interpretations are provided taking into account data with low contamination

level or not (estimated colonies number)

For all food products the regression straight line is the following

log (Alt) = 1040 log Ref ndash 0182

The Listeria PrecisTM method shows satisfying relative accuracy

All products

All products (Level 2)

Thermo Fisher Scientific

ADRIA Deacuteveloppement 1738 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

2113 Detection limit (LOD) and quantification limit (LOQ)

The critical level is the defined as the smallest amount which can be detected (not null) but not

quantified as an exact value Below this value it cannot be sure that the true value is not null

The detection limit is defined as being higher than the critical level because it involves a power the

probability 1- which has to be well over 50 for example 95

The quantification limit is defined as the smallest amount of analyte (that is the lowest actual number of

organisms) which can be measured and quantified with defined precision and accuracy under the

experimental conditions by the method under validation

Protocol

The detection limit of the alternative method was realised with pure cultures

Three different inoculation levels were tested with six replicates per level ie

18 analyses were done by the alternative method

Listeria monocytogenes 18 was used for the detection limit of the alternative

method

Quantification limit was calculated for six independent blank samples

determinations

Results

These data are intrinsic to the method used and are presented in the

following tables

Table 8 ndash Inoculation 1 ml on 3 plates

Level (CFUml) Positive samples number Standard deviation Bias

0 06

05 26 1211 0

1 36 0816 05

5 66 2483 85

Thermo Fisher Scientific

ADRIA Deacuteveloppement 1838 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Table 9 ndash Inoculation 01 ml on 1 plate

Level (CFUml) Positive samples number Standard deviation Bias

0 06

5 36 0548 05

10 46 1414 2

50 66 2258 55

Table 10

Formulas Obtained values

LC 165 S0 + X0 18

LOD 33 S0 + X0 32

LOQ 10 S0 + X0 87

2114 Relative Sensitivity

The relative sensitivity is defined as the ability of the alternative method to detect two different amounts

of analyte measured by the reference method within a given matrix at a specified average value or

over the whole measurement range that is it is the minimal quantity variation (increase of the analyte

concentration x) which gives a significant variation of the measured signal (response y)

These data are intrinsic to the used method and are obtained from the results

of the linearity study

Sensitivity patterns obtained for tested (matrixstrain) pairs are presented in

figure 5

Thermo Fisher Scientific

ADRIA Deacuteveloppement 1938 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Figure 5 ndash Sensitivity patterns for the tested (matrixstrain) pairs

2115 SpecificitySelectivity

The specificity is defined as the degree to which a method is affected (or not) by the other components

present in a multi-component sample That is the ability of a method to measure exactly a given

analyte or its amount within the sample without interference from non-target components such as a

matrix effect or background noise

The selectivity is defined as a measure of the degree of non-interference in the presence of non-target

analytes A method is selective if it can be used to detect the analyte under examination and that a

guarantee can be provided that the detected signal can only be a product by that specific analyte

The specificity and selectivity study was performed for the Listeria PrecisTM

detection method no additional test was realised for the enumeration

method The results were the following

- Among the Listeria monocytogenes strains tested 2 strains (Listeria

monocytogenes CIP 7834 and Listeria monocytogenes CIP 105459) gave

negative results These 2 strains were tested twice by running 48 h

incubation time of the compared agars Positive results were then

observed by the alternative method Pale characteristic colonies were

observed on the ISO agar by streaking the Half Fraser broth only

- No cross reaction was observed with the non-target strains except with

Listeria ivanovii which gave characteristic colonies on Brilliancetrade Listeria

Agar but the confirmatory tests provide a clear discrimination between

Listeria monocytogenes and Listeria ivanovi species

Rillettes

Lettuce

Raw milk

Smoked salmon

Raw egg

Process water

x (CFUg)

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2038 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

212 Practicability

1 Packaging Ready to use Brilliance Listeria agar plates are available

(x 10 plates)

2 Reagent volume

3 Storage conditionsndash

Shelf life of products

not opened

The plates are stored at 6 - 12degC in darkness conditions The

expiration date is mentioned on the package and on the plates It

is around 3 months

4 Modalities of use

after first use

5 Required

equipments No specific equipment is required

6 Reagents Ready to use plates

7 Training 05 day

8 Real time handling

Reference method

9 min for one sample

55 min for 10 samples

Alternative method

7 min for one sample

35 minutes for 10 samples

9 Time to result

Negative results

Reference method Day 2

Alternative method Day 2

Negative results after confirmation or positive results

Reference method Day 4 ndash Day 7

Alternative method Day 4 ndash Day 7

Positive results

Reference method Day 4 ndash Day 7

Alternative method Day 2 ndash Day 3 (with OBIS Mono Test)

or Day 4 ndash Day 7 (by the classical tests described in the

standardised method)

10 Technician skill Identical to that required for the reference method

11 Steps common to

the reference method Mother suspension revivification step and decimal dilutions

12 Traceability

13 Maintenance

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2138 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

213 Inter-laboratory study

2131 Organisation

12 laboratories participated to this study Pasteurised milk was inoculated by

Listeria monocytogenes 12a isolated from raw milk The targeted

inoculation levels were

- 0 CFUml

- 50 CFU ml

- 500 CFU ml

- 5 000 CFU ml

Each laboratory received eight flasks of 10 ml sample ie two flasks per

inoculation level Furthermore one non-inoculated sample has been added

to the package for total viable count microflora (NF ISO 4833 method)

Detailed instructions were transmitted to the collaborators by the expert

laboratory

Collaborative study laboratories and the expert laboratory carried out the

analyses with the alternative and reference methods

2132 Verification of experimental parameters

Strain stability during transport

In order to evaluate the Listeria monocytogenes strain stability during

transport bacterial count of all inoculated flasks were checked at different

time ie inoculation time after 24 h of conservation at 2deg - 8degC Results are

reported in table 11

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2238 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Table 11 ndash Listeria monocytogenes count

by ISO 11290-2A1 method (log CFUml)

Day of the analyses Level contamination Reference method

Day 0

(29052006)

1 45

2 440

3 4 500

Day 1

(30052006)

1 60

2 430

3 4 900

No evolution of the strain was observed after storage for 24 h at 2degC ndash 8degC

Contamination level

The real contamination levels are provided in table 12

Table 12 ndash Real contamination level (CFUml)

Target inoculation

(CFU ml) Samples Ndeg

Real contamination levels

(CFUml)

0 1 - 8 0

50 5 - 6 45

500 3 - 7 440

5000 2 - 4 4 500

Sample temperature at receipt

Measured temperatures at receipt are listed in table 13

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2338 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Table 13 - Sample temperature on receipt

Laboratories Receipt

day

Hour

receipt

Measured temperature

at receipt (degC)

Measured temperature by the

temperature probe (degC)

A

B

C

D

E

F

G

H

I

J

K

L

M

30052006

10h45

10h15

11h15

10h00

09h30

8h30

11h50

10h00

08h30

14h45

08h30

09h45

08h45

82

55

45

63

60

62

81

38

48

-

14

67

76

03

03

-13

03

04

-14

05

04

01

05

-13

04

Conclusion

The temperatures during the transport were correct for all the labs except for

Lab J which didnrsquot provide the information

All the labs received their packages with several broken flasks

Three labs (B D and L) received some damaged samples but proceeded to

analyses

All the labs started the analyses at Day 1 except Lab J

2133 Results

Aerobic mesophilic flora

The count of aerobic mesophilic flora varied from 94 102 CFUml to

33 103 CFUml

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2438 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Expert lab results

The results obtained by the expert lab are the following

Table 14

Target inoculation level (CFUml)

Reference method ISO 11290-2

Alternative method Listeria Precis Enumeration

Rep 1 Rep 2 Rep 1 Rep 2

lt 10 lt 10 lt 10 lt 10 lt10

50 50 (Ne) 60 (Ne) 50 (Ne) 30 (Ne)

500 470 390 490 500

5 000 4 700 5 100 5 900 3 400

Ne estimated number

The obtained results are in agreement with those expected

Collaborator Labs results

A synthesis of the results obtained by the collaborator labs is provided

Table 15 (CFUml) and Table 16 (log CFUml)

Table 15 ndash Synthesis of results obtained by the alternative

and the reference methods (CFUml)

Labs Level 1 Level 2 Level 2 bis Level 3

Reference method

Alternative method

Reference method

Alternative method

Reference method

Alternative method

Reference method

Alternative method

A 40 40 70 40 480 410 210 340 1000 300 400 300 4900 4600 4900 5100

B 40 15 40 20 350 300 360 450 350 650 800 600 4100 4700 5100 3700

C 50 20 20 70 440 430 480 450 300 650 300 500 4600 5400 4600 5000

D 40 60 40 40 490 460 600 540 250 450 600 1000 4900 4900 4400 4600

E 50 40 50 20 490 520 530 560 750 550 400 200 5400 4500 4100 5400

F 60 20 60 50 370 250 300 130 300 350 600 300 4400 4700 4900 5900

G 70 35 120 50 440 390 570 370 450 550 700 700 5000 4500 4000 4400

H 110 40 100 90 520 440 450 540 150 650 300 300 4200 4700 5600 4800

L 35 60 20 40 390 390 170 380 650 350 800 200 5300 4600 5600 5900

M 45 45 30 40 450 490 550 410 400 550 800 180 4900 4900 6400 4800

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2538 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Table 16 ndash Synthesis of results obtained by the alternative

and the reference methods (log CFUml)

Labs Level 1 Level 2 Level 2 bis Level 3

Reference method

Alternative method

Reference method

Alternative method

Reference method

Alternative method

Reference method

Alternative method

A 1602 1602 1845 1602 2681 2613 2322 2531 3000 2477 2602 2477 3690 3663 3690 3708

B 1602 1176 1602 1301 2544 2477 2556 2653 2544 2813 2903 2778 3613 3672 3708 3568

C 1699 1301 1301 1845 2643 2633 2681 2653 2477 2813 2477 2699 3663 3732 3663 3699

D 1602 1778 1602 1602 2690 2663 2778 2732 2398 2653 2778 3000 3690 3690 3643 3663

E 1699 1602 1699 1301 2690 2716 2724 2748 2875 2740 2602 2301 3732 3653 3613 3732

F 1778 1301 1778 1699 2568 2398 2477 2114 2477 2544 2778 2477 3643 3672 3690 3771

G 1845 1544 2079 1699 2643 2591 2756 2568 2653 2740 2845 2845 3699 3653 3602 3643

H 2041 1602 2000 1954 2716 2643 2653 2732 2176 2813 2477 2477 3623 3672 3748 3681

L 1544 1778 1301 1602 2591 2591 2230 2580 2813 2544 2903 2301 3724 3663 3748 3771

M 1653 1653 1477 1602 2653 2690 2740 2613 2602 2740 2903 2255 3690 3690 3806 3681

Level 2 results obtained taking account the 3 plates inoculated with the mother-suspension

Level 2 bis results obtained taking account the plates inoculated with 01 ml of the mother-suspension

Scrutiny of the measurement results for consistency

In order to identify the measurement results or laboratories that could be

inconsistent two graphical consistency techniques were realised Mandelrsquos

h-ank-statistics Mandel indicators h and k at 5 significance highlighted

some possible inconsistent data (See tables and graphics in Appendix 3 and

Appendix 4)

Mandelrsquos values Number of values observed over the threshold

Reference method Alternative method

h gt 1 Lab F Level 2 Lab F Level 2

h gt 5 Lab F Level 2

Lab B Levels 1 and 2

Lab A Level 2

Lab D Level 3

Lab F Level 2

Lab L Level 2

kgt 1 Lab F Level 3

k gt 5 Lab F Level 2 Lab C Level 2

Lab F Level 3

Lab L Levels 1 and 3

Lab M Level 1

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2638 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

The emphasized results appear (i) coherent with the targeted contamination

levels (ii) concordant between both methods

All the results were thus kept for the statistical analysis

Comparison of the trueness and precision characteristics of

the reference method and alternative methods

The statistical values are summarised hereafter

Level p

Reference method Alternative method

Repeatability ratio

Reproducibility ratio Median

Repeatability standard deviation

Reproducibility standard deviation

Median Repeatability

standard deviation

Reproducibility standard deviation

1 10 16519 02415 02425 16021 02045 02433 0847 1007

2 10 26551 00543 00659 26719 00914 01325 1682 2011

2 bis 10 26581 02607 02607 25951 01900 01953 0729 0749

3 10 18671 00378 00378 18671 00478 00611 1264 1617

Bias of the alternative method

In order to estimate the bias of the alternative method with regard to the reference method for every

level Dij and t are calculated according to the following equations

RefAlt ijYijYDij

Diffp

Dijit

)2(

)(median

If the t value is superior to 2 the alternative method is significantly biased

with regard to the reference method

The values are given in table 17

Table 17 ndash t (d) values obtained by level

Level Bias t (d) Conclusion

1 0068 114 Non significant bias

2 0021 082 Non significant bias

2 bis - 0037 035 Non significant bias

3 - 0037 043 Non significant bias

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2738 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

The statistical test concludes to a non significant bias for all the inoculation

levels The bias varies from ndash 0037 to 0068 log CFUml

Comparison of the repeatability standard deviations

If the ratio Srj Alt Srj Ref of the repeatability standard deviations of the

alternative method and the reference method is larger than 2 the precision

under repeatability conditions of the alternative method is considered to be

lower than that of the reference method If this ratio is smaller than 05 the

precision under repeatability conditions of the alternative method is

considered to be greater than that of the reference method

The ratio values are given in table 18

Table 18

Level

Repeatability standard deviation

Reference method Alternative method Alternative method

reference method ratio

1 0242 0205 0847

2 0054 0091 1682

2 bis 0261 0190 0729

3 0038 0048 1264

Level Repeatability limit

Reference method Alternative method

1 0676 0573

2 0152 0256

2 bis 0730 0532

3 0106 0134

The ratios of the repeatability standard deviations are below 2 in all cases

The precision under repeatability conditions of the alternative method is

equivalent to that of the reference method

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2838 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Comparison of the reproducibility standard deviations

If the ratio Srj Alt Srj Ref of the reproducibility standard deviations of the alternative method and the

reference method is larger than 2 the precision under reproducibility conditions of the alternative

method is considered to be lower than that of the reference method If this ratio is smaller than 05 the

precision under reproducibility conditions of the alternative method is considered to be greater than

that of the reference method

The ratio values are given in table 19

Table 19

Level

Reproducibility standard deviation

Reference method Alternative method Alternative method

reference method ratio

1 0242 0243 1007

2 0066 0133 2011

2 bis 0261 0195 0749

3 0038 0061 1617

Level Reproducibility limit

Reference method Alternative method

1 0676 0681

2 0185 0371

2 bis 0730 0547

3 0106 0171

The ratios of the reproducibility standard deviations are below or close to 2

The precision under reproducibility conditions of the alternative method is

equivalent to that of the reference method

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2938 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

214 Conclusion

Method comparison study

The Listeria PrecisTM enumeration method shows satisfying linearity and

relative accuracy

The alternative method shows satisfying inclusivity and exclusivity results

Two Listeria monocytogenes strains gave negative results with the

alternative method These strains grew weakly on the reference method

media (OampA Palcam and Oxford)

The limits of repeatability of the alternative method are similar to those of the

reference method

The bias between both methods are low between - 0133 Log CFUg and

- 0008 Log CFUg

The Listeria PrecisTM enumeration method offers the saving for confirmatory

tests

Positive results are obtained with the alternative method in 2-3 days (with

OBIS Mono test) or in 4-7 days (by the classical tests described in the

standardized method) against 4-7 days for the reference method

Inter-laboratory study

The bias between the reference and the alternative methods shows satisfying

and correct values

- level 1 (100 - 1 000 CFUg) bias = + 007 log CFUg

- level 2 (1 000 - 10 000 CFUg) bias = 002 log CFUg

- level 2 bis (1 000 - 10 000 CFUg) bias = - 004 log CFUg

- level 3 (10 000 - 100 000 CFUg) bias = - 004 log CFUg

The repeatability and the reproducibility of the alternative method look similar

to those of the reference method

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3038 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

22 Extension study (realised by ASEPT 2007)

An extension study was performed in 2007 for a new confirmatory test OBIS

MONO

150 Listeria monocytogenes strains and 100 non-target strains were isolated

onto TSAYE and BrillianceTM Listeria Agar plates All the Listeria

monocytogenes and the non-target strains gave the expected results

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3138 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Appendix 1 ndash Flow diagram of the Listeria PrecisTM method

x g sample + 9 x ml buffered peptone water

Revivification step for 1 h plusmn 5 min at 20degC plusmn 1degC

Streaking 1 ml onto 3 Brilliance Listeria Agar plates (mother suspension)

or 01 ml onto 1 Brilliance Listeria Agar plates (mother suspension and decimal dilutions)

Incubation for 48 h plusmn 3 h at 37degC plusmn 1degC

Confirmatory tests on at least one typical colony

- by the tests described in the ISO method

- by applying the OBIS mono test on an isolated colony

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3238 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Appendix 2 ndash ISO 11290-2 (1998) amp ISO 11290-2A1 (2004) Horizontal

method for the detection and enumeration of Listeria monocytogenes -

Part 2 enumeration of Listeria monocytogenes in foods

10 g cheese + 90 ml buffered peptone water or Half Fraser Broth (without supplements) (110 dilution)

Stomach 1 min 30 seconds

Keep at 20degC plusmn 2degC for 1 h plusmn 5 min

Decimal dilutions in 9 ml peptone salt tubes

Dilution 110

Dilution 1100

Dilution 11000

1 ml on 3 Chromogenic Listeria medium (ISO) plates (x 2) (-1)

01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-2)

01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-3)

01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-4)

Incubation at 37degC plusmn 1degC

24 h - 48 h

Numerate typical colonies at 24 h

(bluegreen surrounded by an opaque halo)

Numerate at 48 h

Streak typical colonies on TSYEA

(5 coloniesplate 2 successive dilutions)

Confirmation tests

Catalase

Gram stain

For confirmation of L monocytogenes

Haemolysis

Carbohydrate (rhamnose and xylose) utilisation

CAMP Test

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3338 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Appendix 3 ndash Mandelrsquos graphics

Laboratoire h Ref

Niveau 1 h Ref

Niveau 2 h Ref

Niveau 3 h Alt

Niveau 1 h Alt

Niveau 2 h Alt

Niveau 3 h5 h1 h5 h1

A -050 -015 083 062 -212 -039 198 299 -198 -299

B -261 -270 021 -077 -058 173 198 299 -198 -299

C -151 -031 -014 -015 -004 -005 198 299 -198 -299

D 038 040 -137 000 072 207 198 299 -198 -299

E -001 090 155 -052 056 -101 198 299 -198 -299

F -112 -321 -153 070 -325 023 198 299 -198 -299

G 042 -071 040 147 -009 176 198 299 -198 -299

H 169 046 -169 192 018 -083 198 299 -198 -299

L 009 -120 021 -077 -231 005 198 299 -198 -299

M 001 031 014 -032 004 -011 198 299 -198 -299

-300

-200

-100

000

100

200

300

A B C D E F G H L M

Vale

urs

h d

e M

an

del(

rob

uste

s)

Laboratoires

Meacutethode de reacutefeacuterence analyse robuste

h Ref Niveau 1

h Ref Niveau 2

h Ref Niveau 3

h 5=198

h 1=299

-300

-200

-100

000

100

200

300

A B C D E F G H L M

Vale

urs

de h

de M

an

del(

rob

uste

s)

Laboratoires

Meacutethode alternative analyse robuste

h Alt Niveau 1

h Alt Niveau 2

h Alt Niveau 2

h 1=299

h 5=198

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3438 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Laboratoire k Ref

Niveau 1 k Ref

Niveau 2 k Ref

Niveau 3 k Alt

Niveau 1 k Alt

Niveau 2 k Alt

Niveau 3 k1 k5

A 000 089 142 084 162 046 259 181

B 125 087 073 104 075 046 259 181

C 117 013 091 188 022 083 259 181

D 052 036 069 000 035 083 259 181

E 028 034 037 138 019 112 259 181

F 140 222 018 027 281 112 259 181

G 088 068 024 131 145 000 259 181

H 129 094 173 016 061 000 259 181

L 069 000 073 104 270 224 259 181

M 000 048 038 043 099 241 259 181

000

050

100

150

200

250

300

350

400

A B C D E F G H L M

Vale

urs

k d

e M

an

del(

rob

uste

s)

Laboratoires

Meacutethode de reacutefeacuterence analyse robuste

k Ref Niveau 1k Ref Niveau 2k Ref Niveau 3

k 1=259

k 5=181

000

050

100

150

200

250

300

A B C D E F G H L M

Vale

urs

k d

e M

an

del

(ro

bu

ste

s)

Laboratoires

Meacutethode alternative analyse robuste

k Alt Niveau 3

k Alt Niveau 1

k Alt Niveau 2

k 1=259

k 5=181

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3538 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Appendix 4 ndash Statistical results

Level 1

Nombre de laboratoires (p) 10

A 1602 1602 1602 000 0000 0000 -0495 0000 A 1845 1602 1724 017 0122 -0122 0621 0840 0122

B 1602 1176 1389 030 0213 -0213 -2613 1247 B 1602 1301 1452 021 0151 -0151 -0769 1041 0062

C 1699 1301 1500 028 0199 -0199 -1510 1165 C 1301 1845 1573 038 -0272 0272 -0148 1881 0073

D 1602 1778 1690 012 -0088 0088 0380 0516 D 1602 1602 1602 000 0000 0000 0000 0000 -0088

E 1699 1602 1651 007 0048 -0048 -0013 0284 E 1699 1301 1500 028 0199 -0199 -0522 1376 -0151

F 1778 1301 1540 034 0239 -0239 -1116 1397 F 1778 1699 1739 006 0040 -0040 0698 0274 0199

G 1845 1544 1695 021 0151 -0151 0425 0881 G 2079 1699 1889 027 0190 -0190 1467 1315 0194

H 2041 1602 1822 031 0220 -0220 1689 1286 H 2000 1954 1977 003 0023 -0023 1917 0158 0155

L 1544 1778 1661 017 -0117 0117 0092 0685 L 1301 1602 1452 021 -0151 0151 -0769 1041 -0210

M 1653 1653 1653 000 0000 0000 0013 0000 M 1477 1602 1540 009 -0062 0062 -0319 0432 -0114

m 165186 m 160206 mediane D 00678

Cn(2p) 1867 Cn(2p) 1867 Cp 16101

Cn(p) 16101 Cn(p) 16101 Qn(D) 0092926

n=2p n 20 n=2p n 20 Qdiff 01496169

f 11 f 11 t 114

l 55 l 55

n=p n 10 n=p n 10

f 6 f 6

l 15 l 15

Qn(2p) 0091465 Qn(2p) 007745

Qn(p) 0062469 Qn(p) 0121519

Qintra 017078 Qintra 014461

Qinter 010058 Qinter 019565

Sr(eacutecart-type de reacutepeacutetabiliteacute) 02415 Sr 02045

RSDr(coefficient de variation de la reacutepeacutetabiliteacute)1462 RSDr 1277

r(repeatability limit)) 0676 r 0573

SL(eacutecart-type interlaboratoire) -001905 SLlt0 SL 001737

SR(eacutecart-type de reproductibiliteacute) 02415 SR 02433

RSDR(coefficient de variation de la reproductibiliteacute)0146 RSDR 0152

R(limite de reproductibiliteacute)) 06762 R(limite de reproductibiliteacute))068123

h k h MoyenneLaboratoires

Niveau 1

si LaboratoiresDeviationMoyenne Dk DeviationsiAlt

Niveau 1

Ref

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3638 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Level 2

Nombre de laboratoires (p) 10

A 2681 2613 2647 005 0034 -0034 -0151 0891 A 2322 2531 2427 015 -0105 0105 -2119 1620 -0220

B 2544 2477 2511 005 0033 -0033 -2699 0872 B 2556 2653 2605 007 -0048 0048 -0581 0750 0094

C 2643 2633 2638 001 0005 -0005 -0311 0130 C 2681 2653 2667 002 0014 -0014 -0040 0217 0029

D 2690 2663 2676 002 0014 -0014 0400 0357 D 2778 2732 2755 003 0023 -0023 0721 0354 0079

E 2690 2716 2703 002 -0013 0013 0897 0336 E 2724 2748 2736 002 -0012 0012 0556 0185 0033

F 2568 2398 2483 012 0085 -0085 -3214 2217 F 2477 2114 2296 026 0182 -0182 -3255 2811 -0188

G 2643 2591 2617 004 0026 -0026 -0707 0682 G 2756 2568 2662 013 0094 -0094 -0085 1453 0045

H 2716 2643 2680 005 0036 -0036 0460 0945 H 2653 2732 2693 006 -0040 0040 0181 0613 0013

L 2591 2591 2591 000 0000 0000 -1196 0000 L 2230 2580 2405 025 -0175 0175 -2307 2704 -0186

M 2653 2690 2672 003 -0018 0018 0311 0481 M 2740 2613 2677 009 0064 -0064 0040 0987 0005

m 2655082 m 26719001 mediane D 00209

Cn(2p) 1867 Cn(2p) 1867 Cp 16101

Cn(p) 16101 Cn(p) 16101 Qn(D) 0039911

n=2p n 20 n=2p n 20 Qdiff 00642591

f 11 f 11 t 082

l 55 l 55

n=p n 10 n=p n 10

f 6 f 6

l 15 l 15

Qn(2p) 0020570 Qn(2p) 00345985

Qn(p) 00332437 Qn(p) 00718158

Qintra 003841 Qintra 006460

Qinter 005352 Qinter 011563

Sr(eacutecart-type de reacutepeacutetabiliteacute) 00543 Sr 00914

RSDr(coefficient de variation de la reacutepeacutetabiliteacute)205 RSDr 342

r(repeatability limit)) 0152 r 0256

SL(eacutecart-type interlaboratoire) 000139 SL 000920

SR(eacutecart-type de reproductibiliteacute) 00659 SR 01325

RSDR(coefficient de variation de la reproductibiliteacute)0025 RSDR 0050

R(limite de reproductibiliteacute)) 018446 R(limite de reproductibiliteacute))03708618

Laboratoires

Niveau 2

Moyenne si Laboratoires

Niveau 2

DRef Alt Moyenne si Deviation h Deviation h k k

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3738 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Level 2 bis

Nombre de laboratoires (p) 10

A 3000 2477 2739 037 0261 -0261 0832 1418 A 2602 2477 2540 009 0062 -0062 -0391 0465 -0199

B 2544 2813 2678 019 -0134 0134 0211 0729 B 2903 2778 2841 009 0062 -0062 1732 0465 0162

C 2477 2813 2645 024 -0168 0168 -0135 0911 C 2477 2699 2588 016 -0111 0111 -0049 0826 -0057

D 2398 2653 2526 018 -0128 0128 -1370 0692 D 2778 3000 2889 016 -0111 0111 2074 0826 0363

E 2875 2740 2808 010 0067 -0067 1547 0365 E 2602 2301 2452 021 0151 -0151 -1012 1120 -0356

F 2477 2544 2511 005 -0033 0033 -1525 0182 F 2778 2477 2628 021 0151 -0151 0230 1120 0117

G 2653 2740 2697 006 -0044 0044 0400 0236 G 2845 2845 2845 000 0000 0000 1764 0000 0148

H 2176 2813 2495 045 -0318 0318 -1692 1727 H 2477 2477 2477 000 0000 0000 -0832 0000 -0017

L 2813 2544 2678 019 0134 -0134 0211 0729 L 2903 2301 2602 043 0301 -0301 0049 2241 -0076

M 2602 2740 2671 010 -0069 0069 0135 0375 M 2903 2255 2579 046 0324 -0324 -0112 2411 -0092

m 2658114 m 259505 mediane D -00372

Cn(2p) 1867 Cn(2p) 1867 Cp 16101

Cn(p) 16101 Cn(p) 16101 Qn(D) 01656915

n=2p n 20 n=2p n 20 Qdiff 02667753

f 11 f 11 t 035

l 55 l 55

n=p n 10 n=p n 10

f 6 f 6

l 15 l 15

Qn(2p) 0098745 Qn(2p) 007195

Qn(p) 006007 Qn(p) 008805

Qintra 018437 Qintra 013435

Qinter 009672 Qinter 014176

Sr(eacutecart-type de reacutepeacutetabiliteacute) 02607 Sr 01900

RSDr(coefficient de variation de la reacutepeacutetabiliteacute)981 RSDr 732

r(repeatability limit)) 0730 r 0532

SL(eacutecart-type interlaboratoire) -002464 SLlt0 SL 000205

SR(eacutecart-type de reproductibiliteacute) 02607 SR 01953

RSDR(coefficient de variation de la reproductibiliteacute)0098 RSDR 0075

R(limite de reproductibiliteacute)) 072996 R(limite de reproductibiliteacute))054686

Laboratoires

Niveau 2 bis

Moyenne si Laboratoires

Niveau 2 bis

DRef Alt Moyenne si Deviation h Deviation h k k

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3838 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Level 3

Nombre de laboratoires (p) 10

A 3690 3663 3676 002 0014 -0014 -0311 0513 A 3690 3708 3699 001 -0009 0009 0354 0257 0022

B 3613 3672 3642 004 -0030 0030 -1851 1109 B 3708 3568 3638 010 0070 -0070 -0844 2061 -0005

C 3663 3732 3698 005 -0035 0035 0645 1302 C 3663 3699 3681 003 -0018 0018 0000 0536 -0017

D 3690 3690 3690 000 0000 0000 0311 0000 D 3643 3663 3653 001 -0010 0010 -0545 0286 -0037

E 3732 3653 3693 006 0040 -0040 0429 1480 E 3613 3732 3673 008 -0060 0060 -0162 1769 -0020

F 3643 3672 3658 002 -0014 0014 -1157 0536 F 3690 3771 3731 006 -0040 0040 0975 1193 0073

G 3699 3653 3676 003 0023 -0023 -0328 0856 G 3602 3643 3623 003 -0021 0021 -1141 0612 -0053

H 3623 3672 3648 003 -0024 0024 -1615 0913 H 3748 3681 3715 005 0033 -0033 0664 0990 0067

L 3724 3663 3694 004 0031 -0031 0461 1150 L 3748 3771 3760 002 -0011 0011 1544 0335 0066

M 3690 3690 3690 000 0000 0000 0311 0000 M 3806 3681 3744 009 0062 -0062 1234 1848 0054

m 368334 m 368086 mediane D 00089

Cn(2p) 1867 Cn(2p) 1867 Cp 16101

Cn(p) 16101 Cn(p) 16101 Qn(D) 00325359

n=2p n 20 n=2p n 20 Qdiff 00523852

f 11 f 11 t 043

l 55 l 55

n=p n 10 n=p n 10

f 6 f 6

l 15 l 15

Qn(2p) 0014323 Qn(2p) 001811

Qn(p) 0013719 Qn(p) 003164

Qintra 002674 Qintra 003381

Qinter 002209 Qinter 005094

Sr(eacutecart-type de reacutepeacutetabiliteacute) 00378 Sr 00478

RSDr(coefficient de variation de la reacutepeacutetabiliteacute)103 RSDr 130

r(repeatability limit)) 0106 r 0134

SL(eacutecart-type interlaboratoire) -000023 SLlt0 SL 000145

SR(eacutecart-type de reproductibiliteacute) 00378 SR 00611

RSDR(coefficient de variation de la reproductibiliteacute)0010 RSDR 0017

R(limite de reproductibiliteacute)) 0105894 R(limite de reproductibiliteacute))017119

Ref Alt DLaboratoires

Niveau 3

Moyenne si Deviation h k Laboratoires

Niveau 3

Moyenne si Deviation h k

Page 9: ÞÀ¿¸7 ú Y5zñLjÜ< /u +æü t¬Á ... - NF Validation · NF VALIDATION Validation of alternative analytical methods Application in food microbiology ... 500 – 1 000 1 000 –

Thermo Fisher Scientific

ADRIA Deacuteveloppement 938 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Discussion

The linearity is accepted in all cases with

P values 5 and correlation coefficients superior to 099

For the matrix ldquoRillettesldquo the linearity test is significant (P = 4) with a high

correlation coefficient value (0996)

The Listeria PrecisTM method shows satisfying linearity

2112 Relative accuracy

The accuracy is the closeness of agreement between a test result and the accepted reference value

The bias is the difference between the expectation of the test results and an accepted reference value

Number and nature of the samples

The repartition per category and types is provided in table 3

Table 3 ndash Number and nature of the samples

Categories Types Number of samples

Analysed Exploited (1)

Meat products

Raw meat products 6 4

Ready to cook products 4 2

Ready to eat delicatessen 10 4

Total 20 10

Dairy products

Raw milk cheeses 7 4

Raw milks 4 3

Other dairy products 5 5

Total 16 12

Fishery products

Raw fishes 5 4

Processed fishes 7 2

Ready-to-eat products 6 4

Total 18 10

Vegetables

Raw vegetables 5 4

Frozen vegetables 3 3

Lettuce raw vegetables 5 5

Total 13 12

Egg products

Raw egg 5 5

Pastries 3 3

Pasteurised egg products 4 3

Total 12 11

Environmental samples

Wipes swabs 8 4

Process water 3 3

Dusts 3 3

Total 14 10

TOTAL 93 65

(1) Some data cannot be exploited because enumeration was under the detection level for at least one of the

method

Thermo Fisher Scientific

ADRIA Deacuteveloppement 1038 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Artificial contamination of the samples

Artificial contaminations were realised by spiking after performing injury

protocols on pure cultures or by cross-contaminations

56 samples were artificially contaminated 53 gave exploitable results among

a total of 65 samples 8150 of the samples were artificially contaminated

The injury protocol and the strains inoculated are provided in Table 4

Table 4

Stress Ndeg Injury protocol Strain Origin

A 45 min at 50 degC

48

LM -H123

LM-H170

Smoked salmon

Lettuce

Lettuce

B 30 min at 50 degC

and 2 h at ndash20degC

18

43

LM-O1

Raw milk (environment)

Environment

Egg product

C 30 min at 55degC

18

48

LM-H123

Raw milk

Smoked salmon

Lettuce

D 30 min at 50degC

et 2 h at ndash80degC

34

LM-H171

LM-O1

Rillettes

Lettuce

Egg product

E 30 min at 60 degC LM-O1 Egg product

Confirmatory tests

For the reference and the alternative methods confirmations were realised

by the tests described in the ISO 12290-2A1 standard For the alternative

method only one colony was confirmed while 5 colonies were tested for the

reference method

Results

Samples were analysed in duplicate by the reference and the alternative

methods

The contamination range is provided in table 5 per tested category

Thermo Fisher Scientific

ADRIA Deacuteveloppement 1138 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Table 5 ndash Contamination range

Food category Contamination level (log CFUg)

Meat products 100 ndash 404

Dairy products 148 ndash 408

Fishery products 100 ndash 384

Vegetables 170 ndash 511

Egg products 130 ndash 499

Environmental samples 170 ndash 511

All categories 100 ndash 511

Bi-dimensional graphs are given figure 3

Figure 3 ndash Bi-dimensional graphs

Graphe bi-dimensionnel Produits carneacutes

Fishery products

Vegetables

Egg products

Meat products

Thermo Fisher Scientific

ADRIA Deacuteveloppement 1238 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Statistical interpretation according to EN ISO16140 method

The results of the statistical interpretation are given in Table 6

Table 6 ndash Statistical interpretation results

Category n R Regression

used a t(a) b t(b) Critical T

P

Ordinate

at 0

Slope

at 1

Meat products 10 158 GMFR -0455 3321 115 3123 2306 001 001

Dairy products 12 065 GMFR -0027 0385 1002 0108 2228 092 071

Fishery products 10 130 GMFR -0305 1024 1034 0312 2306 076 034

Vegetables 12 086 GMFR -0015 0128 0993 0191 2228 085 090

Egg products 11 158 GMFR -0038 0866 1001 0090 2262 093 041

Environmental

samples 10 176 GMFR -0139 265 1034 231 2306 005 003

All products 65 118 GMFR -0182 3042 1040 2145 2000 004 000

All products

(estimated number) 65 118 GMFR -0182 3042 1040 2145 2000 004 000

All products (non

estimated results) 20 115 GMFR -0367 151 1138 1141 2101 027 015

Repeatability limits obtained for the alternative and reference methods as

well as bias obtained are presented in Table 7

Dairy products Environmental samples

Log (Ref) Log (Ref)

All categories

Thermo Fisher Scientific

ADRIA Deacuteveloppement 1338 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Table 7

Categories Bias

Repeatability limits

Rob r

Ref Alt

Meat products 0008 0314 0343

Dairy products -0031 0136 0130

Fishery products -0133 0220 0214

Vegetables -0041 0180 0319

Egg products -0026 0219 0206

Environmental samples -0007 0198 0119

All products -0032 0196 0215

Regression straight lines (graph and equation representations) for each food

category and for all products are presented figure 4

Thermo Fisher Scientific

ADRIA Deacuteveloppement 1438 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Figure 4 ndash Regression straight lines

Meat products

Fishery products

Dairy products

Vegetables

Thermo Fisher Scientific

ADRIA Deacuteveloppement 1538 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Environmental samples

Egg products

All products (estimated number)

Thermo Fisher Scientific

ADRIA Deacuteveloppement 1638 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

The hypotheses that the ordinates are close to 0 and the slopes close to 1

are approved in all cases except for

- Meat products (slope P = 1 ordinate P = 1)

- Environmental samples (ordinate P = 3)

- All products (ordinate P = 0)

The bias between the two methods vary from ndash 0133 to 0008 log CFU For

all categories the bias is ndash 0032 log CFU The repeatability limits of the

alternative and the reference method are similar For all products the values

are respectively 0215 and 0196 log CFU For all products these

interpretations are provided taking into account data with low contamination

level or not (estimated colonies number)

For all food products the regression straight line is the following

log (Alt) = 1040 log Ref ndash 0182

The Listeria PrecisTM method shows satisfying relative accuracy

All products

All products (Level 2)

Thermo Fisher Scientific

ADRIA Deacuteveloppement 1738 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

2113 Detection limit (LOD) and quantification limit (LOQ)

The critical level is the defined as the smallest amount which can be detected (not null) but not

quantified as an exact value Below this value it cannot be sure that the true value is not null

The detection limit is defined as being higher than the critical level because it involves a power the

probability 1- which has to be well over 50 for example 95

The quantification limit is defined as the smallest amount of analyte (that is the lowest actual number of

organisms) which can be measured and quantified with defined precision and accuracy under the

experimental conditions by the method under validation

Protocol

The detection limit of the alternative method was realised with pure cultures

Three different inoculation levels were tested with six replicates per level ie

18 analyses were done by the alternative method

Listeria monocytogenes 18 was used for the detection limit of the alternative

method

Quantification limit was calculated for six independent blank samples

determinations

Results

These data are intrinsic to the method used and are presented in the

following tables

Table 8 ndash Inoculation 1 ml on 3 plates

Level (CFUml) Positive samples number Standard deviation Bias

0 06

05 26 1211 0

1 36 0816 05

5 66 2483 85

Thermo Fisher Scientific

ADRIA Deacuteveloppement 1838 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Table 9 ndash Inoculation 01 ml on 1 plate

Level (CFUml) Positive samples number Standard deviation Bias

0 06

5 36 0548 05

10 46 1414 2

50 66 2258 55

Table 10

Formulas Obtained values

LC 165 S0 + X0 18

LOD 33 S0 + X0 32

LOQ 10 S0 + X0 87

2114 Relative Sensitivity

The relative sensitivity is defined as the ability of the alternative method to detect two different amounts

of analyte measured by the reference method within a given matrix at a specified average value or

over the whole measurement range that is it is the minimal quantity variation (increase of the analyte

concentration x) which gives a significant variation of the measured signal (response y)

These data are intrinsic to the used method and are obtained from the results

of the linearity study

Sensitivity patterns obtained for tested (matrixstrain) pairs are presented in

figure 5

Thermo Fisher Scientific

ADRIA Deacuteveloppement 1938 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Figure 5 ndash Sensitivity patterns for the tested (matrixstrain) pairs

2115 SpecificitySelectivity

The specificity is defined as the degree to which a method is affected (or not) by the other components

present in a multi-component sample That is the ability of a method to measure exactly a given

analyte or its amount within the sample without interference from non-target components such as a

matrix effect or background noise

The selectivity is defined as a measure of the degree of non-interference in the presence of non-target

analytes A method is selective if it can be used to detect the analyte under examination and that a

guarantee can be provided that the detected signal can only be a product by that specific analyte

The specificity and selectivity study was performed for the Listeria PrecisTM

detection method no additional test was realised for the enumeration

method The results were the following

- Among the Listeria monocytogenes strains tested 2 strains (Listeria

monocytogenes CIP 7834 and Listeria monocytogenes CIP 105459) gave

negative results These 2 strains were tested twice by running 48 h

incubation time of the compared agars Positive results were then

observed by the alternative method Pale characteristic colonies were

observed on the ISO agar by streaking the Half Fraser broth only

- No cross reaction was observed with the non-target strains except with

Listeria ivanovii which gave characteristic colonies on Brilliancetrade Listeria

Agar but the confirmatory tests provide a clear discrimination between

Listeria monocytogenes and Listeria ivanovi species

Rillettes

Lettuce

Raw milk

Smoked salmon

Raw egg

Process water

x (CFUg)

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2038 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

212 Practicability

1 Packaging Ready to use Brilliance Listeria agar plates are available

(x 10 plates)

2 Reagent volume

3 Storage conditionsndash

Shelf life of products

not opened

The plates are stored at 6 - 12degC in darkness conditions The

expiration date is mentioned on the package and on the plates It

is around 3 months

4 Modalities of use

after first use

5 Required

equipments No specific equipment is required

6 Reagents Ready to use plates

7 Training 05 day

8 Real time handling

Reference method

9 min for one sample

55 min for 10 samples

Alternative method

7 min for one sample

35 minutes for 10 samples

9 Time to result

Negative results

Reference method Day 2

Alternative method Day 2

Negative results after confirmation or positive results

Reference method Day 4 ndash Day 7

Alternative method Day 4 ndash Day 7

Positive results

Reference method Day 4 ndash Day 7

Alternative method Day 2 ndash Day 3 (with OBIS Mono Test)

or Day 4 ndash Day 7 (by the classical tests described in the

standardised method)

10 Technician skill Identical to that required for the reference method

11 Steps common to

the reference method Mother suspension revivification step and decimal dilutions

12 Traceability

13 Maintenance

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2138 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

213 Inter-laboratory study

2131 Organisation

12 laboratories participated to this study Pasteurised milk was inoculated by

Listeria monocytogenes 12a isolated from raw milk The targeted

inoculation levels were

- 0 CFUml

- 50 CFU ml

- 500 CFU ml

- 5 000 CFU ml

Each laboratory received eight flasks of 10 ml sample ie two flasks per

inoculation level Furthermore one non-inoculated sample has been added

to the package for total viable count microflora (NF ISO 4833 method)

Detailed instructions were transmitted to the collaborators by the expert

laboratory

Collaborative study laboratories and the expert laboratory carried out the

analyses with the alternative and reference methods

2132 Verification of experimental parameters

Strain stability during transport

In order to evaluate the Listeria monocytogenes strain stability during

transport bacterial count of all inoculated flasks were checked at different

time ie inoculation time after 24 h of conservation at 2deg - 8degC Results are

reported in table 11

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2238 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Table 11 ndash Listeria monocytogenes count

by ISO 11290-2A1 method (log CFUml)

Day of the analyses Level contamination Reference method

Day 0

(29052006)

1 45

2 440

3 4 500

Day 1

(30052006)

1 60

2 430

3 4 900

No evolution of the strain was observed after storage for 24 h at 2degC ndash 8degC

Contamination level

The real contamination levels are provided in table 12

Table 12 ndash Real contamination level (CFUml)

Target inoculation

(CFU ml) Samples Ndeg

Real contamination levels

(CFUml)

0 1 - 8 0

50 5 - 6 45

500 3 - 7 440

5000 2 - 4 4 500

Sample temperature at receipt

Measured temperatures at receipt are listed in table 13

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2338 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Table 13 - Sample temperature on receipt

Laboratories Receipt

day

Hour

receipt

Measured temperature

at receipt (degC)

Measured temperature by the

temperature probe (degC)

A

B

C

D

E

F

G

H

I

J

K

L

M

30052006

10h45

10h15

11h15

10h00

09h30

8h30

11h50

10h00

08h30

14h45

08h30

09h45

08h45

82

55

45

63

60

62

81

38

48

-

14

67

76

03

03

-13

03

04

-14

05

04

01

05

-13

04

Conclusion

The temperatures during the transport were correct for all the labs except for

Lab J which didnrsquot provide the information

All the labs received their packages with several broken flasks

Three labs (B D and L) received some damaged samples but proceeded to

analyses

All the labs started the analyses at Day 1 except Lab J

2133 Results

Aerobic mesophilic flora

The count of aerobic mesophilic flora varied from 94 102 CFUml to

33 103 CFUml

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2438 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Expert lab results

The results obtained by the expert lab are the following

Table 14

Target inoculation level (CFUml)

Reference method ISO 11290-2

Alternative method Listeria Precis Enumeration

Rep 1 Rep 2 Rep 1 Rep 2

lt 10 lt 10 lt 10 lt 10 lt10

50 50 (Ne) 60 (Ne) 50 (Ne) 30 (Ne)

500 470 390 490 500

5 000 4 700 5 100 5 900 3 400

Ne estimated number

The obtained results are in agreement with those expected

Collaborator Labs results

A synthesis of the results obtained by the collaborator labs is provided

Table 15 (CFUml) and Table 16 (log CFUml)

Table 15 ndash Synthesis of results obtained by the alternative

and the reference methods (CFUml)

Labs Level 1 Level 2 Level 2 bis Level 3

Reference method

Alternative method

Reference method

Alternative method

Reference method

Alternative method

Reference method

Alternative method

A 40 40 70 40 480 410 210 340 1000 300 400 300 4900 4600 4900 5100

B 40 15 40 20 350 300 360 450 350 650 800 600 4100 4700 5100 3700

C 50 20 20 70 440 430 480 450 300 650 300 500 4600 5400 4600 5000

D 40 60 40 40 490 460 600 540 250 450 600 1000 4900 4900 4400 4600

E 50 40 50 20 490 520 530 560 750 550 400 200 5400 4500 4100 5400

F 60 20 60 50 370 250 300 130 300 350 600 300 4400 4700 4900 5900

G 70 35 120 50 440 390 570 370 450 550 700 700 5000 4500 4000 4400

H 110 40 100 90 520 440 450 540 150 650 300 300 4200 4700 5600 4800

L 35 60 20 40 390 390 170 380 650 350 800 200 5300 4600 5600 5900

M 45 45 30 40 450 490 550 410 400 550 800 180 4900 4900 6400 4800

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2538 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Table 16 ndash Synthesis of results obtained by the alternative

and the reference methods (log CFUml)

Labs Level 1 Level 2 Level 2 bis Level 3

Reference method

Alternative method

Reference method

Alternative method

Reference method

Alternative method

Reference method

Alternative method

A 1602 1602 1845 1602 2681 2613 2322 2531 3000 2477 2602 2477 3690 3663 3690 3708

B 1602 1176 1602 1301 2544 2477 2556 2653 2544 2813 2903 2778 3613 3672 3708 3568

C 1699 1301 1301 1845 2643 2633 2681 2653 2477 2813 2477 2699 3663 3732 3663 3699

D 1602 1778 1602 1602 2690 2663 2778 2732 2398 2653 2778 3000 3690 3690 3643 3663

E 1699 1602 1699 1301 2690 2716 2724 2748 2875 2740 2602 2301 3732 3653 3613 3732

F 1778 1301 1778 1699 2568 2398 2477 2114 2477 2544 2778 2477 3643 3672 3690 3771

G 1845 1544 2079 1699 2643 2591 2756 2568 2653 2740 2845 2845 3699 3653 3602 3643

H 2041 1602 2000 1954 2716 2643 2653 2732 2176 2813 2477 2477 3623 3672 3748 3681

L 1544 1778 1301 1602 2591 2591 2230 2580 2813 2544 2903 2301 3724 3663 3748 3771

M 1653 1653 1477 1602 2653 2690 2740 2613 2602 2740 2903 2255 3690 3690 3806 3681

Level 2 results obtained taking account the 3 plates inoculated with the mother-suspension

Level 2 bis results obtained taking account the plates inoculated with 01 ml of the mother-suspension

Scrutiny of the measurement results for consistency

In order to identify the measurement results or laboratories that could be

inconsistent two graphical consistency techniques were realised Mandelrsquos

h-ank-statistics Mandel indicators h and k at 5 significance highlighted

some possible inconsistent data (See tables and graphics in Appendix 3 and

Appendix 4)

Mandelrsquos values Number of values observed over the threshold

Reference method Alternative method

h gt 1 Lab F Level 2 Lab F Level 2

h gt 5 Lab F Level 2

Lab B Levels 1 and 2

Lab A Level 2

Lab D Level 3

Lab F Level 2

Lab L Level 2

kgt 1 Lab F Level 3

k gt 5 Lab F Level 2 Lab C Level 2

Lab F Level 3

Lab L Levels 1 and 3

Lab M Level 1

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2638 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

The emphasized results appear (i) coherent with the targeted contamination

levels (ii) concordant between both methods

All the results were thus kept for the statistical analysis

Comparison of the trueness and precision characteristics of

the reference method and alternative methods

The statistical values are summarised hereafter

Level p

Reference method Alternative method

Repeatability ratio

Reproducibility ratio Median

Repeatability standard deviation

Reproducibility standard deviation

Median Repeatability

standard deviation

Reproducibility standard deviation

1 10 16519 02415 02425 16021 02045 02433 0847 1007

2 10 26551 00543 00659 26719 00914 01325 1682 2011

2 bis 10 26581 02607 02607 25951 01900 01953 0729 0749

3 10 18671 00378 00378 18671 00478 00611 1264 1617

Bias of the alternative method

In order to estimate the bias of the alternative method with regard to the reference method for every

level Dij and t are calculated according to the following equations

RefAlt ijYijYDij

Diffp

Dijit

)2(

)(median

If the t value is superior to 2 the alternative method is significantly biased

with regard to the reference method

The values are given in table 17

Table 17 ndash t (d) values obtained by level

Level Bias t (d) Conclusion

1 0068 114 Non significant bias

2 0021 082 Non significant bias

2 bis - 0037 035 Non significant bias

3 - 0037 043 Non significant bias

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2738 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

The statistical test concludes to a non significant bias for all the inoculation

levels The bias varies from ndash 0037 to 0068 log CFUml

Comparison of the repeatability standard deviations

If the ratio Srj Alt Srj Ref of the repeatability standard deviations of the

alternative method and the reference method is larger than 2 the precision

under repeatability conditions of the alternative method is considered to be

lower than that of the reference method If this ratio is smaller than 05 the

precision under repeatability conditions of the alternative method is

considered to be greater than that of the reference method

The ratio values are given in table 18

Table 18

Level

Repeatability standard deviation

Reference method Alternative method Alternative method

reference method ratio

1 0242 0205 0847

2 0054 0091 1682

2 bis 0261 0190 0729

3 0038 0048 1264

Level Repeatability limit

Reference method Alternative method

1 0676 0573

2 0152 0256

2 bis 0730 0532

3 0106 0134

The ratios of the repeatability standard deviations are below 2 in all cases

The precision under repeatability conditions of the alternative method is

equivalent to that of the reference method

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2838 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Comparison of the reproducibility standard deviations

If the ratio Srj Alt Srj Ref of the reproducibility standard deviations of the alternative method and the

reference method is larger than 2 the precision under reproducibility conditions of the alternative

method is considered to be lower than that of the reference method If this ratio is smaller than 05 the

precision under reproducibility conditions of the alternative method is considered to be greater than

that of the reference method

The ratio values are given in table 19

Table 19

Level

Reproducibility standard deviation

Reference method Alternative method Alternative method

reference method ratio

1 0242 0243 1007

2 0066 0133 2011

2 bis 0261 0195 0749

3 0038 0061 1617

Level Reproducibility limit

Reference method Alternative method

1 0676 0681

2 0185 0371

2 bis 0730 0547

3 0106 0171

The ratios of the reproducibility standard deviations are below or close to 2

The precision under reproducibility conditions of the alternative method is

equivalent to that of the reference method

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2938 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

214 Conclusion

Method comparison study

The Listeria PrecisTM enumeration method shows satisfying linearity and

relative accuracy

The alternative method shows satisfying inclusivity and exclusivity results

Two Listeria monocytogenes strains gave negative results with the

alternative method These strains grew weakly on the reference method

media (OampA Palcam and Oxford)

The limits of repeatability of the alternative method are similar to those of the

reference method

The bias between both methods are low between - 0133 Log CFUg and

- 0008 Log CFUg

The Listeria PrecisTM enumeration method offers the saving for confirmatory

tests

Positive results are obtained with the alternative method in 2-3 days (with

OBIS Mono test) or in 4-7 days (by the classical tests described in the

standardized method) against 4-7 days for the reference method

Inter-laboratory study

The bias between the reference and the alternative methods shows satisfying

and correct values

- level 1 (100 - 1 000 CFUg) bias = + 007 log CFUg

- level 2 (1 000 - 10 000 CFUg) bias = 002 log CFUg

- level 2 bis (1 000 - 10 000 CFUg) bias = - 004 log CFUg

- level 3 (10 000 - 100 000 CFUg) bias = - 004 log CFUg

The repeatability and the reproducibility of the alternative method look similar

to those of the reference method

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3038 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

22 Extension study (realised by ASEPT 2007)

An extension study was performed in 2007 for a new confirmatory test OBIS

MONO

150 Listeria monocytogenes strains and 100 non-target strains were isolated

onto TSAYE and BrillianceTM Listeria Agar plates All the Listeria

monocytogenes and the non-target strains gave the expected results

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3138 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Appendix 1 ndash Flow diagram of the Listeria PrecisTM method

x g sample + 9 x ml buffered peptone water

Revivification step for 1 h plusmn 5 min at 20degC plusmn 1degC

Streaking 1 ml onto 3 Brilliance Listeria Agar plates (mother suspension)

or 01 ml onto 1 Brilliance Listeria Agar plates (mother suspension and decimal dilutions)

Incubation for 48 h plusmn 3 h at 37degC plusmn 1degC

Confirmatory tests on at least one typical colony

- by the tests described in the ISO method

- by applying the OBIS mono test on an isolated colony

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3238 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Appendix 2 ndash ISO 11290-2 (1998) amp ISO 11290-2A1 (2004) Horizontal

method for the detection and enumeration of Listeria monocytogenes -

Part 2 enumeration of Listeria monocytogenes in foods

10 g cheese + 90 ml buffered peptone water or Half Fraser Broth (without supplements) (110 dilution)

Stomach 1 min 30 seconds

Keep at 20degC plusmn 2degC for 1 h plusmn 5 min

Decimal dilutions in 9 ml peptone salt tubes

Dilution 110

Dilution 1100

Dilution 11000

1 ml on 3 Chromogenic Listeria medium (ISO) plates (x 2) (-1)

01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-2)

01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-3)

01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-4)

Incubation at 37degC plusmn 1degC

24 h - 48 h

Numerate typical colonies at 24 h

(bluegreen surrounded by an opaque halo)

Numerate at 48 h

Streak typical colonies on TSYEA

(5 coloniesplate 2 successive dilutions)

Confirmation tests

Catalase

Gram stain

For confirmation of L monocytogenes

Haemolysis

Carbohydrate (rhamnose and xylose) utilisation

CAMP Test

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3338 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Appendix 3 ndash Mandelrsquos graphics

Laboratoire h Ref

Niveau 1 h Ref

Niveau 2 h Ref

Niveau 3 h Alt

Niveau 1 h Alt

Niveau 2 h Alt

Niveau 3 h5 h1 h5 h1

A -050 -015 083 062 -212 -039 198 299 -198 -299

B -261 -270 021 -077 -058 173 198 299 -198 -299

C -151 -031 -014 -015 -004 -005 198 299 -198 -299

D 038 040 -137 000 072 207 198 299 -198 -299

E -001 090 155 -052 056 -101 198 299 -198 -299

F -112 -321 -153 070 -325 023 198 299 -198 -299

G 042 -071 040 147 -009 176 198 299 -198 -299

H 169 046 -169 192 018 -083 198 299 -198 -299

L 009 -120 021 -077 -231 005 198 299 -198 -299

M 001 031 014 -032 004 -011 198 299 -198 -299

-300

-200

-100

000

100

200

300

A B C D E F G H L M

Vale

urs

h d

e M

an

del(

rob

uste

s)

Laboratoires

Meacutethode de reacutefeacuterence analyse robuste

h Ref Niveau 1

h Ref Niveau 2

h Ref Niveau 3

h 5=198

h 1=299

-300

-200

-100

000

100

200

300

A B C D E F G H L M

Vale

urs

de h

de M

an

del(

rob

uste

s)

Laboratoires

Meacutethode alternative analyse robuste

h Alt Niveau 1

h Alt Niveau 2

h Alt Niveau 2

h 1=299

h 5=198

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3438 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Laboratoire k Ref

Niveau 1 k Ref

Niveau 2 k Ref

Niveau 3 k Alt

Niveau 1 k Alt

Niveau 2 k Alt

Niveau 3 k1 k5

A 000 089 142 084 162 046 259 181

B 125 087 073 104 075 046 259 181

C 117 013 091 188 022 083 259 181

D 052 036 069 000 035 083 259 181

E 028 034 037 138 019 112 259 181

F 140 222 018 027 281 112 259 181

G 088 068 024 131 145 000 259 181

H 129 094 173 016 061 000 259 181

L 069 000 073 104 270 224 259 181

M 000 048 038 043 099 241 259 181

000

050

100

150

200

250

300

350

400

A B C D E F G H L M

Vale

urs

k d

e M

an

del(

rob

uste

s)

Laboratoires

Meacutethode de reacutefeacuterence analyse robuste

k Ref Niveau 1k Ref Niveau 2k Ref Niveau 3

k 1=259

k 5=181

000

050

100

150

200

250

300

A B C D E F G H L M

Vale

urs

k d

e M

an

del

(ro

bu

ste

s)

Laboratoires

Meacutethode alternative analyse robuste

k Alt Niveau 3

k Alt Niveau 1

k Alt Niveau 2

k 1=259

k 5=181

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3538 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Appendix 4 ndash Statistical results

Level 1

Nombre de laboratoires (p) 10

A 1602 1602 1602 000 0000 0000 -0495 0000 A 1845 1602 1724 017 0122 -0122 0621 0840 0122

B 1602 1176 1389 030 0213 -0213 -2613 1247 B 1602 1301 1452 021 0151 -0151 -0769 1041 0062

C 1699 1301 1500 028 0199 -0199 -1510 1165 C 1301 1845 1573 038 -0272 0272 -0148 1881 0073

D 1602 1778 1690 012 -0088 0088 0380 0516 D 1602 1602 1602 000 0000 0000 0000 0000 -0088

E 1699 1602 1651 007 0048 -0048 -0013 0284 E 1699 1301 1500 028 0199 -0199 -0522 1376 -0151

F 1778 1301 1540 034 0239 -0239 -1116 1397 F 1778 1699 1739 006 0040 -0040 0698 0274 0199

G 1845 1544 1695 021 0151 -0151 0425 0881 G 2079 1699 1889 027 0190 -0190 1467 1315 0194

H 2041 1602 1822 031 0220 -0220 1689 1286 H 2000 1954 1977 003 0023 -0023 1917 0158 0155

L 1544 1778 1661 017 -0117 0117 0092 0685 L 1301 1602 1452 021 -0151 0151 -0769 1041 -0210

M 1653 1653 1653 000 0000 0000 0013 0000 M 1477 1602 1540 009 -0062 0062 -0319 0432 -0114

m 165186 m 160206 mediane D 00678

Cn(2p) 1867 Cn(2p) 1867 Cp 16101

Cn(p) 16101 Cn(p) 16101 Qn(D) 0092926

n=2p n 20 n=2p n 20 Qdiff 01496169

f 11 f 11 t 114

l 55 l 55

n=p n 10 n=p n 10

f 6 f 6

l 15 l 15

Qn(2p) 0091465 Qn(2p) 007745

Qn(p) 0062469 Qn(p) 0121519

Qintra 017078 Qintra 014461

Qinter 010058 Qinter 019565

Sr(eacutecart-type de reacutepeacutetabiliteacute) 02415 Sr 02045

RSDr(coefficient de variation de la reacutepeacutetabiliteacute)1462 RSDr 1277

r(repeatability limit)) 0676 r 0573

SL(eacutecart-type interlaboratoire) -001905 SLlt0 SL 001737

SR(eacutecart-type de reproductibiliteacute) 02415 SR 02433

RSDR(coefficient de variation de la reproductibiliteacute)0146 RSDR 0152

R(limite de reproductibiliteacute)) 06762 R(limite de reproductibiliteacute))068123

h k h MoyenneLaboratoires

Niveau 1

si LaboratoiresDeviationMoyenne Dk DeviationsiAlt

Niveau 1

Ref

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3638 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Level 2

Nombre de laboratoires (p) 10

A 2681 2613 2647 005 0034 -0034 -0151 0891 A 2322 2531 2427 015 -0105 0105 -2119 1620 -0220

B 2544 2477 2511 005 0033 -0033 -2699 0872 B 2556 2653 2605 007 -0048 0048 -0581 0750 0094

C 2643 2633 2638 001 0005 -0005 -0311 0130 C 2681 2653 2667 002 0014 -0014 -0040 0217 0029

D 2690 2663 2676 002 0014 -0014 0400 0357 D 2778 2732 2755 003 0023 -0023 0721 0354 0079

E 2690 2716 2703 002 -0013 0013 0897 0336 E 2724 2748 2736 002 -0012 0012 0556 0185 0033

F 2568 2398 2483 012 0085 -0085 -3214 2217 F 2477 2114 2296 026 0182 -0182 -3255 2811 -0188

G 2643 2591 2617 004 0026 -0026 -0707 0682 G 2756 2568 2662 013 0094 -0094 -0085 1453 0045

H 2716 2643 2680 005 0036 -0036 0460 0945 H 2653 2732 2693 006 -0040 0040 0181 0613 0013

L 2591 2591 2591 000 0000 0000 -1196 0000 L 2230 2580 2405 025 -0175 0175 -2307 2704 -0186

M 2653 2690 2672 003 -0018 0018 0311 0481 M 2740 2613 2677 009 0064 -0064 0040 0987 0005

m 2655082 m 26719001 mediane D 00209

Cn(2p) 1867 Cn(2p) 1867 Cp 16101

Cn(p) 16101 Cn(p) 16101 Qn(D) 0039911

n=2p n 20 n=2p n 20 Qdiff 00642591

f 11 f 11 t 082

l 55 l 55

n=p n 10 n=p n 10

f 6 f 6

l 15 l 15

Qn(2p) 0020570 Qn(2p) 00345985

Qn(p) 00332437 Qn(p) 00718158

Qintra 003841 Qintra 006460

Qinter 005352 Qinter 011563

Sr(eacutecart-type de reacutepeacutetabiliteacute) 00543 Sr 00914

RSDr(coefficient de variation de la reacutepeacutetabiliteacute)205 RSDr 342

r(repeatability limit)) 0152 r 0256

SL(eacutecart-type interlaboratoire) 000139 SL 000920

SR(eacutecart-type de reproductibiliteacute) 00659 SR 01325

RSDR(coefficient de variation de la reproductibiliteacute)0025 RSDR 0050

R(limite de reproductibiliteacute)) 018446 R(limite de reproductibiliteacute))03708618

Laboratoires

Niveau 2

Moyenne si Laboratoires

Niveau 2

DRef Alt Moyenne si Deviation h Deviation h k k

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3738 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Level 2 bis

Nombre de laboratoires (p) 10

A 3000 2477 2739 037 0261 -0261 0832 1418 A 2602 2477 2540 009 0062 -0062 -0391 0465 -0199

B 2544 2813 2678 019 -0134 0134 0211 0729 B 2903 2778 2841 009 0062 -0062 1732 0465 0162

C 2477 2813 2645 024 -0168 0168 -0135 0911 C 2477 2699 2588 016 -0111 0111 -0049 0826 -0057

D 2398 2653 2526 018 -0128 0128 -1370 0692 D 2778 3000 2889 016 -0111 0111 2074 0826 0363

E 2875 2740 2808 010 0067 -0067 1547 0365 E 2602 2301 2452 021 0151 -0151 -1012 1120 -0356

F 2477 2544 2511 005 -0033 0033 -1525 0182 F 2778 2477 2628 021 0151 -0151 0230 1120 0117

G 2653 2740 2697 006 -0044 0044 0400 0236 G 2845 2845 2845 000 0000 0000 1764 0000 0148

H 2176 2813 2495 045 -0318 0318 -1692 1727 H 2477 2477 2477 000 0000 0000 -0832 0000 -0017

L 2813 2544 2678 019 0134 -0134 0211 0729 L 2903 2301 2602 043 0301 -0301 0049 2241 -0076

M 2602 2740 2671 010 -0069 0069 0135 0375 M 2903 2255 2579 046 0324 -0324 -0112 2411 -0092

m 2658114 m 259505 mediane D -00372

Cn(2p) 1867 Cn(2p) 1867 Cp 16101

Cn(p) 16101 Cn(p) 16101 Qn(D) 01656915

n=2p n 20 n=2p n 20 Qdiff 02667753

f 11 f 11 t 035

l 55 l 55

n=p n 10 n=p n 10

f 6 f 6

l 15 l 15

Qn(2p) 0098745 Qn(2p) 007195

Qn(p) 006007 Qn(p) 008805

Qintra 018437 Qintra 013435

Qinter 009672 Qinter 014176

Sr(eacutecart-type de reacutepeacutetabiliteacute) 02607 Sr 01900

RSDr(coefficient de variation de la reacutepeacutetabiliteacute)981 RSDr 732

r(repeatability limit)) 0730 r 0532

SL(eacutecart-type interlaboratoire) -002464 SLlt0 SL 000205

SR(eacutecart-type de reproductibiliteacute) 02607 SR 01953

RSDR(coefficient de variation de la reproductibiliteacute)0098 RSDR 0075

R(limite de reproductibiliteacute)) 072996 R(limite de reproductibiliteacute))054686

Laboratoires

Niveau 2 bis

Moyenne si Laboratoires

Niveau 2 bis

DRef Alt Moyenne si Deviation h Deviation h k k

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3838 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Level 3

Nombre de laboratoires (p) 10

A 3690 3663 3676 002 0014 -0014 -0311 0513 A 3690 3708 3699 001 -0009 0009 0354 0257 0022

B 3613 3672 3642 004 -0030 0030 -1851 1109 B 3708 3568 3638 010 0070 -0070 -0844 2061 -0005

C 3663 3732 3698 005 -0035 0035 0645 1302 C 3663 3699 3681 003 -0018 0018 0000 0536 -0017

D 3690 3690 3690 000 0000 0000 0311 0000 D 3643 3663 3653 001 -0010 0010 -0545 0286 -0037

E 3732 3653 3693 006 0040 -0040 0429 1480 E 3613 3732 3673 008 -0060 0060 -0162 1769 -0020

F 3643 3672 3658 002 -0014 0014 -1157 0536 F 3690 3771 3731 006 -0040 0040 0975 1193 0073

G 3699 3653 3676 003 0023 -0023 -0328 0856 G 3602 3643 3623 003 -0021 0021 -1141 0612 -0053

H 3623 3672 3648 003 -0024 0024 -1615 0913 H 3748 3681 3715 005 0033 -0033 0664 0990 0067

L 3724 3663 3694 004 0031 -0031 0461 1150 L 3748 3771 3760 002 -0011 0011 1544 0335 0066

M 3690 3690 3690 000 0000 0000 0311 0000 M 3806 3681 3744 009 0062 -0062 1234 1848 0054

m 368334 m 368086 mediane D 00089

Cn(2p) 1867 Cn(2p) 1867 Cp 16101

Cn(p) 16101 Cn(p) 16101 Qn(D) 00325359

n=2p n 20 n=2p n 20 Qdiff 00523852

f 11 f 11 t 043

l 55 l 55

n=p n 10 n=p n 10

f 6 f 6

l 15 l 15

Qn(2p) 0014323 Qn(2p) 001811

Qn(p) 0013719 Qn(p) 003164

Qintra 002674 Qintra 003381

Qinter 002209 Qinter 005094

Sr(eacutecart-type de reacutepeacutetabiliteacute) 00378 Sr 00478

RSDr(coefficient de variation de la reacutepeacutetabiliteacute)103 RSDr 130

r(repeatability limit)) 0106 r 0134

SL(eacutecart-type interlaboratoire) -000023 SLlt0 SL 000145

SR(eacutecart-type de reproductibiliteacute) 00378 SR 00611

RSDR(coefficient de variation de la reproductibiliteacute)0010 RSDR 0017

R(limite de reproductibiliteacute)) 0105894 R(limite de reproductibiliteacute))017119

Ref Alt DLaboratoires

Niveau 3

Moyenne si Deviation h k Laboratoires

Niveau 3

Moyenne si Deviation h k

Page 10: ÞÀ¿¸7 ú Y5zñLjÜ< /u +æü t¬Á ... - NF Validation · NF VALIDATION Validation of alternative analytical methods Application in food microbiology ... 500 – 1 000 1 000 –

Thermo Fisher Scientific

ADRIA Deacuteveloppement 1038 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Artificial contamination of the samples

Artificial contaminations were realised by spiking after performing injury

protocols on pure cultures or by cross-contaminations

56 samples were artificially contaminated 53 gave exploitable results among

a total of 65 samples 8150 of the samples were artificially contaminated

The injury protocol and the strains inoculated are provided in Table 4

Table 4

Stress Ndeg Injury protocol Strain Origin

A 45 min at 50 degC

48

LM -H123

LM-H170

Smoked salmon

Lettuce

Lettuce

B 30 min at 50 degC

and 2 h at ndash20degC

18

43

LM-O1

Raw milk (environment)

Environment

Egg product

C 30 min at 55degC

18

48

LM-H123

Raw milk

Smoked salmon

Lettuce

D 30 min at 50degC

et 2 h at ndash80degC

34

LM-H171

LM-O1

Rillettes

Lettuce

Egg product

E 30 min at 60 degC LM-O1 Egg product

Confirmatory tests

For the reference and the alternative methods confirmations were realised

by the tests described in the ISO 12290-2A1 standard For the alternative

method only one colony was confirmed while 5 colonies were tested for the

reference method

Results

Samples were analysed in duplicate by the reference and the alternative

methods

The contamination range is provided in table 5 per tested category

Thermo Fisher Scientific

ADRIA Deacuteveloppement 1138 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Table 5 ndash Contamination range

Food category Contamination level (log CFUg)

Meat products 100 ndash 404

Dairy products 148 ndash 408

Fishery products 100 ndash 384

Vegetables 170 ndash 511

Egg products 130 ndash 499

Environmental samples 170 ndash 511

All categories 100 ndash 511

Bi-dimensional graphs are given figure 3

Figure 3 ndash Bi-dimensional graphs

Graphe bi-dimensionnel Produits carneacutes

Fishery products

Vegetables

Egg products

Meat products

Thermo Fisher Scientific

ADRIA Deacuteveloppement 1238 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Statistical interpretation according to EN ISO16140 method

The results of the statistical interpretation are given in Table 6

Table 6 ndash Statistical interpretation results

Category n R Regression

used a t(a) b t(b) Critical T

P

Ordinate

at 0

Slope

at 1

Meat products 10 158 GMFR -0455 3321 115 3123 2306 001 001

Dairy products 12 065 GMFR -0027 0385 1002 0108 2228 092 071

Fishery products 10 130 GMFR -0305 1024 1034 0312 2306 076 034

Vegetables 12 086 GMFR -0015 0128 0993 0191 2228 085 090

Egg products 11 158 GMFR -0038 0866 1001 0090 2262 093 041

Environmental

samples 10 176 GMFR -0139 265 1034 231 2306 005 003

All products 65 118 GMFR -0182 3042 1040 2145 2000 004 000

All products

(estimated number) 65 118 GMFR -0182 3042 1040 2145 2000 004 000

All products (non

estimated results) 20 115 GMFR -0367 151 1138 1141 2101 027 015

Repeatability limits obtained for the alternative and reference methods as

well as bias obtained are presented in Table 7

Dairy products Environmental samples

Log (Ref) Log (Ref)

All categories

Thermo Fisher Scientific

ADRIA Deacuteveloppement 1338 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Table 7

Categories Bias

Repeatability limits

Rob r

Ref Alt

Meat products 0008 0314 0343

Dairy products -0031 0136 0130

Fishery products -0133 0220 0214

Vegetables -0041 0180 0319

Egg products -0026 0219 0206

Environmental samples -0007 0198 0119

All products -0032 0196 0215

Regression straight lines (graph and equation representations) for each food

category and for all products are presented figure 4

Thermo Fisher Scientific

ADRIA Deacuteveloppement 1438 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Figure 4 ndash Regression straight lines

Meat products

Fishery products

Dairy products

Vegetables

Thermo Fisher Scientific

ADRIA Deacuteveloppement 1538 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Environmental samples

Egg products

All products (estimated number)

Thermo Fisher Scientific

ADRIA Deacuteveloppement 1638 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

The hypotheses that the ordinates are close to 0 and the slopes close to 1

are approved in all cases except for

- Meat products (slope P = 1 ordinate P = 1)

- Environmental samples (ordinate P = 3)

- All products (ordinate P = 0)

The bias between the two methods vary from ndash 0133 to 0008 log CFU For

all categories the bias is ndash 0032 log CFU The repeatability limits of the

alternative and the reference method are similar For all products the values

are respectively 0215 and 0196 log CFU For all products these

interpretations are provided taking into account data with low contamination

level or not (estimated colonies number)

For all food products the regression straight line is the following

log (Alt) = 1040 log Ref ndash 0182

The Listeria PrecisTM method shows satisfying relative accuracy

All products

All products (Level 2)

Thermo Fisher Scientific

ADRIA Deacuteveloppement 1738 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

2113 Detection limit (LOD) and quantification limit (LOQ)

The critical level is the defined as the smallest amount which can be detected (not null) but not

quantified as an exact value Below this value it cannot be sure that the true value is not null

The detection limit is defined as being higher than the critical level because it involves a power the

probability 1- which has to be well over 50 for example 95

The quantification limit is defined as the smallest amount of analyte (that is the lowest actual number of

organisms) which can be measured and quantified with defined precision and accuracy under the

experimental conditions by the method under validation

Protocol

The detection limit of the alternative method was realised with pure cultures

Three different inoculation levels were tested with six replicates per level ie

18 analyses were done by the alternative method

Listeria monocytogenes 18 was used for the detection limit of the alternative

method

Quantification limit was calculated for six independent blank samples

determinations

Results

These data are intrinsic to the method used and are presented in the

following tables

Table 8 ndash Inoculation 1 ml on 3 plates

Level (CFUml) Positive samples number Standard deviation Bias

0 06

05 26 1211 0

1 36 0816 05

5 66 2483 85

Thermo Fisher Scientific

ADRIA Deacuteveloppement 1838 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Table 9 ndash Inoculation 01 ml on 1 plate

Level (CFUml) Positive samples number Standard deviation Bias

0 06

5 36 0548 05

10 46 1414 2

50 66 2258 55

Table 10

Formulas Obtained values

LC 165 S0 + X0 18

LOD 33 S0 + X0 32

LOQ 10 S0 + X0 87

2114 Relative Sensitivity

The relative sensitivity is defined as the ability of the alternative method to detect two different amounts

of analyte measured by the reference method within a given matrix at a specified average value or

over the whole measurement range that is it is the minimal quantity variation (increase of the analyte

concentration x) which gives a significant variation of the measured signal (response y)

These data are intrinsic to the used method and are obtained from the results

of the linearity study

Sensitivity patterns obtained for tested (matrixstrain) pairs are presented in

figure 5

Thermo Fisher Scientific

ADRIA Deacuteveloppement 1938 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Figure 5 ndash Sensitivity patterns for the tested (matrixstrain) pairs

2115 SpecificitySelectivity

The specificity is defined as the degree to which a method is affected (or not) by the other components

present in a multi-component sample That is the ability of a method to measure exactly a given

analyte or its amount within the sample without interference from non-target components such as a

matrix effect or background noise

The selectivity is defined as a measure of the degree of non-interference in the presence of non-target

analytes A method is selective if it can be used to detect the analyte under examination and that a

guarantee can be provided that the detected signal can only be a product by that specific analyte

The specificity and selectivity study was performed for the Listeria PrecisTM

detection method no additional test was realised for the enumeration

method The results were the following

- Among the Listeria monocytogenes strains tested 2 strains (Listeria

monocytogenes CIP 7834 and Listeria monocytogenes CIP 105459) gave

negative results These 2 strains were tested twice by running 48 h

incubation time of the compared agars Positive results were then

observed by the alternative method Pale characteristic colonies were

observed on the ISO agar by streaking the Half Fraser broth only

- No cross reaction was observed with the non-target strains except with

Listeria ivanovii which gave characteristic colonies on Brilliancetrade Listeria

Agar but the confirmatory tests provide a clear discrimination between

Listeria monocytogenes and Listeria ivanovi species

Rillettes

Lettuce

Raw milk

Smoked salmon

Raw egg

Process water

x (CFUg)

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2038 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

212 Practicability

1 Packaging Ready to use Brilliance Listeria agar plates are available

(x 10 plates)

2 Reagent volume

3 Storage conditionsndash

Shelf life of products

not opened

The plates are stored at 6 - 12degC in darkness conditions The

expiration date is mentioned on the package and on the plates It

is around 3 months

4 Modalities of use

after first use

5 Required

equipments No specific equipment is required

6 Reagents Ready to use plates

7 Training 05 day

8 Real time handling

Reference method

9 min for one sample

55 min for 10 samples

Alternative method

7 min for one sample

35 minutes for 10 samples

9 Time to result

Negative results

Reference method Day 2

Alternative method Day 2

Negative results after confirmation or positive results

Reference method Day 4 ndash Day 7

Alternative method Day 4 ndash Day 7

Positive results

Reference method Day 4 ndash Day 7

Alternative method Day 2 ndash Day 3 (with OBIS Mono Test)

or Day 4 ndash Day 7 (by the classical tests described in the

standardised method)

10 Technician skill Identical to that required for the reference method

11 Steps common to

the reference method Mother suspension revivification step and decimal dilutions

12 Traceability

13 Maintenance

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2138 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

213 Inter-laboratory study

2131 Organisation

12 laboratories participated to this study Pasteurised milk was inoculated by

Listeria monocytogenes 12a isolated from raw milk The targeted

inoculation levels were

- 0 CFUml

- 50 CFU ml

- 500 CFU ml

- 5 000 CFU ml

Each laboratory received eight flasks of 10 ml sample ie two flasks per

inoculation level Furthermore one non-inoculated sample has been added

to the package for total viable count microflora (NF ISO 4833 method)

Detailed instructions were transmitted to the collaborators by the expert

laboratory

Collaborative study laboratories and the expert laboratory carried out the

analyses with the alternative and reference methods

2132 Verification of experimental parameters

Strain stability during transport

In order to evaluate the Listeria monocytogenes strain stability during

transport bacterial count of all inoculated flasks were checked at different

time ie inoculation time after 24 h of conservation at 2deg - 8degC Results are

reported in table 11

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2238 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Table 11 ndash Listeria monocytogenes count

by ISO 11290-2A1 method (log CFUml)

Day of the analyses Level contamination Reference method

Day 0

(29052006)

1 45

2 440

3 4 500

Day 1

(30052006)

1 60

2 430

3 4 900

No evolution of the strain was observed after storage for 24 h at 2degC ndash 8degC

Contamination level

The real contamination levels are provided in table 12

Table 12 ndash Real contamination level (CFUml)

Target inoculation

(CFU ml) Samples Ndeg

Real contamination levels

(CFUml)

0 1 - 8 0

50 5 - 6 45

500 3 - 7 440

5000 2 - 4 4 500

Sample temperature at receipt

Measured temperatures at receipt are listed in table 13

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2338 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Table 13 - Sample temperature on receipt

Laboratories Receipt

day

Hour

receipt

Measured temperature

at receipt (degC)

Measured temperature by the

temperature probe (degC)

A

B

C

D

E

F

G

H

I

J

K

L

M

30052006

10h45

10h15

11h15

10h00

09h30

8h30

11h50

10h00

08h30

14h45

08h30

09h45

08h45

82

55

45

63

60

62

81

38

48

-

14

67

76

03

03

-13

03

04

-14

05

04

01

05

-13

04

Conclusion

The temperatures during the transport were correct for all the labs except for

Lab J which didnrsquot provide the information

All the labs received their packages with several broken flasks

Three labs (B D and L) received some damaged samples but proceeded to

analyses

All the labs started the analyses at Day 1 except Lab J

2133 Results

Aerobic mesophilic flora

The count of aerobic mesophilic flora varied from 94 102 CFUml to

33 103 CFUml

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2438 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Expert lab results

The results obtained by the expert lab are the following

Table 14

Target inoculation level (CFUml)

Reference method ISO 11290-2

Alternative method Listeria Precis Enumeration

Rep 1 Rep 2 Rep 1 Rep 2

lt 10 lt 10 lt 10 lt 10 lt10

50 50 (Ne) 60 (Ne) 50 (Ne) 30 (Ne)

500 470 390 490 500

5 000 4 700 5 100 5 900 3 400

Ne estimated number

The obtained results are in agreement with those expected

Collaborator Labs results

A synthesis of the results obtained by the collaborator labs is provided

Table 15 (CFUml) and Table 16 (log CFUml)

Table 15 ndash Synthesis of results obtained by the alternative

and the reference methods (CFUml)

Labs Level 1 Level 2 Level 2 bis Level 3

Reference method

Alternative method

Reference method

Alternative method

Reference method

Alternative method

Reference method

Alternative method

A 40 40 70 40 480 410 210 340 1000 300 400 300 4900 4600 4900 5100

B 40 15 40 20 350 300 360 450 350 650 800 600 4100 4700 5100 3700

C 50 20 20 70 440 430 480 450 300 650 300 500 4600 5400 4600 5000

D 40 60 40 40 490 460 600 540 250 450 600 1000 4900 4900 4400 4600

E 50 40 50 20 490 520 530 560 750 550 400 200 5400 4500 4100 5400

F 60 20 60 50 370 250 300 130 300 350 600 300 4400 4700 4900 5900

G 70 35 120 50 440 390 570 370 450 550 700 700 5000 4500 4000 4400

H 110 40 100 90 520 440 450 540 150 650 300 300 4200 4700 5600 4800

L 35 60 20 40 390 390 170 380 650 350 800 200 5300 4600 5600 5900

M 45 45 30 40 450 490 550 410 400 550 800 180 4900 4900 6400 4800

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2538 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Table 16 ndash Synthesis of results obtained by the alternative

and the reference methods (log CFUml)

Labs Level 1 Level 2 Level 2 bis Level 3

Reference method

Alternative method

Reference method

Alternative method

Reference method

Alternative method

Reference method

Alternative method

A 1602 1602 1845 1602 2681 2613 2322 2531 3000 2477 2602 2477 3690 3663 3690 3708

B 1602 1176 1602 1301 2544 2477 2556 2653 2544 2813 2903 2778 3613 3672 3708 3568

C 1699 1301 1301 1845 2643 2633 2681 2653 2477 2813 2477 2699 3663 3732 3663 3699

D 1602 1778 1602 1602 2690 2663 2778 2732 2398 2653 2778 3000 3690 3690 3643 3663

E 1699 1602 1699 1301 2690 2716 2724 2748 2875 2740 2602 2301 3732 3653 3613 3732

F 1778 1301 1778 1699 2568 2398 2477 2114 2477 2544 2778 2477 3643 3672 3690 3771

G 1845 1544 2079 1699 2643 2591 2756 2568 2653 2740 2845 2845 3699 3653 3602 3643

H 2041 1602 2000 1954 2716 2643 2653 2732 2176 2813 2477 2477 3623 3672 3748 3681

L 1544 1778 1301 1602 2591 2591 2230 2580 2813 2544 2903 2301 3724 3663 3748 3771

M 1653 1653 1477 1602 2653 2690 2740 2613 2602 2740 2903 2255 3690 3690 3806 3681

Level 2 results obtained taking account the 3 plates inoculated with the mother-suspension

Level 2 bis results obtained taking account the plates inoculated with 01 ml of the mother-suspension

Scrutiny of the measurement results for consistency

In order to identify the measurement results or laboratories that could be

inconsistent two graphical consistency techniques were realised Mandelrsquos

h-ank-statistics Mandel indicators h and k at 5 significance highlighted

some possible inconsistent data (See tables and graphics in Appendix 3 and

Appendix 4)

Mandelrsquos values Number of values observed over the threshold

Reference method Alternative method

h gt 1 Lab F Level 2 Lab F Level 2

h gt 5 Lab F Level 2

Lab B Levels 1 and 2

Lab A Level 2

Lab D Level 3

Lab F Level 2

Lab L Level 2

kgt 1 Lab F Level 3

k gt 5 Lab F Level 2 Lab C Level 2

Lab F Level 3

Lab L Levels 1 and 3

Lab M Level 1

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2638 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

The emphasized results appear (i) coherent with the targeted contamination

levels (ii) concordant between both methods

All the results were thus kept for the statistical analysis

Comparison of the trueness and precision characteristics of

the reference method and alternative methods

The statistical values are summarised hereafter

Level p

Reference method Alternative method

Repeatability ratio

Reproducibility ratio Median

Repeatability standard deviation

Reproducibility standard deviation

Median Repeatability

standard deviation

Reproducibility standard deviation

1 10 16519 02415 02425 16021 02045 02433 0847 1007

2 10 26551 00543 00659 26719 00914 01325 1682 2011

2 bis 10 26581 02607 02607 25951 01900 01953 0729 0749

3 10 18671 00378 00378 18671 00478 00611 1264 1617

Bias of the alternative method

In order to estimate the bias of the alternative method with regard to the reference method for every

level Dij and t are calculated according to the following equations

RefAlt ijYijYDij

Diffp

Dijit

)2(

)(median

If the t value is superior to 2 the alternative method is significantly biased

with regard to the reference method

The values are given in table 17

Table 17 ndash t (d) values obtained by level

Level Bias t (d) Conclusion

1 0068 114 Non significant bias

2 0021 082 Non significant bias

2 bis - 0037 035 Non significant bias

3 - 0037 043 Non significant bias

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2738 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

The statistical test concludes to a non significant bias for all the inoculation

levels The bias varies from ndash 0037 to 0068 log CFUml

Comparison of the repeatability standard deviations

If the ratio Srj Alt Srj Ref of the repeatability standard deviations of the

alternative method and the reference method is larger than 2 the precision

under repeatability conditions of the alternative method is considered to be

lower than that of the reference method If this ratio is smaller than 05 the

precision under repeatability conditions of the alternative method is

considered to be greater than that of the reference method

The ratio values are given in table 18

Table 18

Level

Repeatability standard deviation

Reference method Alternative method Alternative method

reference method ratio

1 0242 0205 0847

2 0054 0091 1682

2 bis 0261 0190 0729

3 0038 0048 1264

Level Repeatability limit

Reference method Alternative method

1 0676 0573

2 0152 0256

2 bis 0730 0532

3 0106 0134

The ratios of the repeatability standard deviations are below 2 in all cases

The precision under repeatability conditions of the alternative method is

equivalent to that of the reference method

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2838 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Comparison of the reproducibility standard deviations

If the ratio Srj Alt Srj Ref of the reproducibility standard deviations of the alternative method and the

reference method is larger than 2 the precision under reproducibility conditions of the alternative

method is considered to be lower than that of the reference method If this ratio is smaller than 05 the

precision under reproducibility conditions of the alternative method is considered to be greater than

that of the reference method

The ratio values are given in table 19

Table 19

Level

Reproducibility standard deviation

Reference method Alternative method Alternative method

reference method ratio

1 0242 0243 1007

2 0066 0133 2011

2 bis 0261 0195 0749

3 0038 0061 1617

Level Reproducibility limit

Reference method Alternative method

1 0676 0681

2 0185 0371

2 bis 0730 0547

3 0106 0171

The ratios of the reproducibility standard deviations are below or close to 2

The precision under reproducibility conditions of the alternative method is

equivalent to that of the reference method

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2938 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

214 Conclusion

Method comparison study

The Listeria PrecisTM enumeration method shows satisfying linearity and

relative accuracy

The alternative method shows satisfying inclusivity and exclusivity results

Two Listeria monocytogenes strains gave negative results with the

alternative method These strains grew weakly on the reference method

media (OampA Palcam and Oxford)

The limits of repeatability of the alternative method are similar to those of the

reference method

The bias between both methods are low between - 0133 Log CFUg and

- 0008 Log CFUg

The Listeria PrecisTM enumeration method offers the saving for confirmatory

tests

Positive results are obtained with the alternative method in 2-3 days (with

OBIS Mono test) or in 4-7 days (by the classical tests described in the

standardized method) against 4-7 days for the reference method

Inter-laboratory study

The bias between the reference and the alternative methods shows satisfying

and correct values

- level 1 (100 - 1 000 CFUg) bias = + 007 log CFUg

- level 2 (1 000 - 10 000 CFUg) bias = 002 log CFUg

- level 2 bis (1 000 - 10 000 CFUg) bias = - 004 log CFUg

- level 3 (10 000 - 100 000 CFUg) bias = - 004 log CFUg

The repeatability and the reproducibility of the alternative method look similar

to those of the reference method

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3038 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

22 Extension study (realised by ASEPT 2007)

An extension study was performed in 2007 for a new confirmatory test OBIS

MONO

150 Listeria monocytogenes strains and 100 non-target strains were isolated

onto TSAYE and BrillianceTM Listeria Agar plates All the Listeria

monocytogenes and the non-target strains gave the expected results

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3138 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Appendix 1 ndash Flow diagram of the Listeria PrecisTM method

x g sample + 9 x ml buffered peptone water

Revivification step for 1 h plusmn 5 min at 20degC plusmn 1degC

Streaking 1 ml onto 3 Brilliance Listeria Agar plates (mother suspension)

or 01 ml onto 1 Brilliance Listeria Agar plates (mother suspension and decimal dilutions)

Incubation for 48 h plusmn 3 h at 37degC plusmn 1degC

Confirmatory tests on at least one typical colony

- by the tests described in the ISO method

- by applying the OBIS mono test on an isolated colony

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3238 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Appendix 2 ndash ISO 11290-2 (1998) amp ISO 11290-2A1 (2004) Horizontal

method for the detection and enumeration of Listeria monocytogenes -

Part 2 enumeration of Listeria monocytogenes in foods

10 g cheese + 90 ml buffered peptone water or Half Fraser Broth (without supplements) (110 dilution)

Stomach 1 min 30 seconds

Keep at 20degC plusmn 2degC for 1 h plusmn 5 min

Decimal dilutions in 9 ml peptone salt tubes

Dilution 110

Dilution 1100

Dilution 11000

1 ml on 3 Chromogenic Listeria medium (ISO) plates (x 2) (-1)

01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-2)

01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-3)

01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-4)

Incubation at 37degC plusmn 1degC

24 h - 48 h

Numerate typical colonies at 24 h

(bluegreen surrounded by an opaque halo)

Numerate at 48 h

Streak typical colonies on TSYEA

(5 coloniesplate 2 successive dilutions)

Confirmation tests

Catalase

Gram stain

For confirmation of L monocytogenes

Haemolysis

Carbohydrate (rhamnose and xylose) utilisation

CAMP Test

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3338 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Appendix 3 ndash Mandelrsquos graphics

Laboratoire h Ref

Niveau 1 h Ref

Niveau 2 h Ref

Niveau 3 h Alt

Niveau 1 h Alt

Niveau 2 h Alt

Niveau 3 h5 h1 h5 h1

A -050 -015 083 062 -212 -039 198 299 -198 -299

B -261 -270 021 -077 -058 173 198 299 -198 -299

C -151 -031 -014 -015 -004 -005 198 299 -198 -299

D 038 040 -137 000 072 207 198 299 -198 -299

E -001 090 155 -052 056 -101 198 299 -198 -299

F -112 -321 -153 070 -325 023 198 299 -198 -299

G 042 -071 040 147 -009 176 198 299 -198 -299

H 169 046 -169 192 018 -083 198 299 -198 -299

L 009 -120 021 -077 -231 005 198 299 -198 -299

M 001 031 014 -032 004 -011 198 299 -198 -299

-300

-200

-100

000

100

200

300

A B C D E F G H L M

Vale

urs

h d

e M

an

del(

rob

uste

s)

Laboratoires

Meacutethode de reacutefeacuterence analyse robuste

h Ref Niveau 1

h Ref Niveau 2

h Ref Niveau 3

h 5=198

h 1=299

-300

-200

-100

000

100

200

300

A B C D E F G H L M

Vale

urs

de h

de M

an

del(

rob

uste

s)

Laboratoires

Meacutethode alternative analyse robuste

h Alt Niveau 1

h Alt Niveau 2

h Alt Niveau 2

h 1=299

h 5=198

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3438 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Laboratoire k Ref

Niveau 1 k Ref

Niveau 2 k Ref

Niveau 3 k Alt

Niveau 1 k Alt

Niveau 2 k Alt

Niveau 3 k1 k5

A 000 089 142 084 162 046 259 181

B 125 087 073 104 075 046 259 181

C 117 013 091 188 022 083 259 181

D 052 036 069 000 035 083 259 181

E 028 034 037 138 019 112 259 181

F 140 222 018 027 281 112 259 181

G 088 068 024 131 145 000 259 181

H 129 094 173 016 061 000 259 181

L 069 000 073 104 270 224 259 181

M 000 048 038 043 099 241 259 181

000

050

100

150

200

250

300

350

400

A B C D E F G H L M

Vale

urs

k d

e M

an

del(

rob

uste

s)

Laboratoires

Meacutethode de reacutefeacuterence analyse robuste

k Ref Niveau 1k Ref Niveau 2k Ref Niveau 3

k 1=259

k 5=181

000

050

100

150

200

250

300

A B C D E F G H L M

Vale

urs

k d

e M

an

del

(ro

bu

ste

s)

Laboratoires

Meacutethode alternative analyse robuste

k Alt Niveau 3

k Alt Niveau 1

k Alt Niveau 2

k 1=259

k 5=181

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3538 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Appendix 4 ndash Statistical results

Level 1

Nombre de laboratoires (p) 10

A 1602 1602 1602 000 0000 0000 -0495 0000 A 1845 1602 1724 017 0122 -0122 0621 0840 0122

B 1602 1176 1389 030 0213 -0213 -2613 1247 B 1602 1301 1452 021 0151 -0151 -0769 1041 0062

C 1699 1301 1500 028 0199 -0199 -1510 1165 C 1301 1845 1573 038 -0272 0272 -0148 1881 0073

D 1602 1778 1690 012 -0088 0088 0380 0516 D 1602 1602 1602 000 0000 0000 0000 0000 -0088

E 1699 1602 1651 007 0048 -0048 -0013 0284 E 1699 1301 1500 028 0199 -0199 -0522 1376 -0151

F 1778 1301 1540 034 0239 -0239 -1116 1397 F 1778 1699 1739 006 0040 -0040 0698 0274 0199

G 1845 1544 1695 021 0151 -0151 0425 0881 G 2079 1699 1889 027 0190 -0190 1467 1315 0194

H 2041 1602 1822 031 0220 -0220 1689 1286 H 2000 1954 1977 003 0023 -0023 1917 0158 0155

L 1544 1778 1661 017 -0117 0117 0092 0685 L 1301 1602 1452 021 -0151 0151 -0769 1041 -0210

M 1653 1653 1653 000 0000 0000 0013 0000 M 1477 1602 1540 009 -0062 0062 -0319 0432 -0114

m 165186 m 160206 mediane D 00678

Cn(2p) 1867 Cn(2p) 1867 Cp 16101

Cn(p) 16101 Cn(p) 16101 Qn(D) 0092926

n=2p n 20 n=2p n 20 Qdiff 01496169

f 11 f 11 t 114

l 55 l 55

n=p n 10 n=p n 10

f 6 f 6

l 15 l 15

Qn(2p) 0091465 Qn(2p) 007745

Qn(p) 0062469 Qn(p) 0121519

Qintra 017078 Qintra 014461

Qinter 010058 Qinter 019565

Sr(eacutecart-type de reacutepeacutetabiliteacute) 02415 Sr 02045

RSDr(coefficient de variation de la reacutepeacutetabiliteacute)1462 RSDr 1277

r(repeatability limit)) 0676 r 0573

SL(eacutecart-type interlaboratoire) -001905 SLlt0 SL 001737

SR(eacutecart-type de reproductibiliteacute) 02415 SR 02433

RSDR(coefficient de variation de la reproductibiliteacute)0146 RSDR 0152

R(limite de reproductibiliteacute)) 06762 R(limite de reproductibiliteacute))068123

h k h MoyenneLaboratoires

Niveau 1

si LaboratoiresDeviationMoyenne Dk DeviationsiAlt

Niveau 1

Ref

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3638 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Level 2

Nombre de laboratoires (p) 10

A 2681 2613 2647 005 0034 -0034 -0151 0891 A 2322 2531 2427 015 -0105 0105 -2119 1620 -0220

B 2544 2477 2511 005 0033 -0033 -2699 0872 B 2556 2653 2605 007 -0048 0048 -0581 0750 0094

C 2643 2633 2638 001 0005 -0005 -0311 0130 C 2681 2653 2667 002 0014 -0014 -0040 0217 0029

D 2690 2663 2676 002 0014 -0014 0400 0357 D 2778 2732 2755 003 0023 -0023 0721 0354 0079

E 2690 2716 2703 002 -0013 0013 0897 0336 E 2724 2748 2736 002 -0012 0012 0556 0185 0033

F 2568 2398 2483 012 0085 -0085 -3214 2217 F 2477 2114 2296 026 0182 -0182 -3255 2811 -0188

G 2643 2591 2617 004 0026 -0026 -0707 0682 G 2756 2568 2662 013 0094 -0094 -0085 1453 0045

H 2716 2643 2680 005 0036 -0036 0460 0945 H 2653 2732 2693 006 -0040 0040 0181 0613 0013

L 2591 2591 2591 000 0000 0000 -1196 0000 L 2230 2580 2405 025 -0175 0175 -2307 2704 -0186

M 2653 2690 2672 003 -0018 0018 0311 0481 M 2740 2613 2677 009 0064 -0064 0040 0987 0005

m 2655082 m 26719001 mediane D 00209

Cn(2p) 1867 Cn(2p) 1867 Cp 16101

Cn(p) 16101 Cn(p) 16101 Qn(D) 0039911

n=2p n 20 n=2p n 20 Qdiff 00642591

f 11 f 11 t 082

l 55 l 55

n=p n 10 n=p n 10

f 6 f 6

l 15 l 15

Qn(2p) 0020570 Qn(2p) 00345985

Qn(p) 00332437 Qn(p) 00718158

Qintra 003841 Qintra 006460

Qinter 005352 Qinter 011563

Sr(eacutecart-type de reacutepeacutetabiliteacute) 00543 Sr 00914

RSDr(coefficient de variation de la reacutepeacutetabiliteacute)205 RSDr 342

r(repeatability limit)) 0152 r 0256

SL(eacutecart-type interlaboratoire) 000139 SL 000920

SR(eacutecart-type de reproductibiliteacute) 00659 SR 01325

RSDR(coefficient de variation de la reproductibiliteacute)0025 RSDR 0050

R(limite de reproductibiliteacute)) 018446 R(limite de reproductibiliteacute))03708618

Laboratoires

Niveau 2

Moyenne si Laboratoires

Niveau 2

DRef Alt Moyenne si Deviation h Deviation h k k

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3738 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Level 2 bis

Nombre de laboratoires (p) 10

A 3000 2477 2739 037 0261 -0261 0832 1418 A 2602 2477 2540 009 0062 -0062 -0391 0465 -0199

B 2544 2813 2678 019 -0134 0134 0211 0729 B 2903 2778 2841 009 0062 -0062 1732 0465 0162

C 2477 2813 2645 024 -0168 0168 -0135 0911 C 2477 2699 2588 016 -0111 0111 -0049 0826 -0057

D 2398 2653 2526 018 -0128 0128 -1370 0692 D 2778 3000 2889 016 -0111 0111 2074 0826 0363

E 2875 2740 2808 010 0067 -0067 1547 0365 E 2602 2301 2452 021 0151 -0151 -1012 1120 -0356

F 2477 2544 2511 005 -0033 0033 -1525 0182 F 2778 2477 2628 021 0151 -0151 0230 1120 0117

G 2653 2740 2697 006 -0044 0044 0400 0236 G 2845 2845 2845 000 0000 0000 1764 0000 0148

H 2176 2813 2495 045 -0318 0318 -1692 1727 H 2477 2477 2477 000 0000 0000 -0832 0000 -0017

L 2813 2544 2678 019 0134 -0134 0211 0729 L 2903 2301 2602 043 0301 -0301 0049 2241 -0076

M 2602 2740 2671 010 -0069 0069 0135 0375 M 2903 2255 2579 046 0324 -0324 -0112 2411 -0092

m 2658114 m 259505 mediane D -00372

Cn(2p) 1867 Cn(2p) 1867 Cp 16101

Cn(p) 16101 Cn(p) 16101 Qn(D) 01656915

n=2p n 20 n=2p n 20 Qdiff 02667753

f 11 f 11 t 035

l 55 l 55

n=p n 10 n=p n 10

f 6 f 6

l 15 l 15

Qn(2p) 0098745 Qn(2p) 007195

Qn(p) 006007 Qn(p) 008805

Qintra 018437 Qintra 013435

Qinter 009672 Qinter 014176

Sr(eacutecart-type de reacutepeacutetabiliteacute) 02607 Sr 01900

RSDr(coefficient de variation de la reacutepeacutetabiliteacute)981 RSDr 732

r(repeatability limit)) 0730 r 0532

SL(eacutecart-type interlaboratoire) -002464 SLlt0 SL 000205

SR(eacutecart-type de reproductibiliteacute) 02607 SR 01953

RSDR(coefficient de variation de la reproductibiliteacute)0098 RSDR 0075

R(limite de reproductibiliteacute)) 072996 R(limite de reproductibiliteacute))054686

Laboratoires

Niveau 2 bis

Moyenne si Laboratoires

Niveau 2 bis

DRef Alt Moyenne si Deviation h Deviation h k k

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3838 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Level 3

Nombre de laboratoires (p) 10

A 3690 3663 3676 002 0014 -0014 -0311 0513 A 3690 3708 3699 001 -0009 0009 0354 0257 0022

B 3613 3672 3642 004 -0030 0030 -1851 1109 B 3708 3568 3638 010 0070 -0070 -0844 2061 -0005

C 3663 3732 3698 005 -0035 0035 0645 1302 C 3663 3699 3681 003 -0018 0018 0000 0536 -0017

D 3690 3690 3690 000 0000 0000 0311 0000 D 3643 3663 3653 001 -0010 0010 -0545 0286 -0037

E 3732 3653 3693 006 0040 -0040 0429 1480 E 3613 3732 3673 008 -0060 0060 -0162 1769 -0020

F 3643 3672 3658 002 -0014 0014 -1157 0536 F 3690 3771 3731 006 -0040 0040 0975 1193 0073

G 3699 3653 3676 003 0023 -0023 -0328 0856 G 3602 3643 3623 003 -0021 0021 -1141 0612 -0053

H 3623 3672 3648 003 -0024 0024 -1615 0913 H 3748 3681 3715 005 0033 -0033 0664 0990 0067

L 3724 3663 3694 004 0031 -0031 0461 1150 L 3748 3771 3760 002 -0011 0011 1544 0335 0066

M 3690 3690 3690 000 0000 0000 0311 0000 M 3806 3681 3744 009 0062 -0062 1234 1848 0054

m 368334 m 368086 mediane D 00089

Cn(2p) 1867 Cn(2p) 1867 Cp 16101

Cn(p) 16101 Cn(p) 16101 Qn(D) 00325359

n=2p n 20 n=2p n 20 Qdiff 00523852

f 11 f 11 t 043

l 55 l 55

n=p n 10 n=p n 10

f 6 f 6

l 15 l 15

Qn(2p) 0014323 Qn(2p) 001811

Qn(p) 0013719 Qn(p) 003164

Qintra 002674 Qintra 003381

Qinter 002209 Qinter 005094

Sr(eacutecart-type de reacutepeacutetabiliteacute) 00378 Sr 00478

RSDr(coefficient de variation de la reacutepeacutetabiliteacute)103 RSDr 130

r(repeatability limit)) 0106 r 0134

SL(eacutecart-type interlaboratoire) -000023 SLlt0 SL 000145

SR(eacutecart-type de reproductibiliteacute) 00378 SR 00611

RSDR(coefficient de variation de la reproductibiliteacute)0010 RSDR 0017

R(limite de reproductibiliteacute)) 0105894 R(limite de reproductibiliteacute))017119

Ref Alt DLaboratoires

Niveau 3

Moyenne si Deviation h k Laboratoires

Niveau 3

Moyenne si Deviation h k

Page 11: ÞÀ¿¸7 ú Y5zñLjÜ< /u +æü t¬Á ... - NF Validation · NF VALIDATION Validation of alternative analytical methods Application in food microbiology ... 500 – 1 000 1 000 –

Thermo Fisher Scientific

ADRIA Deacuteveloppement 1138 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Table 5 ndash Contamination range

Food category Contamination level (log CFUg)

Meat products 100 ndash 404

Dairy products 148 ndash 408

Fishery products 100 ndash 384

Vegetables 170 ndash 511

Egg products 130 ndash 499

Environmental samples 170 ndash 511

All categories 100 ndash 511

Bi-dimensional graphs are given figure 3

Figure 3 ndash Bi-dimensional graphs

Graphe bi-dimensionnel Produits carneacutes

Fishery products

Vegetables

Egg products

Meat products

Thermo Fisher Scientific

ADRIA Deacuteveloppement 1238 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Statistical interpretation according to EN ISO16140 method

The results of the statistical interpretation are given in Table 6

Table 6 ndash Statistical interpretation results

Category n R Regression

used a t(a) b t(b) Critical T

P

Ordinate

at 0

Slope

at 1

Meat products 10 158 GMFR -0455 3321 115 3123 2306 001 001

Dairy products 12 065 GMFR -0027 0385 1002 0108 2228 092 071

Fishery products 10 130 GMFR -0305 1024 1034 0312 2306 076 034

Vegetables 12 086 GMFR -0015 0128 0993 0191 2228 085 090

Egg products 11 158 GMFR -0038 0866 1001 0090 2262 093 041

Environmental

samples 10 176 GMFR -0139 265 1034 231 2306 005 003

All products 65 118 GMFR -0182 3042 1040 2145 2000 004 000

All products

(estimated number) 65 118 GMFR -0182 3042 1040 2145 2000 004 000

All products (non

estimated results) 20 115 GMFR -0367 151 1138 1141 2101 027 015

Repeatability limits obtained for the alternative and reference methods as

well as bias obtained are presented in Table 7

Dairy products Environmental samples

Log (Ref) Log (Ref)

All categories

Thermo Fisher Scientific

ADRIA Deacuteveloppement 1338 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Table 7

Categories Bias

Repeatability limits

Rob r

Ref Alt

Meat products 0008 0314 0343

Dairy products -0031 0136 0130

Fishery products -0133 0220 0214

Vegetables -0041 0180 0319

Egg products -0026 0219 0206

Environmental samples -0007 0198 0119

All products -0032 0196 0215

Regression straight lines (graph and equation representations) for each food

category and for all products are presented figure 4

Thermo Fisher Scientific

ADRIA Deacuteveloppement 1438 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Figure 4 ndash Regression straight lines

Meat products

Fishery products

Dairy products

Vegetables

Thermo Fisher Scientific

ADRIA Deacuteveloppement 1538 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Environmental samples

Egg products

All products (estimated number)

Thermo Fisher Scientific

ADRIA Deacuteveloppement 1638 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

The hypotheses that the ordinates are close to 0 and the slopes close to 1

are approved in all cases except for

- Meat products (slope P = 1 ordinate P = 1)

- Environmental samples (ordinate P = 3)

- All products (ordinate P = 0)

The bias between the two methods vary from ndash 0133 to 0008 log CFU For

all categories the bias is ndash 0032 log CFU The repeatability limits of the

alternative and the reference method are similar For all products the values

are respectively 0215 and 0196 log CFU For all products these

interpretations are provided taking into account data with low contamination

level or not (estimated colonies number)

For all food products the regression straight line is the following

log (Alt) = 1040 log Ref ndash 0182

The Listeria PrecisTM method shows satisfying relative accuracy

All products

All products (Level 2)

Thermo Fisher Scientific

ADRIA Deacuteveloppement 1738 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

2113 Detection limit (LOD) and quantification limit (LOQ)

The critical level is the defined as the smallest amount which can be detected (not null) but not

quantified as an exact value Below this value it cannot be sure that the true value is not null

The detection limit is defined as being higher than the critical level because it involves a power the

probability 1- which has to be well over 50 for example 95

The quantification limit is defined as the smallest amount of analyte (that is the lowest actual number of

organisms) which can be measured and quantified with defined precision and accuracy under the

experimental conditions by the method under validation

Protocol

The detection limit of the alternative method was realised with pure cultures

Three different inoculation levels were tested with six replicates per level ie

18 analyses were done by the alternative method

Listeria monocytogenes 18 was used for the detection limit of the alternative

method

Quantification limit was calculated for six independent blank samples

determinations

Results

These data are intrinsic to the method used and are presented in the

following tables

Table 8 ndash Inoculation 1 ml on 3 plates

Level (CFUml) Positive samples number Standard deviation Bias

0 06

05 26 1211 0

1 36 0816 05

5 66 2483 85

Thermo Fisher Scientific

ADRIA Deacuteveloppement 1838 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Table 9 ndash Inoculation 01 ml on 1 plate

Level (CFUml) Positive samples number Standard deviation Bias

0 06

5 36 0548 05

10 46 1414 2

50 66 2258 55

Table 10

Formulas Obtained values

LC 165 S0 + X0 18

LOD 33 S0 + X0 32

LOQ 10 S0 + X0 87

2114 Relative Sensitivity

The relative sensitivity is defined as the ability of the alternative method to detect two different amounts

of analyte measured by the reference method within a given matrix at a specified average value or

over the whole measurement range that is it is the minimal quantity variation (increase of the analyte

concentration x) which gives a significant variation of the measured signal (response y)

These data are intrinsic to the used method and are obtained from the results

of the linearity study

Sensitivity patterns obtained for tested (matrixstrain) pairs are presented in

figure 5

Thermo Fisher Scientific

ADRIA Deacuteveloppement 1938 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Figure 5 ndash Sensitivity patterns for the tested (matrixstrain) pairs

2115 SpecificitySelectivity

The specificity is defined as the degree to which a method is affected (or not) by the other components

present in a multi-component sample That is the ability of a method to measure exactly a given

analyte or its amount within the sample without interference from non-target components such as a

matrix effect or background noise

The selectivity is defined as a measure of the degree of non-interference in the presence of non-target

analytes A method is selective if it can be used to detect the analyte under examination and that a

guarantee can be provided that the detected signal can only be a product by that specific analyte

The specificity and selectivity study was performed for the Listeria PrecisTM

detection method no additional test was realised for the enumeration

method The results were the following

- Among the Listeria monocytogenes strains tested 2 strains (Listeria

monocytogenes CIP 7834 and Listeria monocytogenes CIP 105459) gave

negative results These 2 strains were tested twice by running 48 h

incubation time of the compared agars Positive results were then

observed by the alternative method Pale characteristic colonies were

observed on the ISO agar by streaking the Half Fraser broth only

- No cross reaction was observed with the non-target strains except with

Listeria ivanovii which gave characteristic colonies on Brilliancetrade Listeria

Agar but the confirmatory tests provide a clear discrimination between

Listeria monocytogenes and Listeria ivanovi species

Rillettes

Lettuce

Raw milk

Smoked salmon

Raw egg

Process water

x (CFUg)

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2038 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

212 Practicability

1 Packaging Ready to use Brilliance Listeria agar plates are available

(x 10 plates)

2 Reagent volume

3 Storage conditionsndash

Shelf life of products

not opened

The plates are stored at 6 - 12degC in darkness conditions The

expiration date is mentioned on the package and on the plates It

is around 3 months

4 Modalities of use

after first use

5 Required

equipments No specific equipment is required

6 Reagents Ready to use plates

7 Training 05 day

8 Real time handling

Reference method

9 min for one sample

55 min for 10 samples

Alternative method

7 min for one sample

35 minutes for 10 samples

9 Time to result

Negative results

Reference method Day 2

Alternative method Day 2

Negative results after confirmation or positive results

Reference method Day 4 ndash Day 7

Alternative method Day 4 ndash Day 7

Positive results

Reference method Day 4 ndash Day 7

Alternative method Day 2 ndash Day 3 (with OBIS Mono Test)

or Day 4 ndash Day 7 (by the classical tests described in the

standardised method)

10 Technician skill Identical to that required for the reference method

11 Steps common to

the reference method Mother suspension revivification step and decimal dilutions

12 Traceability

13 Maintenance

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2138 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

213 Inter-laboratory study

2131 Organisation

12 laboratories participated to this study Pasteurised milk was inoculated by

Listeria monocytogenes 12a isolated from raw milk The targeted

inoculation levels were

- 0 CFUml

- 50 CFU ml

- 500 CFU ml

- 5 000 CFU ml

Each laboratory received eight flasks of 10 ml sample ie two flasks per

inoculation level Furthermore one non-inoculated sample has been added

to the package for total viable count microflora (NF ISO 4833 method)

Detailed instructions were transmitted to the collaborators by the expert

laboratory

Collaborative study laboratories and the expert laboratory carried out the

analyses with the alternative and reference methods

2132 Verification of experimental parameters

Strain stability during transport

In order to evaluate the Listeria monocytogenes strain stability during

transport bacterial count of all inoculated flasks were checked at different

time ie inoculation time after 24 h of conservation at 2deg - 8degC Results are

reported in table 11

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2238 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Table 11 ndash Listeria monocytogenes count

by ISO 11290-2A1 method (log CFUml)

Day of the analyses Level contamination Reference method

Day 0

(29052006)

1 45

2 440

3 4 500

Day 1

(30052006)

1 60

2 430

3 4 900

No evolution of the strain was observed after storage for 24 h at 2degC ndash 8degC

Contamination level

The real contamination levels are provided in table 12

Table 12 ndash Real contamination level (CFUml)

Target inoculation

(CFU ml) Samples Ndeg

Real contamination levels

(CFUml)

0 1 - 8 0

50 5 - 6 45

500 3 - 7 440

5000 2 - 4 4 500

Sample temperature at receipt

Measured temperatures at receipt are listed in table 13

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2338 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Table 13 - Sample temperature on receipt

Laboratories Receipt

day

Hour

receipt

Measured temperature

at receipt (degC)

Measured temperature by the

temperature probe (degC)

A

B

C

D

E

F

G

H

I

J

K

L

M

30052006

10h45

10h15

11h15

10h00

09h30

8h30

11h50

10h00

08h30

14h45

08h30

09h45

08h45

82

55

45

63

60

62

81

38

48

-

14

67

76

03

03

-13

03

04

-14

05

04

01

05

-13

04

Conclusion

The temperatures during the transport were correct for all the labs except for

Lab J which didnrsquot provide the information

All the labs received their packages with several broken flasks

Three labs (B D and L) received some damaged samples but proceeded to

analyses

All the labs started the analyses at Day 1 except Lab J

2133 Results

Aerobic mesophilic flora

The count of aerobic mesophilic flora varied from 94 102 CFUml to

33 103 CFUml

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2438 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Expert lab results

The results obtained by the expert lab are the following

Table 14

Target inoculation level (CFUml)

Reference method ISO 11290-2

Alternative method Listeria Precis Enumeration

Rep 1 Rep 2 Rep 1 Rep 2

lt 10 lt 10 lt 10 lt 10 lt10

50 50 (Ne) 60 (Ne) 50 (Ne) 30 (Ne)

500 470 390 490 500

5 000 4 700 5 100 5 900 3 400

Ne estimated number

The obtained results are in agreement with those expected

Collaborator Labs results

A synthesis of the results obtained by the collaborator labs is provided

Table 15 (CFUml) and Table 16 (log CFUml)

Table 15 ndash Synthesis of results obtained by the alternative

and the reference methods (CFUml)

Labs Level 1 Level 2 Level 2 bis Level 3

Reference method

Alternative method

Reference method

Alternative method

Reference method

Alternative method

Reference method

Alternative method

A 40 40 70 40 480 410 210 340 1000 300 400 300 4900 4600 4900 5100

B 40 15 40 20 350 300 360 450 350 650 800 600 4100 4700 5100 3700

C 50 20 20 70 440 430 480 450 300 650 300 500 4600 5400 4600 5000

D 40 60 40 40 490 460 600 540 250 450 600 1000 4900 4900 4400 4600

E 50 40 50 20 490 520 530 560 750 550 400 200 5400 4500 4100 5400

F 60 20 60 50 370 250 300 130 300 350 600 300 4400 4700 4900 5900

G 70 35 120 50 440 390 570 370 450 550 700 700 5000 4500 4000 4400

H 110 40 100 90 520 440 450 540 150 650 300 300 4200 4700 5600 4800

L 35 60 20 40 390 390 170 380 650 350 800 200 5300 4600 5600 5900

M 45 45 30 40 450 490 550 410 400 550 800 180 4900 4900 6400 4800

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2538 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Table 16 ndash Synthesis of results obtained by the alternative

and the reference methods (log CFUml)

Labs Level 1 Level 2 Level 2 bis Level 3

Reference method

Alternative method

Reference method

Alternative method

Reference method

Alternative method

Reference method

Alternative method

A 1602 1602 1845 1602 2681 2613 2322 2531 3000 2477 2602 2477 3690 3663 3690 3708

B 1602 1176 1602 1301 2544 2477 2556 2653 2544 2813 2903 2778 3613 3672 3708 3568

C 1699 1301 1301 1845 2643 2633 2681 2653 2477 2813 2477 2699 3663 3732 3663 3699

D 1602 1778 1602 1602 2690 2663 2778 2732 2398 2653 2778 3000 3690 3690 3643 3663

E 1699 1602 1699 1301 2690 2716 2724 2748 2875 2740 2602 2301 3732 3653 3613 3732

F 1778 1301 1778 1699 2568 2398 2477 2114 2477 2544 2778 2477 3643 3672 3690 3771

G 1845 1544 2079 1699 2643 2591 2756 2568 2653 2740 2845 2845 3699 3653 3602 3643

H 2041 1602 2000 1954 2716 2643 2653 2732 2176 2813 2477 2477 3623 3672 3748 3681

L 1544 1778 1301 1602 2591 2591 2230 2580 2813 2544 2903 2301 3724 3663 3748 3771

M 1653 1653 1477 1602 2653 2690 2740 2613 2602 2740 2903 2255 3690 3690 3806 3681

Level 2 results obtained taking account the 3 plates inoculated with the mother-suspension

Level 2 bis results obtained taking account the plates inoculated with 01 ml of the mother-suspension

Scrutiny of the measurement results for consistency

In order to identify the measurement results or laboratories that could be

inconsistent two graphical consistency techniques were realised Mandelrsquos

h-ank-statistics Mandel indicators h and k at 5 significance highlighted

some possible inconsistent data (See tables and graphics in Appendix 3 and

Appendix 4)

Mandelrsquos values Number of values observed over the threshold

Reference method Alternative method

h gt 1 Lab F Level 2 Lab F Level 2

h gt 5 Lab F Level 2

Lab B Levels 1 and 2

Lab A Level 2

Lab D Level 3

Lab F Level 2

Lab L Level 2

kgt 1 Lab F Level 3

k gt 5 Lab F Level 2 Lab C Level 2

Lab F Level 3

Lab L Levels 1 and 3

Lab M Level 1

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2638 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

The emphasized results appear (i) coherent with the targeted contamination

levels (ii) concordant between both methods

All the results were thus kept for the statistical analysis

Comparison of the trueness and precision characteristics of

the reference method and alternative methods

The statistical values are summarised hereafter

Level p

Reference method Alternative method

Repeatability ratio

Reproducibility ratio Median

Repeatability standard deviation

Reproducibility standard deviation

Median Repeatability

standard deviation

Reproducibility standard deviation

1 10 16519 02415 02425 16021 02045 02433 0847 1007

2 10 26551 00543 00659 26719 00914 01325 1682 2011

2 bis 10 26581 02607 02607 25951 01900 01953 0729 0749

3 10 18671 00378 00378 18671 00478 00611 1264 1617

Bias of the alternative method

In order to estimate the bias of the alternative method with regard to the reference method for every

level Dij and t are calculated according to the following equations

RefAlt ijYijYDij

Diffp

Dijit

)2(

)(median

If the t value is superior to 2 the alternative method is significantly biased

with regard to the reference method

The values are given in table 17

Table 17 ndash t (d) values obtained by level

Level Bias t (d) Conclusion

1 0068 114 Non significant bias

2 0021 082 Non significant bias

2 bis - 0037 035 Non significant bias

3 - 0037 043 Non significant bias

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2738 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

The statistical test concludes to a non significant bias for all the inoculation

levels The bias varies from ndash 0037 to 0068 log CFUml

Comparison of the repeatability standard deviations

If the ratio Srj Alt Srj Ref of the repeatability standard deviations of the

alternative method and the reference method is larger than 2 the precision

under repeatability conditions of the alternative method is considered to be

lower than that of the reference method If this ratio is smaller than 05 the

precision under repeatability conditions of the alternative method is

considered to be greater than that of the reference method

The ratio values are given in table 18

Table 18

Level

Repeatability standard deviation

Reference method Alternative method Alternative method

reference method ratio

1 0242 0205 0847

2 0054 0091 1682

2 bis 0261 0190 0729

3 0038 0048 1264

Level Repeatability limit

Reference method Alternative method

1 0676 0573

2 0152 0256

2 bis 0730 0532

3 0106 0134

The ratios of the repeatability standard deviations are below 2 in all cases

The precision under repeatability conditions of the alternative method is

equivalent to that of the reference method

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2838 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Comparison of the reproducibility standard deviations

If the ratio Srj Alt Srj Ref of the reproducibility standard deviations of the alternative method and the

reference method is larger than 2 the precision under reproducibility conditions of the alternative

method is considered to be lower than that of the reference method If this ratio is smaller than 05 the

precision under reproducibility conditions of the alternative method is considered to be greater than

that of the reference method

The ratio values are given in table 19

Table 19

Level

Reproducibility standard deviation

Reference method Alternative method Alternative method

reference method ratio

1 0242 0243 1007

2 0066 0133 2011

2 bis 0261 0195 0749

3 0038 0061 1617

Level Reproducibility limit

Reference method Alternative method

1 0676 0681

2 0185 0371

2 bis 0730 0547

3 0106 0171

The ratios of the reproducibility standard deviations are below or close to 2

The precision under reproducibility conditions of the alternative method is

equivalent to that of the reference method

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2938 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

214 Conclusion

Method comparison study

The Listeria PrecisTM enumeration method shows satisfying linearity and

relative accuracy

The alternative method shows satisfying inclusivity and exclusivity results

Two Listeria monocytogenes strains gave negative results with the

alternative method These strains grew weakly on the reference method

media (OampA Palcam and Oxford)

The limits of repeatability of the alternative method are similar to those of the

reference method

The bias between both methods are low between - 0133 Log CFUg and

- 0008 Log CFUg

The Listeria PrecisTM enumeration method offers the saving for confirmatory

tests

Positive results are obtained with the alternative method in 2-3 days (with

OBIS Mono test) or in 4-7 days (by the classical tests described in the

standardized method) against 4-7 days for the reference method

Inter-laboratory study

The bias between the reference and the alternative methods shows satisfying

and correct values

- level 1 (100 - 1 000 CFUg) bias = + 007 log CFUg

- level 2 (1 000 - 10 000 CFUg) bias = 002 log CFUg

- level 2 bis (1 000 - 10 000 CFUg) bias = - 004 log CFUg

- level 3 (10 000 - 100 000 CFUg) bias = - 004 log CFUg

The repeatability and the reproducibility of the alternative method look similar

to those of the reference method

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3038 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

22 Extension study (realised by ASEPT 2007)

An extension study was performed in 2007 for a new confirmatory test OBIS

MONO

150 Listeria monocytogenes strains and 100 non-target strains were isolated

onto TSAYE and BrillianceTM Listeria Agar plates All the Listeria

monocytogenes and the non-target strains gave the expected results

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3138 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Appendix 1 ndash Flow diagram of the Listeria PrecisTM method

x g sample + 9 x ml buffered peptone water

Revivification step for 1 h plusmn 5 min at 20degC plusmn 1degC

Streaking 1 ml onto 3 Brilliance Listeria Agar plates (mother suspension)

or 01 ml onto 1 Brilliance Listeria Agar plates (mother suspension and decimal dilutions)

Incubation for 48 h plusmn 3 h at 37degC plusmn 1degC

Confirmatory tests on at least one typical colony

- by the tests described in the ISO method

- by applying the OBIS mono test on an isolated colony

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3238 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Appendix 2 ndash ISO 11290-2 (1998) amp ISO 11290-2A1 (2004) Horizontal

method for the detection and enumeration of Listeria monocytogenes -

Part 2 enumeration of Listeria monocytogenes in foods

10 g cheese + 90 ml buffered peptone water or Half Fraser Broth (without supplements) (110 dilution)

Stomach 1 min 30 seconds

Keep at 20degC plusmn 2degC for 1 h plusmn 5 min

Decimal dilutions in 9 ml peptone salt tubes

Dilution 110

Dilution 1100

Dilution 11000

1 ml on 3 Chromogenic Listeria medium (ISO) plates (x 2) (-1)

01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-2)

01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-3)

01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-4)

Incubation at 37degC plusmn 1degC

24 h - 48 h

Numerate typical colonies at 24 h

(bluegreen surrounded by an opaque halo)

Numerate at 48 h

Streak typical colonies on TSYEA

(5 coloniesplate 2 successive dilutions)

Confirmation tests

Catalase

Gram stain

For confirmation of L monocytogenes

Haemolysis

Carbohydrate (rhamnose and xylose) utilisation

CAMP Test

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3338 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Appendix 3 ndash Mandelrsquos graphics

Laboratoire h Ref

Niveau 1 h Ref

Niveau 2 h Ref

Niveau 3 h Alt

Niveau 1 h Alt

Niveau 2 h Alt

Niveau 3 h5 h1 h5 h1

A -050 -015 083 062 -212 -039 198 299 -198 -299

B -261 -270 021 -077 -058 173 198 299 -198 -299

C -151 -031 -014 -015 -004 -005 198 299 -198 -299

D 038 040 -137 000 072 207 198 299 -198 -299

E -001 090 155 -052 056 -101 198 299 -198 -299

F -112 -321 -153 070 -325 023 198 299 -198 -299

G 042 -071 040 147 -009 176 198 299 -198 -299

H 169 046 -169 192 018 -083 198 299 -198 -299

L 009 -120 021 -077 -231 005 198 299 -198 -299

M 001 031 014 -032 004 -011 198 299 -198 -299

-300

-200

-100

000

100

200

300

A B C D E F G H L M

Vale

urs

h d

e M

an

del(

rob

uste

s)

Laboratoires

Meacutethode de reacutefeacuterence analyse robuste

h Ref Niveau 1

h Ref Niveau 2

h Ref Niveau 3

h 5=198

h 1=299

-300

-200

-100

000

100

200

300

A B C D E F G H L M

Vale

urs

de h

de M

an

del(

rob

uste

s)

Laboratoires

Meacutethode alternative analyse robuste

h Alt Niveau 1

h Alt Niveau 2

h Alt Niveau 2

h 1=299

h 5=198

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3438 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Laboratoire k Ref

Niveau 1 k Ref

Niveau 2 k Ref

Niveau 3 k Alt

Niveau 1 k Alt

Niveau 2 k Alt

Niveau 3 k1 k5

A 000 089 142 084 162 046 259 181

B 125 087 073 104 075 046 259 181

C 117 013 091 188 022 083 259 181

D 052 036 069 000 035 083 259 181

E 028 034 037 138 019 112 259 181

F 140 222 018 027 281 112 259 181

G 088 068 024 131 145 000 259 181

H 129 094 173 016 061 000 259 181

L 069 000 073 104 270 224 259 181

M 000 048 038 043 099 241 259 181

000

050

100

150

200

250

300

350

400

A B C D E F G H L M

Vale

urs

k d

e M

an

del(

rob

uste

s)

Laboratoires

Meacutethode de reacutefeacuterence analyse robuste

k Ref Niveau 1k Ref Niveau 2k Ref Niveau 3

k 1=259

k 5=181

000

050

100

150

200

250

300

A B C D E F G H L M

Vale

urs

k d

e M

an

del

(ro

bu

ste

s)

Laboratoires

Meacutethode alternative analyse robuste

k Alt Niveau 3

k Alt Niveau 1

k Alt Niveau 2

k 1=259

k 5=181

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3538 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Appendix 4 ndash Statistical results

Level 1

Nombre de laboratoires (p) 10

A 1602 1602 1602 000 0000 0000 -0495 0000 A 1845 1602 1724 017 0122 -0122 0621 0840 0122

B 1602 1176 1389 030 0213 -0213 -2613 1247 B 1602 1301 1452 021 0151 -0151 -0769 1041 0062

C 1699 1301 1500 028 0199 -0199 -1510 1165 C 1301 1845 1573 038 -0272 0272 -0148 1881 0073

D 1602 1778 1690 012 -0088 0088 0380 0516 D 1602 1602 1602 000 0000 0000 0000 0000 -0088

E 1699 1602 1651 007 0048 -0048 -0013 0284 E 1699 1301 1500 028 0199 -0199 -0522 1376 -0151

F 1778 1301 1540 034 0239 -0239 -1116 1397 F 1778 1699 1739 006 0040 -0040 0698 0274 0199

G 1845 1544 1695 021 0151 -0151 0425 0881 G 2079 1699 1889 027 0190 -0190 1467 1315 0194

H 2041 1602 1822 031 0220 -0220 1689 1286 H 2000 1954 1977 003 0023 -0023 1917 0158 0155

L 1544 1778 1661 017 -0117 0117 0092 0685 L 1301 1602 1452 021 -0151 0151 -0769 1041 -0210

M 1653 1653 1653 000 0000 0000 0013 0000 M 1477 1602 1540 009 -0062 0062 -0319 0432 -0114

m 165186 m 160206 mediane D 00678

Cn(2p) 1867 Cn(2p) 1867 Cp 16101

Cn(p) 16101 Cn(p) 16101 Qn(D) 0092926

n=2p n 20 n=2p n 20 Qdiff 01496169

f 11 f 11 t 114

l 55 l 55

n=p n 10 n=p n 10

f 6 f 6

l 15 l 15

Qn(2p) 0091465 Qn(2p) 007745

Qn(p) 0062469 Qn(p) 0121519

Qintra 017078 Qintra 014461

Qinter 010058 Qinter 019565

Sr(eacutecart-type de reacutepeacutetabiliteacute) 02415 Sr 02045

RSDr(coefficient de variation de la reacutepeacutetabiliteacute)1462 RSDr 1277

r(repeatability limit)) 0676 r 0573

SL(eacutecart-type interlaboratoire) -001905 SLlt0 SL 001737

SR(eacutecart-type de reproductibiliteacute) 02415 SR 02433

RSDR(coefficient de variation de la reproductibiliteacute)0146 RSDR 0152

R(limite de reproductibiliteacute)) 06762 R(limite de reproductibiliteacute))068123

h k h MoyenneLaboratoires

Niveau 1

si LaboratoiresDeviationMoyenne Dk DeviationsiAlt

Niveau 1

Ref

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3638 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Level 2

Nombre de laboratoires (p) 10

A 2681 2613 2647 005 0034 -0034 -0151 0891 A 2322 2531 2427 015 -0105 0105 -2119 1620 -0220

B 2544 2477 2511 005 0033 -0033 -2699 0872 B 2556 2653 2605 007 -0048 0048 -0581 0750 0094

C 2643 2633 2638 001 0005 -0005 -0311 0130 C 2681 2653 2667 002 0014 -0014 -0040 0217 0029

D 2690 2663 2676 002 0014 -0014 0400 0357 D 2778 2732 2755 003 0023 -0023 0721 0354 0079

E 2690 2716 2703 002 -0013 0013 0897 0336 E 2724 2748 2736 002 -0012 0012 0556 0185 0033

F 2568 2398 2483 012 0085 -0085 -3214 2217 F 2477 2114 2296 026 0182 -0182 -3255 2811 -0188

G 2643 2591 2617 004 0026 -0026 -0707 0682 G 2756 2568 2662 013 0094 -0094 -0085 1453 0045

H 2716 2643 2680 005 0036 -0036 0460 0945 H 2653 2732 2693 006 -0040 0040 0181 0613 0013

L 2591 2591 2591 000 0000 0000 -1196 0000 L 2230 2580 2405 025 -0175 0175 -2307 2704 -0186

M 2653 2690 2672 003 -0018 0018 0311 0481 M 2740 2613 2677 009 0064 -0064 0040 0987 0005

m 2655082 m 26719001 mediane D 00209

Cn(2p) 1867 Cn(2p) 1867 Cp 16101

Cn(p) 16101 Cn(p) 16101 Qn(D) 0039911

n=2p n 20 n=2p n 20 Qdiff 00642591

f 11 f 11 t 082

l 55 l 55

n=p n 10 n=p n 10

f 6 f 6

l 15 l 15

Qn(2p) 0020570 Qn(2p) 00345985

Qn(p) 00332437 Qn(p) 00718158

Qintra 003841 Qintra 006460

Qinter 005352 Qinter 011563

Sr(eacutecart-type de reacutepeacutetabiliteacute) 00543 Sr 00914

RSDr(coefficient de variation de la reacutepeacutetabiliteacute)205 RSDr 342

r(repeatability limit)) 0152 r 0256

SL(eacutecart-type interlaboratoire) 000139 SL 000920

SR(eacutecart-type de reproductibiliteacute) 00659 SR 01325

RSDR(coefficient de variation de la reproductibiliteacute)0025 RSDR 0050

R(limite de reproductibiliteacute)) 018446 R(limite de reproductibiliteacute))03708618

Laboratoires

Niveau 2

Moyenne si Laboratoires

Niveau 2

DRef Alt Moyenne si Deviation h Deviation h k k

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3738 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Level 2 bis

Nombre de laboratoires (p) 10

A 3000 2477 2739 037 0261 -0261 0832 1418 A 2602 2477 2540 009 0062 -0062 -0391 0465 -0199

B 2544 2813 2678 019 -0134 0134 0211 0729 B 2903 2778 2841 009 0062 -0062 1732 0465 0162

C 2477 2813 2645 024 -0168 0168 -0135 0911 C 2477 2699 2588 016 -0111 0111 -0049 0826 -0057

D 2398 2653 2526 018 -0128 0128 -1370 0692 D 2778 3000 2889 016 -0111 0111 2074 0826 0363

E 2875 2740 2808 010 0067 -0067 1547 0365 E 2602 2301 2452 021 0151 -0151 -1012 1120 -0356

F 2477 2544 2511 005 -0033 0033 -1525 0182 F 2778 2477 2628 021 0151 -0151 0230 1120 0117

G 2653 2740 2697 006 -0044 0044 0400 0236 G 2845 2845 2845 000 0000 0000 1764 0000 0148

H 2176 2813 2495 045 -0318 0318 -1692 1727 H 2477 2477 2477 000 0000 0000 -0832 0000 -0017

L 2813 2544 2678 019 0134 -0134 0211 0729 L 2903 2301 2602 043 0301 -0301 0049 2241 -0076

M 2602 2740 2671 010 -0069 0069 0135 0375 M 2903 2255 2579 046 0324 -0324 -0112 2411 -0092

m 2658114 m 259505 mediane D -00372

Cn(2p) 1867 Cn(2p) 1867 Cp 16101

Cn(p) 16101 Cn(p) 16101 Qn(D) 01656915

n=2p n 20 n=2p n 20 Qdiff 02667753

f 11 f 11 t 035

l 55 l 55

n=p n 10 n=p n 10

f 6 f 6

l 15 l 15

Qn(2p) 0098745 Qn(2p) 007195

Qn(p) 006007 Qn(p) 008805

Qintra 018437 Qintra 013435

Qinter 009672 Qinter 014176

Sr(eacutecart-type de reacutepeacutetabiliteacute) 02607 Sr 01900

RSDr(coefficient de variation de la reacutepeacutetabiliteacute)981 RSDr 732

r(repeatability limit)) 0730 r 0532

SL(eacutecart-type interlaboratoire) -002464 SLlt0 SL 000205

SR(eacutecart-type de reproductibiliteacute) 02607 SR 01953

RSDR(coefficient de variation de la reproductibiliteacute)0098 RSDR 0075

R(limite de reproductibiliteacute)) 072996 R(limite de reproductibiliteacute))054686

Laboratoires

Niveau 2 bis

Moyenne si Laboratoires

Niveau 2 bis

DRef Alt Moyenne si Deviation h Deviation h k k

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3838 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Level 3

Nombre de laboratoires (p) 10

A 3690 3663 3676 002 0014 -0014 -0311 0513 A 3690 3708 3699 001 -0009 0009 0354 0257 0022

B 3613 3672 3642 004 -0030 0030 -1851 1109 B 3708 3568 3638 010 0070 -0070 -0844 2061 -0005

C 3663 3732 3698 005 -0035 0035 0645 1302 C 3663 3699 3681 003 -0018 0018 0000 0536 -0017

D 3690 3690 3690 000 0000 0000 0311 0000 D 3643 3663 3653 001 -0010 0010 -0545 0286 -0037

E 3732 3653 3693 006 0040 -0040 0429 1480 E 3613 3732 3673 008 -0060 0060 -0162 1769 -0020

F 3643 3672 3658 002 -0014 0014 -1157 0536 F 3690 3771 3731 006 -0040 0040 0975 1193 0073

G 3699 3653 3676 003 0023 -0023 -0328 0856 G 3602 3643 3623 003 -0021 0021 -1141 0612 -0053

H 3623 3672 3648 003 -0024 0024 -1615 0913 H 3748 3681 3715 005 0033 -0033 0664 0990 0067

L 3724 3663 3694 004 0031 -0031 0461 1150 L 3748 3771 3760 002 -0011 0011 1544 0335 0066

M 3690 3690 3690 000 0000 0000 0311 0000 M 3806 3681 3744 009 0062 -0062 1234 1848 0054

m 368334 m 368086 mediane D 00089

Cn(2p) 1867 Cn(2p) 1867 Cp 16101

Cn(p) 16101 Cn(p) 16101 Qn(D) 00325359

n=2p n 20 n=2p n 20 Qdiff 00523852

f 11 f 11 t 043

l 55 l 55

n=p n 10 n=p n 10

f 6 f 6

l 15 l 15

Qn(2p) 0014323 Qn(2p) 001811

Qn(p) 0013719 Qn(p) 003164

Qintra 002674 Qintra 003381

Qinter 002209 Qinter 005094

Sr(eacutecart-type de reacutepeacutetabiliteacute) 00378 Sr 00478

RSDr(coefficient de variation de la reacutepeacutetabiliteacute)103 RSDr 130

r(repeatability limit)) 0106 r 0134

SL(eacutecart-type interlaboratoire) -000023 SLlt0 SL 000145

SR(eacutecart-type de reproductibiliteacute) 00378 SR 00611

RSDR(coefficient de variation de la reproductibiliteacute)0010 RSDR 0017

R(limite de reproductibiliteacute)) 0105894 R(limite de reproductibiliteacute))017119

Ref Alt DLaboratoires

Niveau 3

Moyenne si Deviation h k Laboratoires

Niveau 3

Moyenne si Deviation h k

Page 12: ÞÀ¿¸7 ú Y5zñLjÜ< /u +æü t¬Á ... - NF Validation · NF VALIDATION Validation of alternative analytical methods Application in food microbiology ... 500 – 1 000 1 000 –

Thermo Fisher Scientific

ADRIA Deacuteveloppement 1238 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Statistical interpretation according to EN ISO16140 method

The results of the statistical interpretation are given in Table 6

Table 6 ndash Statistical interpretation results

Category n R Regression

used a t(a) b t(b) Critical T

P

Ordinate

at 0

Slope

at 1

Meat products 10 158 GMFR -0455 3321 115 3123 2306 001 001

Dairy products 12 065 GMFR -0027 0385 1002 0108 2228 092 071

Fishery products 10 130 GMFR -0305 1024 1034 0312 2306 076 034

Vegetables 12 086 GMFR -0015 0128 0993 0191 2228 085 090

Egg products 11 158 GMFR -0038 0866 1001 0090 2262 093 041

Environmental

samples 10 176 GMFR -0139 265 1034 231 2306 005 003

All products 65 118 GMFR -0182 3042 1040 2145 2000 004 000

All products

(estimated number) 65 118 GMFR -0182 3042 1040 2145 2000 004 000

All products (non

estimated results) 20 115 GMFR -0367 151 1138 1141 2101 027 015

Repeatability limits obtained for the alternative and reference methods as

well as bias obtained are presented in Table 7

Dairy products Environmental samples

Log (Ref) Log (Ref)

All categories

Thermo Fisher Scientific

ADRIA Deacuteveloppement 1338 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Table 7

Categories Bias

Repeatability limits

Rob r

Ref Alt

Meat products 0008 0314 0343

Dairy products -0031 0136 0130

Fishery products -0133 0220 0214

Vegetables -0041 0180 0319

Egg products -0026 0219 0206

Environmental samples -0007 0198 0119

All products -0032 0196 0215

Regression straight lines (graph and equation representations) for each food

category and for all products are presented figure 4

Thermo Fisher Scientific

ADRIA Deacuteveloppement 1438 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Figure 4 ndash Regression straight lines

Meat products

Fishery products

Dairy products

Vegetables

Thermo Fisher Scientific

ADRIA Deacuteveloppement 1538 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Environmental samples

Egg products

All products (estimated number)

Thermo Fisher Scientific

ADRIA Deacuteveloppement 1638 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

The hypotheses that the ordinates are close to 0 and the slopes close to 1

are approved in all cases except for

- Meat products (slope P = 1 ordinate P = 1)

- Environmental samples (ordinate P = 3)

- All products (ordinate P = 0)

The bias between the two methods vary from ndash 0133 to 0008 log CFU For

all categories the bias is ndash 0032 log CFU The repeatability limits of the

alternative and the reference method are similar For all products the values

are respectively 0215 and 0196 log CFU For all products these

interpretations are provided taking into account data with low contamination

level or not (estimated colonies number)

For all food products the regression straight line is the following

log (Alt) = 1040 log Ref ndash 0182

The Listeria PrecisTM method shows satisfying relative accuracy

All products

All products (Level 2)

Thermo Fisher Scientific

ADRIA Deacuteveloppement 1738 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

2113 Detection limit (LOD) and quantification limit (LOQ)

The critical level is the defined as the smallest amount which can be detected (not null) but not

quantified as an exact value Below this value it cannot be sure that the true value is not null

The detection limit is defined as being higher than the critical level because it involves a power the

probability 1- which has to be well over 50 for example 95

The quantification limit is defined as the smallest amount of analyte (that is the lowest actual number of

organisms) which can be measured and quantified with defined precision and accuracy under the

experimental conditions by the method under validation

Protocol

The detection limit of the alternative method was realised with pure cultures

Three different inoculation levels were tested with six replicates per level ie

18 analyses were done by the alternative method

Listeria monocytogenes 18 was used for the detection limit of the alternative

method

Quantification limit was calculated for six independent blank samples

determinations

Results

These data are intrinsic to the method used and are presented in the

following tables

Table 8 ndash Inoculation 1 ml on 3 plates

Level (CFUml) Positive samples number Standard deviation Bias

0 06

05 26 1211 0

1 36 0816 05

5 66 2483 85

Thermo Fisher Scientific

ADRIA Deacuteveloppement 1838 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Table 9 ndash Inoculation 01 ml on 1 plate

Level (CFUml) Positive samples number Standard deviation Bias

0 06

5 36 0548 05

10 46 1414 2

50 66 2258 55

Table 10

Formulas Obtained values

LC 165 S0 + X0 18

LOD 33 S0 + X0 32

LOQ 10 S0 + X0 87

2114 Relative Sensitivity

The relative sensitivity is defined as the ability of the alternative method to detect two different amounts

of analyte measured by the reference method within a given matrix at a specified average value or

over the whole measurement range that is it is the minimal quantity variation (increase of the analyte

concentration x) which gives a significant variation of the measured signal (response y)

These data are intrinsic to the used method and are obtained from the results

of the linearity study

Sensitivity patterns obtained for tested (matrixstrain) pairs are presented in

figure 5

Thermo Fisher Scientific

ADRIA Deacuteveloppement 1938 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Figure 5 ndash Sensitivity patterns for the tested (matrixstrain) pairs

2115 SpecificitySelectivity

The specificity is defined as the degree to which a method is affected (or not) by the other components

present in a multi-component sample That is the ability of a method to measure exactly a given

analyte or its amount within the sample without interference from non-target components such as a

matrix effect or background noise

The selectivity is defined as a measure of the degree of non-interference in the presence of non-target

analytes A method is selective if it can be used to detect the analyte under examination and that a

guarantee can be provided that the detected signal can only be a product by that specific analyte

The specificity and selectivity study was performed for the Listeria PrecisTM

detection method no additional test was realised for the enumeration

method The results were the following

- Among the Listeria monocytogenes strains tested 2 strains (Listeria

monocytogenes CIP 7834 and Listeria monocytogenes CIP 105459) gave

negative results These 2 strains were tested twice by running 48 h

incubation time of the compared agars Positive results were then

observed by the alternative method Pale characteristic colonies were

observed on the ISO agar by streaking the Half Fraser broth only

- No cross reaction was observed with the non-target strains except with

Listeria ivanovii which gave characteristic colonies on Brilliancetrade Listeria

Agar but the confirmatory tests provide a clear discrimination between

Listeria monocytogenes and Listeria ivanovi species

Rillettes

Lettuce

Raw milk

Smoked salmon

Raw egg

Process water

x (CFUg)

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2038 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

212 Practicability

1 Packaging Ready to use Brilliance Listeria agar plates are available

(x 10 plates)

2 Reagent volume

3 Storage conditionsndash

Shelf life of products

not opened

The plates are stored at 6 - 12degC in darkness conditions The

expiration date is mentioned on the package and on the plates It

is around 3 months

4 Modalities of use

after first use

5 Required

equipments No specific equipment is required

6 Reagents Ready to use plates

7 Training 05 day

8 Real time handling

Reference method

9 min for one sample

55 min for 10 samples

Alternative method

7 min for one sample

35 minutes for 10 samples

9 Time to result

Negative results

Reference method Day 2

Alternative method Day 2

Negative results after confirmation or positive results

Reference method Day 4 ndash Day 7

Alternative method Day 4 ndash Day 7

Positive results

Reference method Day 4 ndash Day 7

Alternative method Day 2 ndash Day 3 (with OBIS Mono Test)

or Day 4 ndash Day 7 (by the classical tests described in the

standardised method)

10 Technician skill Identical to that required for the reference method

11 Steps common to

the reference method Mother suspension revivification step and decimal dilutions

12 Traceability

13 Maintenance

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2138 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

213 Inter-laboratory study

2131 Organisation

12 laboratories participated to this study Pasteurised milk was inoculated by

Listeria monocytogenes 12a isolated from raw milk The targeted

inoculation levels were

- 0 CFUml

- 50 CFU ml

- 500 CFU ml

- 5 000 CFU ml

Each laboratory received eight flasks of 10 ml sample ie two flasks per

inoculation level Furthermore one non-inoculated sample has been added

to the package for total viable count microflora (NF ISO 4833 method)

Detailed instructions were transmitted to the collaborators by the expert

laboratory

Collaborative study laboratories and the expert laboratory carried out the

analyses with the alternative and reference methods

2132 Verification of experimental parameters

Strain stability during transport

In order to evaluate the Listeria monocytogenes strain stability during

transport bacterial count of all inoculated flasks were checked at different

time ie inoculation time after 24 h of conservation at 2deg - 8degC Results are

reported in table 11

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2238 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Table 11 ndash Listeria monocytogenes count

by ISO 11290-2A1 method (log CFUml)

Day of the analyses Level contamination Reference method

Day 0

(29052006)

1 45

2 440

3 4 500

Day 1

(30052006)

1 60

2 430

3 4 900

No evolution of the strain was observed after storage for 24 h at 2degC ndash 8degC

Contamination level

The real contamination levels are provided in table 12

Table 12 ndash Real contamination level (CFUml)

Target inoculation

(CFU ml) Samples Ndeg

Real contamination levels

(CFUml)

0 1 - 8 0

50 5 - 6 45

500 3 - 7 440

5000 2 - 4 4 500

Sample temperature at receipt

Measured temperatures at receipt are listed in table 13

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2338 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Table 13 - Sample temperature on receipt

Laboratories Receipt

day

Hour

receipt

Measured temperature

at receipt (degC)

Measured temperature by the

temperature probe (degC)

A

B

C

D

E

F

G

H

I

J

K

L

M

30052006

10h45

10h15

11h15

10h00

09h30

8h30

11h50

10h00

08h30

14h45

08h30

09h45

08h45

82

55

45

63

60

62

81

38

48

-

14

67

76

03

03

-13

03

04

-14

05

04

01

05

-13

04

Conclusion

The temperatures during the transport were correct for all the labs except for

Lab J which didnrsquot provide the information

All the labs received their packages with several broken flasks

Three labs (B D and L) received some damaged samples but proceeded to

analyses

All the labs started the analyses at Day 1 except Lab J

2133 Results

Aerobic mesophilic flora

The count of aerobic mesophilic flora varied from 94 102 CFUml to

33 103 CFUml

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2438 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Expert lab results

The results obtained by the expert lab are the following

Table 14

Target inoculation level (CFUml)

Reference method ISO 11290-2

Alternative method Listeria Precis Enumeration

Rep 1 Rep 2 Rep 1 Rep 2

lt 10 lt 10 lt 10 lt 10 lt10

50 50 (Ne) 60 (Ne) 50 (Ne) 30 (Ne)

500 470 390 490 500

5 000 4 700 5 100 5 900 3 400

Ne estimated number

The obtained results are in agreement with those expected

Collaborator Labs results

A synthesis of the results obtained by the collaborator labs is provided

Table 15 (CFUml) and Table 16 (log CFUml)

Table 15 ndash Synthesis of results obtained by the alternative

and the reference methods (CFUml)

Labs Level 1 Level 2 Level 2 bis Level 3

Reference method

Alternative method

Reference method

Alternative method

Reference method

Alternative method

Reference method

Alternative method

A 40 40 70 40 480 410 210 340 1000 300 400 300 4900 4600 4900 5100

B 40 15 40 20 350 300 360 450 350 650 800 600 4100 4700 5100 3700

C 50 20 20 70 440 430 480 450 300 650 300 500 4600 5400 4600 5000

D 40 60 40 40 490 460 600 540 250 450 600 1000 4900 4900 4400 4600

E 50 40 50 20 490 520 530 560 750 550 400 200 5400 4500 4100 5400

F 60 20 60 50 370 250 300 130 300 350 600 300 4400 4700 4900 5900

G 70 35 120 50 440 390 570 370 450 550 700 700 5000 4500 4000 4400

H 110 40 100 90 520 440 450 540 150 650 300 300 4200 4700 5600 4800

L 35 60 20 40 390 390 170 380 650 350 800 200 5300 4600 5600 5900

M 45 45 30 40 450 490 550 410 400 550 800 180 4900 4900 6400 4800

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2538 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Table 16 ndash Synthesis of results obtained by the alternative

and the reference methods (log CFUml)

Labs Level 1 Level 2 Level 2 bis Level 3

Reference method

Alternative method

Reference method

Alternative method

Reference method

Alternative method

Reference method

Alternative method

A 1602 1602 1845 1602 2681 2613 2322 2531 3000 2477 2602 2477 3690 3663 3690 3708

B 1602 1176 1602 1301 2544 2477 2556 2653 2544 2813 2903 2778 3613 3672 3708 3568

C 1699 1301 1301 1845 2643 2633 2681 2653 2477 2813 2477 2699 3663 3732 3663 3699

D 1602 1778 1602 1602 2690 2663 2778 2732 2398 2653 2778 3000 3690 3690 3643 3663

E 1699 1602 1699 1301 2690 2716 2724 2748 2875 2740 2602 2301 3732 3653 3613 3732

F 1778 1301 1778 1699 2568 2398 2477 2114 2477 2544 2778 2477 3643 3672 3690 3771

G 1845 1544 2079 1699 2643 2591 2756 2568 2653 2740 2845 2845 3699 3653 3602 3643

H 2041 1602 2000 1954 2716 2643 2653 2732 2176 2813 2477 2477 3623 3672 3748 3681

L 1544 1778 1301 1602 2591 2591 2230 2580 2813 2544 2903 2301 3724 3663 3748 3771

M 1653 1653 1477 1602 2653 2690 2740 2613 2602 2740 2903 2255 3690 3690 3806 3681

Level 2 results obtained taking account the 3 plates inoculated with the mother-suspension

Level 2 bis results obtained taking account the plates inoculated with 01 ml of the mother-suspension

Scrutiny of the measurement results for consistency

In order to identify the measurement results or laboratories that could be

inconsistent two graphical consistency techniques were realised Mandelrsquos

h-ank-statistics Mandel indicators h and k at 5 significance highlighted

some possible inconsistent data (See tables and graphics in Appendix 3 and

Appendix 4)

Mandelrsquos values Number of values observed over the threshold

Reference method Alternative method

h gt 1 Lab F Level 2 Lab F Level 2

h gt 5 Lab F Level 2

Lab B Levels 1 and 2

Lab A Level 2

Lab D Level 3

Lab F Level 2

Lab L Level 2

kgt 1 Lab F Level 3

k gt 5 Lab F Level 2 Lab C Level 2

Lab F Level 3

Lab L Levels 1 and 3

Lab M Level 1

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2638 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

The emphasized results appear (i) coherent with the targeted contamination

levels (ii) concordant between both methods

All the results were thus kept for the statistical analysis

Comparison of the trueness and precision characteristics of

the reference method and alternative methods

The statistical values are summarised hereafter

Level p

Reference method Alternative method

Repeatability ratio

Reproducibility ratio Median

Repeatability standard deviation

Reproducibility standard deviation

Median Repeatability

standard deviation

Reproducibility standard deviation

1 10 16519 02415 02425 16021 02045 02433 0847 1007

2 10 26551 00543 00659 26719 00914 01325 1682 2011

2 bis 10 26581 02607 02607 25951 01900 01953 0729 0749

3 10 18671 00378 00378 18671 00478 00611 1264 1617

Bias of the alternative method

In order to estimate the bias of the alternative method with regard to the reference method for every

level Dij and t are calculated according to the following equations

RefAlt ijYijYDij

Diffp

Dijit

)2(

)(median

If the t value is superior to 2 the alternative method is significantly biased

with regard to the reference method

The values are given in table 17

Table 17 ndash t (d) values obtained by level

Level Bias t (d) Conclusion

1 0068 114 Non significant bias

2 0021 082 Non significant bias

2 bis - 0037 035 Non significant bias

3 - 0037 043 Non significant bias

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2738 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

The statistical test concludes to a non significant bias for all the inoculation

levels The bias varies from ndash 0037 to 0068 log CFUml

Comparison of the repeatability standard deviations

If the ratio Srj Alt Srj Ref of the repeatability standard deviations of the

alternative method and the reference method is larger than 2 the precision

under repeatability conditions of the alternative method is considered to be

lower than that of the reference method If this ratio is smaller than 05 the

precision under repeatability conditions of the alternative method is

considered to be greater than that of the reference method

The ratio values are given in table 18

Table 18

Level

Repeatability standard deviation

Reference method Alternative method Alternative method

reference method ratio

1 0242 0205 0847

2 0054 0091 1682

2 bis 0261 0190 0729

3 0038 0048 1264

Level Repeatability limit

Reference method Alternative method

1 0676 0573

2 0152 0256

2 bis 0730 0532

3 0106 0134

The ratios of the repeatability standard deviations are below 2 in all cases

The precision under repeatability conditions of the alternative method is

equivalent to that of the reference method

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2838 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Comparison of the reproducibility standard deviations

If the ratio Srj Alt Srj Ref of the reproducibility standard deviations of the alternative method and the

reference method is larger than 2 the precision under reproducibility conditions of the alternative

method is considered to be lower than that of the reference method If this ratio is smaller than 05 the

precision under reproducibility conditions of the alternative method is considered to be greater than

that of the reference method

The ratio values are given in table 19

Table 19

Level

Reproducibility standard deviation

Reference method Alternative method Alternative method

reference method ratio

1 0242 0243 1007

2 0066 0133 2011

2 bis 0261 0195 0749

3 0038 0061 1617

Level Reproducibility limit

Reference method Alternative method

1 0676 0681

2 0185 0371

2 bis 0730 0547

3 0106 0171

The ratios of the reproducibility standard deviations are below or close to 2

The precision under reproducibility conditions of the alternative method is

equivalent to that of the reference method

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2938 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

214 Conclusion

Method comparison study

The Listeria PrecisTM enumeration method shows satisfying linearity and

relative accuracy

The alternative method shows satisfying inclusivity and exclusivity results

Two Listeria monocytogenes strains gave negative results with the

alternative method These strains grew weakly on the reference method

media (OampA Palcam and Oxford)

The limits of repeatability of the alternative method are similar to those of the

reference method

The bias between both methods are low between - 0133 Log CFUg and

- 0008 Log CFUg

The Listeria PrecisTM enumeration method offers the saving for confirmatory

tests

Positive results are obtained with the alternative method in 2-3 days (with

OBIS Mono test) or in 4-7 days (by the classical tests described in the

standardized method) against 4-7 days for the reference method

Inter-laboratory study

The bias between the reference and the alternative methods shows satisfying

and correct values

- level 1 (100 - 1 000 CFUg) bias = + 007 log CFUg

- level 2 (1 000 - 10 000 CFUg) bias = 002 log CFUg

- level 2 bis (1 000 - 10 000 CFUg) bias = - 004 log CFUg

- level 3 (10 000 - 100 000 CFUg) bias = - 004 log CFUg

The repeatability and the reproducibility of the alternative method look similar

to those of the reference method

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3038 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

22 Extension study (realised by ASEPT 2007)

An extension study was performed in 2007 for a new confirmatory test OBIS

MONO

150 Listeria monocytogenes strains and 100 non-target strains were isolated

onto TSAYE and BrillianceTM Listeria Agar plates All the Listeria

monocytogenes and the non-target strains gave the expected results

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3138 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Appendix 1 ndash Flow diagram of the Listeria PrecisTM method

x g sample + 9 x ml buffered peptone water

Revivification step for 1 h plusmn 5 min at 20degC plusmn 1degC

Streaking 1 ml onto 3 Brilliance Listeria Agar plates (mother suspension)

or 01 ml onto 1 Brilliance Listeria Agar plates (mother suspension and decimal dilutions)

Incubation for 48 h plusmn 3 h at 37degC plusmn 1degC

Confirmatory tests on at least one typical colony

- by the tests described in the ISO method

- by applying the OBIS mono test on an isolated colony

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3238 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Appendix 2 ndash ISO 11290-2 (1998) amp ISO 11290-2A1 (2004) Horizontal

method for the detection and enumeration of Listeria monocytogenes -

Part 2 enumeration of Listeria monocytogenes in foods

10 g cheese + 90 ml buffered peptone water or Half Fraser Broth (without supplements) (110 dilution)

Stomach 1 min 30 seconds

Keep at 20degC plusmn 2degC for 1 h plusmn 5 min

Decimal dilutions in 9 ml peptone salt tubes

Dilution 110

Dilution 1100

Dilution 11000

1 ml on 3 Chromogenic Listeria medium (ISO) plates (x 2) (-1)

01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-2)

01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-3)

01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-4)

Incubation at 37degC plusmn 1degC

24 h - 48 h

Numerate typical colonies at 24 h

(bluegreen surrounded by an opaque halo)

Numerate at 48 h

Streak typical colonies on TSYEA

(5 coloniesplate 2 successive dilutions)

Confirmation tests

Catalase

Gram stain

For confirmation of L monocytogenes

Haemolysis

Carbohydrate (rhamnose and xylose) utilisation

CAMP Test

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3338 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Appendix 3 ndash Mandelrsquos graphics

Laboratoire h Ref

Niveau 1 h Ref

Niveau 2 h Ref

Niveau 3 h Alt

Niveau 1 h Alt

Niveau 2 h Alt

Niveau 3 h5 h1 h5 h1

A -050 -015 083 062 -212 -039 198 299 -198 -299

B -261 -270 021 -077 -058 173 198 299 -198 -299

C -151 -031 -014 -015 -004 -005 198 299 -198 -299

D 038 040 -137 000 072 207 198 299 -198 -299

E -001 090 155 -052 056 -101 198 299 -198 -299

F -112 -321 -153 070 -325 023 198 299 -198 -299

G 042 -071 040 147 -009 176 198 299 -198 -299

H 169 046 -169 192 018 -083 198 299 -198 -299

L 009 -120 021 -077 -231 005 198 299 -198 -299

M 001 031 014 -032 004 -011 198 299 -198 -299

-300

-200

-100

000

100

200

300

A B C D E F G H L M

Vale

urs

h d

e M

an

del(

rob

uste

s)

Laboratoires

Meacutethode de reacutefeacuterence analyse robuste

h Ref Niveau 1

h Ref Niveau 2

h Ref Niveau 3

h 5=198

h 1=299

-300

-200

-100

000

100

200

300

A B C D E F G H L M

Vale

urs

de h

de M

an

del(

rob

uste

s)

Laboratoires

Meacutethode alternative analyse robuste

h Alt Niveau 1

h Alt Niveau 2

h Alt Niveau 2

h 1=299

h 5=198

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3438 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Laboratoire k Ref

Niveau 1 k Ref

Niveau 2 k Ref

Niveau 3 k Alt

Niveau 1 k Alt

Niveau 2 k Alt

Niveau 3 k1 k5

A 000 089 142 084 162 046 259 181

B 125 087 073 104 075 046 259 181

C 117 013 091 188 022 083 259 181

D 052 036 069 000 035 083 259 181

E 028 034 037 138 019 112 259 181

F 140 222 018 027 281 112 259 181

G 088 068 024 131 145 000 259 181

H 129 094 173 016 061 000 259 181

L 069 000 073 104 270 224 259 181

M 000 048 038 043 099 241 259 181

000

050

100

150

200

250

300

350

400

A B C D E F G H L M

Vale

urs

k d

e M

an

del(

rob

uste

s)

Laboratoires

Meacutethode de reacutefeacuterence analyse robuste

k Ref Niveau 1k Ref Niveau 2k Ref Niveau 3

k 1=259

k 5=181

000

050

100

150

200

250

300

A B C D E F G H L M

Vale

urs

k d

e M

an

del

(ro

bu

ste

s)

Laboratoires

Meacutethode alternative analyse robuste

k Alt Niveau 3

k Alt Niveau 1

k Alt Niveau 2

k 1=259

k 5=181

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3538 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Appendix 4 ndash Statistical results

Level 1

Nombre de laboratoires (p) 10

A 1602 1602 1602 000 0000 0000 -0495 0000 A 1845 1602 1724 017 0122 -0122 0621 0840 0122

B 1602 1176 1389 030 0213 -0213 -2613 1247 B 1602 1301 1452 021 0151 -0151 -0769 1041 0062

C 1699 1301 1500 028 0199 -0199 -1510 1165 C 1301 1845 1573 038 -0272 0272 -0148 1881 0073

D 1602 1778 1690 012 -0088 0088 0380 0516 D 1602 1602 1602 000 0000 0000 0000 0000 -0088

E 1699 1602 1651 007 0048 -0048 -0013 0284 E 1699 1301 1500 028 0199 -0199 -0522 1376 -0151

F 1778 1301 1540 034 0239 -0239 -1116 1397 F 1778 1699 1739 006 0040 -0040 0698 0274 0199

G 1845 1544 1695 021 0151 -0151 0425 0881 G 2079 1699 1889 027 0190 -0190 1467 1315 0194

H 2041 1602 1822 031 0220 -0220 1689 1286 H 2000 1954 1977 003 0023 -0023 1917 0158 0155

L 1544 1778 1661 017 -0117 0117 0092 0685 L 1301 1602 1452 021 -0151 0151 -0769 1041 -0210

M 1653 1653 1653 000 0000 0000 0013 0000 M 1477 1602 1540 009 -0062 0062 -0319 0432 -0114

m 165186 m 160206 mediane D 00678

Cn(2p) 1867 Cn(2p) 1867 Cp 16101

Cn(p) 16101 Cn(p) 16101 Qn(D) 0092926

n=2p n 20 n=2p n 20 Qdiff 01496169

f 11 f 11 t 114

l 55 l 55

n=p n 10 n=p n 10

f 6 f 6

l 15 l 15

Qn(2p) 0091465 Qn(2p) 007745

Qn(p) 0062469 Qn(p) 0121519

Qintra 017078 Qintra 014461

Qinter 010058 Qinter 019565

Sr(eacutecart-type de reacutepeacutetabiliteacute) 02415 Sr 02045

RSDr(coefficient de variation de la reacutepeacutetabiliteacute)1462 RSDr 1277

r(repeatability limit)) 0676 r 0573

SL(eacutecart-type interlaboratoire) -001905 SLlt0 SL 001737

SR(eacutecart-type de reproductibiliteacute) 02415 SR 02433

RSDR(coefficient de variation de la reproductibiliteacute)0146 RSDR 0152

R(limite de reproductibiliteacute)) 06762 R(limite de reproductibiliteacute))068123

h k h MoyenneLaboratoires

Niveau 1

si LaboratoiresDeviationMoyenne Dk DeviationsiAlt

Niveau 1

Ref

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3638 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Level 2

Nombre de laboratoires (p) 10

A 2681 2613 2647 005 0034 -0034 -0151 0891 A 2322 2531 2427 015 -0105 0105 -2119 1620 -0220

B 2544 2477 2511 005 0033 -0033 -2699 0872 B 2556 2653 2605 007 -0048 0048 -0581 0750 0094

C 2643 2633 2638 001 0005 -0005 -0311 0130 C 2681 2653 2667 002 0014 -0014 -0040 0217 0029

D 2690 2663 2676 002 0014 -0014 0400 0357 D 2778 2732 2755 003 0023 -0023 0721 0354 0079

E 2690 2716 2703 002 -0013 0013 0897 0336 E 2724 2748 2736 002 -0012 0012 0556 0185 0033

F 2568 2398 2483 012 0085 -0085 -3214 2217 F 2477 2114 2296 026 0182 -0182 -3255 2811 -0188

G 2643 2591 2617 004 0026 -0026 -0707 0682 G 2756 2568 2662 013 0094 -0094 -0085 1453 0045

H 2716 2643 2680 005 0036 -0036 0460 0945 H 2653 2732 2693 006 -0040 0040 0181 0613 0013

L 2591 2591 2591 000 0000 0000 -1196 0000 L 2230 2580 2405 025 -0175 0175 -2307 2704 -0186

M 2653 2690 2672 003 -0018 0018 0311 0481 M 2740 2613 2677 009 0064 -0064 0040 0987 0005

m 2655082 m 26719001 mediane D 00209

Cn(2p) 1867 Cn(2p) 1867 Cp 16101

Cn(p) 16101 Cn(p) 16101 Qn(D) 0039911

n=2p n 20 n=2p n 20 Qdiff 00642591

f 11 f 11 t 082

l 55 l 55

n=p n 10 n=p n 10

f 6 f 6

l 15 l 15

Qn(2p) 0020570 Qn(2p) 00345985

Qn(p) 00332437 Qn(p) 00718158

Qintra 003841 Qintra 006460

Qinter 005352 Qinter 011563

Sr(eacutecart-type de reacutepeacutetabiliteacute) 00543 Sr 00914

RSDr(coefficient de variation de la reacutepeacutetabiliteacute)205 RSDr 342

r(repeatability limit)) 0152 r 0256

SL(eacutecart-type interlaboratoire) 000139 SL 000920

SR(eacutecart-type de reproductibiliteacute) 00659 SR 01325

RSDR(coefficient de variation de la reproductibiliteacute)0025 RSDR 0050

R(limite de reproductibiliteacute)) 018446 R(limite de reproductibiliteacute))03708618

Laboratoires

Niveau 2

Moyenne si Laboratoires

Niveau 2

DRef Alt Moyenne si Deviation h Deviation h k k

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3738 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Level 2 bis

Nombre de laboratoires (p) 10

A 3000 2477 2739 037 0261 -0261 0832 1418 A 2602 2477 2540 009 0062 -0062 -0391 0465 -0199

B 2544 2813 2678 019 -0134 0134 0211 0729 B 2903 2778 2841 009 0062 -0062 1732 0465 0162

C 2477 2813 2645 024 -0168 0168 -0135 0911 C 2477 2699 2588 016 -0111 0111 -0049 0826 -0057

D 2398 2653 2526 018 -0128 0128 -1370 0692 D 2778 3000 2889 016 -0111 0111 2074 0826 0363

E 2875 2740 2808 010 0067 -0067 1547 0365 E 2602 2301 2452 021 0151 -0151 -1012 1120 -0356

F 2477 2544 2511 005 -0033 0033 -1525 0182 F 2778 2477 2628 021 0151 -0151 0230 1120 0117

G 2653 2740 2697 006 -0044 0044 0400 0236 G 2845 2845 2845 000 0000 0000 1764 0000 0148

H 2176 2813 2495 045 -0318 0318 -1692 1727 H 2477 2477 2477 000 0000 0000 -0832 0000 -0017

L 2813 2544 2678 019 0134 -0134 0211 0729 L 2903 2301 2602 043 0301 -0301 0049 2241 -0076

M 2602 2740 2671 010 -0069 0069 0135 0375 M 2903 2255 2579 046 0324 -0324 -0112 2411 -0092

m 2658114 m 259505 mediane D -00372

Cn(2p) 1867 Cn(2p) 1867 Cp 16101

Cn(p) 16101 Cn(p) 16101 Qn(D) 01656915

n=2p n 20 n=2p n 20 Qdiff 02667753

f 11 f 11 t 035

l 55 l 55

n=p n 10 n=p n 10

f 6 f 6

l 15 l 15

Qn(2p) 0098745 Qn(2p) 007195

Qn(p) 006007 Qn(p) 008805

Qintra 018437 Qintra 013435

Qinter 009672 Qinter 014176

Sr(eacutecart-type de reacutepeacutetabiliteacute) 02607 Sr 01900

RSDr(coefficient de variation de la reacutepeacutetabiliteacute)981 RSDr 732

r(repeatability limit)) 0730 r 0532

SL(eacutecart-type interlaboratoire) -002464 SLlt0 SL 000205

SR(eacutecart-type de reproductibiliteacute) 02607 SR 01953

RSDR(coefficient de variation de la reproductibiliteacute)0098 RSDR 0075

R(limite de reproductibiliteacute)) 072996 R(limite de reproductibiliteacute))054686

Laboratoires

Niveau 2 bis

Moyenne si Laboratoires

Niveau 2 bis

DRef Alt Moyenne si Deviation h Deviation h k k

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3838 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Level 3

Nombre de laboratoires (p) 10

A 3690 3663 3676 002 0014 -0014 -0311 0513 A 3690 3708 3699 001 -0009 0009 0354 0257 0022

B 3613 3672 3642 004 -0030 0030 -1851 1109 B 3708 3568 3638 010 0070 -0070 -0844 2061 -0005

C 3663 3732 3698 005 -0035 0035 0645 1302 C 3663 3699 3681 003 -0018 0018 0000 0536 -0017

D 3690 3690 3690 000 0000 0000 0311 0000 D 3643 3663 3653 001 -0010 0010 -0545 0286 -0037

E 3732 3653 3693 006 0040 -0040 0429 1480 E 3613 3732 3673 008 -0060 0060 -0162 1769 -0020

F 3643 3672 3658 002 -0014 0014 -1157 0536 F 3690 3771 3731 006 -0040 0040 0975 1193 0073

G 3699 3653 3676 003 0023 -0023 -0328 0856 G 3602 3643 3623 003 -0021 0021 -1141 0612 -0053

H 3623 3672 3648 003 -0024 0024 -1615 0913 H 3748 3681 3715 005 0033 -0033 0664 0990 0067

L 3724 3663 3694 004 0031 -0031 0461 1150 L 3748 3771 3760 002 -0011 0011 1544 0335 0066

M 3690 3690 3690 000 0000 0000 0311 0000 M 3806 3681 3744 009 0062 -0062 1234 1848 0054

m 368334 m 368086 mediane D 00089

Cn(2p) 1867 Cn(2p) 1867 Cp 16101

Cn(p) 16101 Cn(p) 16101 Qn(D) 00325359

n=2p n 20 n=2p n 20 Qdiff 00523852

f 11 f 11 t 043

l 55 l 55

n=p n 10 n=p n 10

f 6 f 6

l 15 l 15

Qn(2p) 0014323 Qn(2p) 001811

Qn(p) 0013719 Qn(p) 003164

Qintra 002674 Qintra 003381

Qinter 002209 Qinter 005094

Sr(eacutecart-type de reacutepeacutetabiliteacute) 00378 Sr 00478

RSDr(coefficient de variation de la reacutepeacutetabiliteacute)103 RSDr 130

r(repeatability limit)) 0106 r 0134

SL(eacutecart-type interlaboratoire) -000023 SLlt0 SL 000145

SR(eacutecart-type de reproductibiliteacute) 00378 SR 00611

RSDR(coefficient de variation de la reproductibiliteacute)0010 RSDR 0017

R(limite de reproductibiliteacute)) 0105894 R(limite de reproductibiliteacute))017119

Ref Alt DLaboratoires

Niveau 3

Moyenne si Deviation h k Laboratoires

Niveau 3

Moyenne si Deviation h k

Page 13: ÞÀ¿¸7 ú Y5zñLjÜ< /u +æü t¬Á ... - NF Validation · NF VALIDATION Validation of alternative analytical methods Application in food microbiology ... 500 – 1 000 1 000 –

Thermo Fisher Scientific

ADRIA Deacuteveloppement 1338 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Table 7

Categories Bias

Repeatability limits

Rob r

Ref Alt

Meat products 0008 0314 0343

Dairy products -0031 0136 0130

Fishery products -0133 0220 0214

Vegetables -0041 0180 0319

Egg products -0026 0219 0206

Environmental samples -0007 0198 0119

All products -0032 0196 0215

Regression straight lines (graph and equation representations) for each food

category and for all products are presented figure 4

Thermo Fisher Scientific

ADRIA Deacuteveloppement 1438 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Figure 4 ndash Regression straight lines

Meat products

Fishery products

Dairy products

Vegetables

Thermo Fisher Scientific

ADRIA Deacuteveloppement 1538 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Environmental samples

Egg products

All products (estimated number)

Thermo Fisher Scientific

ADRIA Deacuteveloppement 1638 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

The hypotheses that the ordinates are close to 0 and the slopes close to 1

are approved in all cases except for

- Meat products (slope P = 1 ordinate P = 1)

- Environmental samples (ordinate P = 3)

- All products (ordinate P = 0)

The bias between the two methods vary from ndash 0133 to 0008 log CFU For

all categories the bias is ndash 0032 log CFU The repeatability limits of the

alternative and the reference method are similar For all products the values

are respectively 0215 and 0196 log CFU For all products these

interpretations are provided taking into account data with low contamination

level or not (estimated colonies number)

For all food products the regression straight line is the following

log (Alt) = 1040 log Ref ndash 0182

The Listeria PrecisTM method shows satisfying relative accuracy

All products

All products (Level 2)

Thermo Fisher Scientific

ADRIA Deacuteveloppement 1738 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

2113 Detection limit (LOD) and quantification limit (LOQ)

The critical level is the defined as the smallest amount which can be detected (not null) but not

quantified as an exact value Below this value it cannot be sure that the true value is not null

The detection limit is defined as being higher than the critical level because it involves a power the

probability 1- which has to be well over 50 for example 95

The quantification limit is defined as the smallest amount of analyte (that is the lowest actual number of

organisms) which can be measured and quantified with defined precision and accuracy under the

experimental conditions by the method under validation

Protocol

The detection limit of the alternative method was realised with pure cultures

Three different inoculation levels were tested with six replicates per level ie

18 analyses were done by the alternative method

Listeria monocytogenes 18 was used for the detection limit of the alternative

method

Quantification limit was calculated for six independent blank samples

determinations

Results

These data are intrinsic to the method used and are presented in the

following tables

Table 8 ndash Inoculation 1 ml on 3 plates

Level (CFUml) Positive samples number Standard deviation Bias

0 06

05 26 1211 0

1 36 0816 05

5 66 2483 85

Thermo Fisher Scientific

ADRIA Deacuteveloppement 1838 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Table 9 ndash Inoculation 01 ml on 1 plate

Level (CFUml) Positive samples number Standard deviation Bias

0 06

5 36 0548 05

10 46 1414 2

50 66 2258 55

Table 10

Formulas Obtained values

LC 165 S0 + X0 18

LOD 33 S0 + X0 32

LOQ 10 S0 + X0 87

2114 Relative Sensitivity

The relative sensitivity is defined as the ability of the alternative method to detect two different amounts

of analyte measured by the reference method within a given matrix at a specified average value or

over the whole measurement range that is it is the minimal quantity variation (increase of the analyte

concentration x) which gives a significant variation of the measured signal (response y)

These data are intrinsic to the used method and are obtained from the results

of the linearity study

Sensitivity patterns obtained for tested (matrixstrain) pairs are presented in

figure 5

Thermo Fisher Scientific

ADRIA Deacuteveloppement 1938 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Figure 5 ndash Sensitivity patterns for the tested (matrixstrain) pairs

2115 SpecificitySelectivity

The specificity is defined as the degree to which a method is affected (or not) by the other components

present in a multi-component sample That is the ability of a method to measure exactly a given

analyte or its amount within the sample without interference from non-target components such as a

matrix effect or background noise

The selectivity is defined as a measure of the degree of non-interference in the presence of non-target

analytes A method is selective if it can be used to detect the analyte under examination and that a

guarantee can be provided that the detected signal can only be a product by that specific analyte

The specificity and selectivity study was performed for the Listeria PrecisTM

detection method no additional test was realised for the enumeration

method The results were the following

- Among the Listeria monocytogenes strains tested 2 strains (Listeria

monocytogenes CIP 7834 and Listeria monocytogenes CIP 105459) gave

negative results These 2 strains were tested twice by running 48 h

incubation time of the compared agars Positive results were then

observed by the alternative method Pale characteristic colonies were

observed on the ISO agar by streaking the Half Fraser broth only

- No cross reaction was observed with the non-target strains except with

Listeria ivanovii which gave characteristic colonies on Brilliancetrade Listeria

Agar but the confirmatory tests provide a clear discrimination between

Listeria monocytogenes and Listeria ivanovi species

Rillettes

Lettuce

Raw milk

Smoked salmon

Raw egg

Process water

x (CFUg)

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2038 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

212 Practicability

1 Packaging Ready to use Brilliance Listeria agar plates are available

(x 10 plates)

2 Reagent volume

3 Storage conditionsndash

Shelf life of products

not opened

The plates are stored at 6 - 12degC in darkness conditions The

expiration date is mentioned on the package and on the plates It

is around 3 months

4 Modalities of use

after first use

5 Required

equipments No specific equipment is required

6 Reagents Ready to use plates

7 Training 05 day

8 Real time handling

Reference method

9 min for one sample

55 min for 10 samples

Alternative method

7 min for one sample

35 minutes for 10 samples

9 Time to result

Negative results

Reference method Day 2

Alternative method Day 2

Negative results after confirmation or positive results

Reference method Day 4 ndash Day 7

Alternative method Day 4 ndash Day 7

Positive results

Reference method Day 4 ndash Day 7

Alternative method Day 2 ndash Day 3 (with OBIS Mono Test)

or Day 4 ndash Day 7 (by the classical tests described in the

standardised method)

10 Technician skill Identical to that required for the reference method

11 Steps common to

the reference method Mother suspension revivification step and decimal dilutions

12 Traceability

13 Maintenance

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2138 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

213 Inter-laboratory study

2131 Organisation

12 laboratories participated to this study Pasteurised milk was inoculated by

Listeria monocytogenes 12a isolated from raw milk The targeted

inoculation levels were

- 0 CFUml

- 50 CFU ml

- 500 CFU ml

- 5 000 CFU ml

Each laboratory received eight flasks of 10 ml sample ie two flasks per

inoculation level Furthermore one non-inoculated sample has been added

to the package for total viable count microflora (NF ISO 4833 method)

Detailed instructions were transmitted to the collaborators by the expert

laboratory

Collaborative study laboratories and the expert laboratory carried out the

analyses with the alternative and reference methods

2132 Verification of experimental parameters

Strain stability during transport

In order to evaluate the Listeria monocytogenes strain stability during

transport bacterial count of all inoculated flasks were checked at different

time ie inoculation time after 24 h of conservation at 2deg - 8degC Results are

reported in table 11

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2238 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Table 11 ndash Listeria monocytogenes count

by ISO 11290-2A1 method (log CFUml)

Day of the analyses Level contamination Reference method

Day 0

(29052006)

1 45

2 440

3 4 500

Day 1

(30052006)

1 60

2 430

3 4 900

No evolution of the strain was observed after storage for 24 h at 2degC ndash 8degC

Contamination level

The real contamination levels are provided in table 12

Table 12 ndash Real contamination level (CFUml)

Target inoculation

(CFU ml) Samples Ndeg

Real contamination levels

(CFUml)

0 1 - 8 0

50 5 - 6 45

500 3 - 7 440

5000 2 - 4 4 500

Sample temperature at receipt

Measured temperatures at receipt are listed in table 13

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2338 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Table 13 - Sample temperature on receipt

Laboratories Receipt

day

Hour

receipt

Measured temperature

at receipt (degC)

Measured temperature by the

temperature probe (degC)

A

B

C

D

E

F

G

H

I

J

K

L

M

30052006

10h45

10h15

11h15

10h00

09h30

8h30

11h50

10h00

08h30

14h45

08h30

09h45

08h45

82

55

45

63

60

62

81

38

48

-

14

67

76

03

03

-13

03

04

-14

05

04

01

05

-13

04

Conclusion

The temperatures during the transport were correct for all the labs except for

Lab J which didnrsquot provide the information

All the labs received their packages with several broken flasks

Three labs (B D and L) received some damaged samples but proceeded to

analyses

All the labs started the analyses at Day 1 except Lab J

2133 Results

Aerobic mesophilic flora

The count of aerobic mesophilic flora varied from 94 102 CFUml to

33 103 CFUml

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2438 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Expert lab results

The results obtained by the expert lab are the following

Table 14

Target inoculation level (CFUml)

Reference method ISO 11290-2

Alternative method Listeria Precis Enumeration

Rep 1 Rep 2 Rep 1 Rep 2

lt 10 lt 10 lt 10 lt 10 lt10

50 50 (Ne) 60 (Ne) 50 (Ne) 30 (Ne)

500 470 390 490 500

5 000 4 700 5 100 5 900 3 400

Ne estimated number

The obtained results are in agreement with those expected

Collaborator Labs results

A synthesis of the results obtained by the collaborator labs is provided

Table 15 (CFUml) and Table 16 (log CFUml)

Table 15 ndash Synthesis of results obtained by the alternative

and the reference methods (CFUml)

Labs Level 1 Level 2 Level 2 bis Level 3

Reference method

Alternative method

Reference method

Alternative method

Reference method

Alternative method

Reference method

Alternative method

A 40 40 70 40 480 410 210 340 1000 300 400 300 4900 4600 4900 5100

B 40 15 40 20 350 300 360 450 350 650 800 600 4100 4700 5100 3700

C 50 20 20 70 440 430 480 450 300 650 300 500 4600 5400 4600 5000

D 40 60 40 40 490 460 600 540 250 450 600 1000 4900 4900 4400 4600

E 50 40 50 20 490 520 530 560 750 550 400 200 5400 4500 4100 5400

F 60 20 60 50 370 250 300 130 300 350 600 300 4400 4700 4900 5900

G 70 35 120 50 440 390 570 370 450 550 700 700 5000 4500 4000 4400

H 110 40 100 90 520 440 450 540 150 650 300 300 4200 4700 5600 4800

L 35 60 20 40 390 390 170 380 650 350 800 200 5300 4600 5600 5900

M 45 45 30 40 450 490 550 410 400 550 800 180 4900 4900 6400 4800

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2538 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Table 16 ndash Synthesis of results obtained by the alternative

and the reference methods (log CFUml)

Labs Level 1 Level 2 Level 2 bis Level 3

Reference method

Alternative method

Reference method

Alternative method

Reference method

Alternative method

Reference method

Alternative method

A 1602 1602 1845 1602 2681 2613 2322 2531 3000 2477 2602 2477 3690 3663 3690 3708

B 1602 1176 1602 1301 2544 2477 2556 2653 2544 2813 2903 2778 3613 3672 3708 3568

C 1699 1301 1301 1845 2643 2633 2681 2653 2477 2813 2477 2699 3663 3732 3663 3699

D 1602 1778 1602 1602 2690 2663 2778 2732 2398 2653 2778 3000 3690 3690 3643 3663

E 1699 1602 1699 1301 2690 2716 2724 2748 2875 2740 2602 2301 3732 3653 3613 3732

F 1778 1301 1778 1699 2568 2398 2477 2114 2477 2544 2778 2477 3643 3672 3690 3771

G 1845 1544 2079 1699 2643 2591 2756 2568 2653 2740 2845 2845 3699 3653 3602 3643

H 2041 1602 2000 1954 2716 2643 2653 2732 2176 2813 2477 2477 3623 3672 3748 3681

L 1544 1778 1301 1602 2591 2591 2230 2580 2813 2544 2903 2301 3724 3663 3748 3771

M 1653 1653 1477 1602 2653 2690 2740 2613 2602 2740 2903 2255 3690 3690 3806 3681

Level 2 results obtained taking account the 3 plates inoculated with the mother-suspension

Level 2 bis results obtained taking account the plates inoculated with 01 ml of the mother-suspension

Scrutiny of the measurement results for consistency

In order to identify the measurement results or laboratories that could be

inconsistent two graphical consistency techniques were realised Mandelrsquos

h-ank-statistics Mandel indicators h and k at 5 significance highlighted

some possible inconsistent data (See tables and graphics in Appendix 3 and

Appendix 4)

Mandelrsquos values Number of values observed over the threshold

Reference method Alternative method

h gt 1 Lab F Level 2 Lab F Level 2

h gt 5 Lab F Level 2

Lab B Levels 1 and 2

Lab A Level 2

Lab D Level 3

Lab F Level 2

Lab L Level 2

kgt 1 Lab F Level 3

k gt 5 Lab F Level 2 Lab C Level 2

Lab F Level 3

Lab L Levels 1 and 3

Lab M Level 1

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2638 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

The emphasized results appear (i) coherent with the targeted contamination

levels (ii) concordant between both methods

All the results were thus kept for the statistical analysis

Comparison of the trueness and precision characteristics of

the reference method and alternative methods

The statistical values are summarised hereafter

Level p

Reference method Alternative method

Repeatability ratio

Reproducibility ratio Median

Repeatability standard deviation

Reproducibility standard deviation

Median Repeatability

standard deviation

Reproducibility standard deviation

1 10 16519 02415 02425 16021 02045 02433 0847 1007

2 10 26551 00543 00659 26719 00914 01325 1682 2011

2 bis 10 26581 02607 02607 25951 01900 01953 0729 0749

3 10 18671 00378 00378 18671 00478 00611 1264 1617

Bias of the alternative method

In order to estimate the bias of the alternative method with regard to the reference method for every

level Dij and t are calculated according to the following equations

RefAlt ijYijYDij

Diffp

Dijit

)2(

)(median

If the t value is superior to 2 the alternative method is significantly biased

with regard to the reference method

The values are given in table 17

Table 17 ndash t (d) values obtained by level

Level Bias t (d) Conclusion

1 0068 114 Non significant bias

2 0021 082 Non significant bias

2 bis - 0037 035 Non significant bias

3 - 0037 043 Non significant bias

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2738 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

The statistical test concludes to a non significant bias for all the inoculation

levels The bias varies from ndash 0037 to 0068 log CFUml

Comparison of the repeatability standard deviations

If the ratio Srj Alt Srj Ref of the repeatability standard deviations of the

alternative method and the reference method is larger than 2 the precision

under repeatability conditions of the alternative method is considered to be

lower than that of the reference method If this ratio is smaller than 05 the

precision under repeatability conditions of the alternative method is

considered to be greater than that of the reference method

The ratio values are given in table 18

Table 18

Level

Repeatability standard deviation

Reference method Alternative method Alternative method

reference method ratio

1 0242 0205 0847

2 0054 0091 1682

2 bis 0261 0190 0729

3 0038 0048 1264

Level Repeatability limit

Reference method Alternative method

1 0676 0573

2 0152 0256

2 bis 0730 0532

3 0106 0134

The ratios of the repeatability standard deviations are below 2 in all cases

The precision under repeatability conditions of the alternative method is

equivalent to that of the reference method

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2838 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Comparison of the reproducibility standard deviations

If the ratio Srj Alt Srj Ref of the reproducibility standard deviations of the alternative method and the

reference method is larger than 2 the precision under reproducibility conditions of the alternative

method is considered to be lower than that of the reference method If this ratio is smaller than 05 the

precision under reproducibility conditions of the alternative method is considered to be greater than

that of the reference method

The ratio values are given in table 19

Table 19

Level

Reproducibility standard deviation

Reference method Alternative method Alternative method

reference method ratio

1 0242 0243 1007

2 0066 0133 2011

2 bis 0261 0195 0749

3 0038 0061 1617

Level Reproducibility limit

Reference method Alternative method

1 0676 0681

2 0185 0371

2 bis 0730 0547

3 0106 0171

The ratios of the reproducibility standard deviations are below or close to 2

The precision under reproducibility conditions of the alternative method is

equivalent to that of the reference method

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2938 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

214 Conclusion

Method comparison study

The Listeria PrecisTM enumeration method shows satisfying linearity and

relative accuracy

The alternative method shows satisfying inclusivity and exclusivity results

Two Listeria monocytogenes strains gave negative results with the

alternative method These strains grew weakly on the reference method

media (OampA Palcam and Oxford)

The limits of repeatability of the alternative method are similar to those of the

reference method

The bias between both methods are low between - 0133 Log CFUg and

- 0008 Log CFUg

The Listeria PrecisTM enumeration method offers the saving for confirmatory

tests

Positive results are obtained with the alternative method in 2-3 days (with

OBIS Mono test) or in 4-7 days (by the classical tests described in the

standardized method) against 4-7 days for the reference method

Inter-laboratory study

The bias between the reference and the alternative methods shows satisfying

and correct values

- level 1 (100 - 1 000 CFUg) bias = + 007 log CFUg

- level 2 (1 000 - 10 000 CFUg) bias = 002 log CFUg

- level 2 bis (1 000 - 10 000 CFUg) bias = - 004 log CFUg

- level 3 (10 000 - 100 000 CFUg) bias = - 004 log CFUg

The repeatability and the reproducibility of the alternative method look similar

to those of the reference method

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3038 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

22 Extension study (realised by ASEPT 2007)

An extension study was performed in 2007 for a new confirmatory test OBIS

MONO

150 Listeria monocytogenes strains and 100 non-target strains were isolated

onto TSAYE and BrillianceTM Listeria Agar plates All the Listeria

monocytogenes and the non-target strains gave the expected results

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3138 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Appendix 1 ndash Flow diagram of the Listeria PrecisTM method

x g sample + 9 x ml buffered peptone water

Revivification step for 1 h plusmn 5 min at 20degC plusmn 1degC

Streaking 1 ml onto 3 Brilliance Listeria Agar plates (mother suspension)

or 01 ml onto 1 Brilliance Listeria Agar plates (mother suspension and decimal dilutions)

Incubation for 48 h plusmn 3 h at 37degC plusmn 1degC

Confirmatory tests on at least one typical colony

- by the tests described in the ISO method

- by applying the OBIS mono test on an isolated colony

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3238 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Appendix 2 ndash ISO 11290-2 (1998) amp ISO 11290-2A1 (2004) Horizontal

method for the detection and enumeration of Listeria monocytogenes -

Part 2 enumeration of Listeria monocytogenes in foods

10 g cheese + 90 ml buffered peptone water or Half Fraser Broth (without supplements) (110 dilution)

Stomach 1 min 30 seconds

Keep at 20degC plusmn 2degC for 1 h plusmn 5 min

Decimal dilutions in 9 ml peptone salt tubes

Dilution 110

Dilution 1100

Dilution 11000

1 ml on 3 Chromogenic Listeria medium (ISO) plates (x 2) (-1)

01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-2)

01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-3)

01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-4)

Incubation at 37degC plusmn 1degC

24 h - 48 h

Numerate typical colonies at 24 h

(bluegreen surrounded by an opaque halo)

Numerate at 48 h

Streak typical colonies on TSYEA

(5 coloniesplate 2 successive dilutions)

Confirmation tests

Catalase

Gram stain

For confirmation of L monocytogenes

Haemolysis

Carbohydrate (rhamnose and xylose) utilisation

CAMP Test

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3338 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Appendix 3 ndash Mandelrsquos graphics

Laboratoire h Ref

Niveau 1 h Ref

Niveau 2 h Ref

Niveau 3 h Alt

Niveau 1 h Alt

Niveau 2 h Alt

Niveau 3 h5 h1 h5 h1

A -050 -015 083 062 -212 -039 198 299 -198 -299

B -261 -270 021 -077 -058 173 198 299 -198 -299

C -151 -031 -014 -015 -004 -005 198 299 -198 -299

D 038 040 -137 000 072 207 198 299 -198 -299

E -001 090 155 -052 056 -101 198 299 -198 -299

F -112 -321 -153 070 -325 023 198 299 -198 -299

G 042 -071 040 147 -009 176 198 299 -198 -299

H 169 046 -169 192 018 -083 198 299 -198 -299

L 009 -120 021 -077 -231 005 198 299 -198 -299

M 001 031 014 -032 004 -011 198 299 -198 -299

-300

-200

-100

000

100

200

300

A B C D E F G H L M

Vale

urs

h d

e M

an

del(

rob

uste

s)

Laboratoires

Meacutethode de reacutefeacuterence analyse robuste

h Ref Niveau 1

h Ref Niveau 2

h Ref Niveau 3

h 5=198

h 1=299

-300

-200

-100

000

100

200

300

A B C D E F G H L M

Vale

urs

de h

de M

an

del(

rob

uste

s)

Laboratoires

Meacutethode alternative analyse robuste

h Alt Niveau 1

h Alt Niveau 2

h Alt Niveau 2

h 1=299

h 5=198

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3438 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Laboratoire k Ref

Niveau 1 k Ref

Niveau 2 k Ref

Niveau 3 k Alt

Niveau 1 k Alt

Niveau 2 k Alt

Niveau 3 k1 k5

A 000 089 142 084 162 046 259 181

B 125 087 073 104 075 046 259 181

C 117 013 091 188 022 083 259 181

D 052 036 069 000 035 083 259 181

E 028 034 037 138 019 112 259 181

F 140 222 018 027 281 112 259 181

G 088 068 024 131 145 000 259 181

H 129 094 173 016 061 000 259 181

L 069 000 073 104 270 224 259 181

M 000 048 038 043 099 241 259 181

000

050

100

150

200

250

300

350

400

A B C D E F G H L M

Vale

urs

k d

e M

an

del(

rob

uste

s)

Laboratoires

Meacutethode de reacutefeacuterence analyse robuste

k Ref Niveau 1k Ref Niveau 2k Ref Niveau 3

k 1=259

k 5=181

000

050

100

150

200

250

300

A B C D E F G H L M

Vale

urs

k d

e M

an

del

(ro

bu

ste

s)

Laboratoires

Meacutethode alternative analyse robuste

k Alt Niveau 3

k Alt Niveau 1

k Alt Niveau 2

k 1=259

k 5=181

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3538 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Appendix 4 ndash Statistical results

Level 1

Nombre de laboratoires (p) 10

A 1602 1602 1602 000 0000 0000 -0495 0000 A 1845 1602 1724 017 0122 -0122 0621 0840 0122

B 1602 1176 1389 030 0213 -0213 -2613 1247 B 1602 1301 1452 021 0151 -0151 -0769 1041 0062

C 1699 1301 1500 028 0199 -0199 -1510 1165 C 1301 1845 1573 038 -0272 0272 -0148 1881 0073

D 1602 1778 1690 012 -0088 0088 0380 0516 D 1602 1602 1602 000 0000 0000 0000 0000 -0088

E 1699 1602 1651 007 0048 -0048 -0013 0284 E 1699 1301 1500 028 0199 -0199 -0522 1376 -0151

F 1778 1301 1540 034 0239 -0239 -1116 1397 F 1778 1699 1739 006 0040 -0040 0698 0274 0199

G 1845 1544 1695 021 0151 -0151 0425 0881 G 2079 1699 1889 027 0190 -0190 1467 1315 0194

H 2041 1602 1822 031 0220 -0220 1689 1286 H 2000 1954 1977 003 0023 -0023 1917 0158 0155

L 1544 1778 1661 017 -0117 0117 0092 0685 L 1301 1602 1452 021 -0151 0151 -0769 1041 -0210

M 1653 1653 1653 000 0000 0000 0013 0000 M 1477 1602 1540 009 -0062 0062 -0319 0432 -0114

m 165186 m 160206 mediane D 00678

Cn(2p) 1867 Cn(2p) 1867 Cp 16101

Cn(p) 16101 Cn(p) 16101 Qn(D) 0092926

n=2p n 20 n=2p n 20 Qdiff 01496169

f 11 f 11 t 114

l 55 l 55

n=p n 10 n=p n 10

f 6 f 6

l 15 l 15

Qn(2p) 0091465 Qn(2p) 007745

Qn(p) 0062469 Qn(p) 0121519

Qintra 017078 Qintra 014461

Qinter 010058 Qinter 019565

Sr(eacutecart-type de reacutepeacutetabiliteacute) 02415 Sr 02045

RSDr(coefficient de variation de la reacutepeacutetabiliteacute)1462 RSDr 1277

r(repeatability limit)) 0676 r 0573

SL(eacutecart-type interlaboratoire) -001905 SLlt0 SL 001737

SR(eacutecart-type de reproductibiliteacute) 02415 SR 02433

RSDR(coefficient de variation de la reproductibiliteacute)0146 RSDR 0152

R(limite de reproductibiliteacute)) 06762 R(limite de reproductibiliteacute))068123

h k h MoyenneLaboratoires

Niveau 1

si LaboratoiresDeviationMoyenne Dk DeviationsiAlt

Niveau 1

Ref

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3638 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Level 2

Nombre de laboratoires (p) 10

A 2681 2613 2647 005 0034 -0034 -0151 0891 A 2322 2531 2427 015 -0105 0105 -2119 1620 -0220

B 2544 2477 2511 005 0033 -0033 -2699 0872 B 2556 2653 2605 007 -0048 0048 -0581 0750 0094

C 2643 2633 2638 001 0005 -0005 -0311 0130 C 2681 2653 2667 002 0014 -0014 -0040 0217 0029

D 2690 2663 2676 002 0014 -0014 0400 0357 D 2778 2732 2755 003 0023 -0023 0721 0354 0079

E 2690 2716 2703 002 -0013 0013 0897 0336 E 2724 2748 2736 002 -0012 0012 0556 0185 0033

F 2568 2398 2483 012 0085 -0085 -3214 2217 F 2477 2114 2296 026 0182 -0182 -3255 2811 -0188

G 2643 2591 2617 004 0026 -0026 -0707 0682 G 2756 2568 2662 013 0094 -0094 -0085 1453 0045

H 2716 2643 2680 005 0036 -0036 0460 0945 H 2653 2732 2693 006 -0040 0040 0181 0613 0013

L 2591 2591 2591 000 0000 0000 -1196 0000 L 2230 2580 2405 025 -0175 0175 -2307 2704 -0186

M 2653 2690 2672 003 -0018 0018 0311 0481 M 2740 2613 2677 009 0064 -0064 0040 0987 0005

m 2655082 m 26719001 mediane D 00209

Cn(2p) 1867 Cn(2p) 1867 Cp 16101

Cn(p) 16101 Cn(p) 16101 Qn(D) 0039911

n=2p n 20 n=2p n 20 Qdiff 00642591

f 11 f 11 t 082

l 55 l 55

n=p n 10 n=p n 10

f 6 f 6

l 15 l 15

Qn(2p) 0020570 Qn(2p) 00345985

Qn(p) 00332437 Qn(p) 00718158

Qintra 003841 Qintra 006460

Qinter 005352 Qinter 011563

Sr(eacutecart-type de reacutepeacutetabiliteacute) 00543 Sr 00914

RSDr(coefficient de variation de la reacutepeacutetabiliteacute)205 RSDr 342

r(repeatability limit)) 0152 r 0256

SL(eacutecart-type interlaboratoire) 000139 SL 000920

SR(eacutecart-type de reproductibiliteacute) 00659 SR 01325

RSDR(coefficient de variation de la reproductibiliteacute)0025 RSDR 0050

R(limite de reproductibiliteacute)) 018446 R(limite de reproductibiliteacute))03708618

Laboratoires

Niveau 2

Moyenne si Laboratoires

Niveau 2

DRef Alt Moyenne si Deviation h Deviation h k k

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3738 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Level 2 bis

Nombre de laboratoires (p) 10

A 3000 2477 2739 037 0261 -0261 0832 1418 A 2602 2477 2540 009 0062 -0062 -0391 0465 -0199

B 2544 2813 2678 019 -0134 0134 0211 0729 B 2903 2778 2841 009 0062 -0062 1732 0465 0162

C 2477 2813 2645 024 -0168 0168 -0135 0911 C 2477 2699 2588 016 -0111 0111 -0049 0826 -0057

D 2398 2653 2526 018 -0128 0128 -1370 0692 D 2778 3000 2889 016 -0111 0111 2074 0826 0363

E 2875 2740 2808 010 0067 -0067 1547 0365 E 2602 2301 2452 021 0151 -0151 -1012 1120 -0356

F 2477 2544 2511 005 -0033 0033 -1525 0182 F 2778 2477 2628 021 0151 -0151 0230 1120 0117

G 2653 2740 2697 006 -0044 0044 0400 0236 G 2845 2845 2845 000 0000 0000 1764 0000 0148

H 2176 2813 2495 045 -0318 0318 -1692 1727 H 2477 2477 2477 000 0000 0000 -0832 0000 -0017

L 2813 2544 2678 019 0134 -0134 0211 0729 L 2903 2301 2602 043 0301 -0301 0049 2241 -0076

M 2602 2740 2671 010 -0069 0069 0135 0375 M 2903 2255 2579 046 0324 -0324 -0112 2411 -0092

m 2658114 m 259505 mediane D -00372

Cn(2p) 1867 Cn(2p) 1867 Cp 16101

Cn(p) 16101 Cn(p) 16101 Qn(D) 01656915

n=2p n 20 n=2p n 20 Qdiff 02667753

f 11 f 11 t 035

l 55 l 55

n=p n 10 n=p n 10

f 6 f 6

l 15 l 15

Qn(2p) 0098745 Qn(2p) 007195

Qn(p) 006007 Qn(p) 008805

Qintra 018437 Qintra 013435

Qinter 009672 Qinter 014176

Sr(eacutecart-type de reacutepeacutetabiliteacute) 02607 Sr 01900

RSDr(coefficient de variation de la reacutepeacutetabiliteacute)981 RSDr 732

r(repeatability limit)) 0730 r 0532

SL(eacutecart-type interlaboratoire) -002464 SLlt0 SL 000205

SR(eacutecart-type de reproductibiliteacute) 02607 SR 01953

RSDR(coefficient de variation de la reproductibiliteacute)0098 RSDR 0075

R(limite de reproductibiliteacute)) 072996 R(limite de reproductibiliteacute))054686

Laboratoires

Niveau 2 bis

Moyenne si Laboratoires

Niveau 2 bis

DRef Alt Moyenne si Deviation h Deviation h k k

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3838 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Level 3

Nombre de laboratoires (p) 10

A 3690 3663 3676 002 0014 -0014 -0311 0513 A 3690 3708 3699 001 -0009 0009 0354 0257 0022

B 3613 3672 3642 004 -0030 0030 -1851 1109 B 3708 3568 3638 010 0070 -0070 -0844 2061 -0005

C 3663 3732 3698 005 -0035 0035 0645 1302 C 3663 3699 3681 003 -0018 0018 0000 0536 -0017

D 3690 3690 3690 000 0000 0000 0311 0000 D 3643 3663 3653 001 -0010 0010 -0545 0286 -0037

E 3732 3653 3693 006 0040 -0040 0429 1480 E 3613 3732 3673 008 -0060 0060 -0162 1769 -0020

F 3643 3672 3658 002 -0014 0014 -1157 0536 F 3690 3771 3731 006 -0040 0040 0975 1193 0073

G 3699 3653 3676 003 0023 -0023 -0328 0856 G 3602 3643 3623 003 -0021 0021 -1141 0612 -0053

H 3623 3672 3648 003 -0024 0024 -1615 0913 H 3748 3681 3715 005 0033 -0033 0664 0990 0067

L 3724 3663 3694 004 0031 -0031 0461 1150 L 3748 3771 3760 002 -0011 0011 1544 0335 0066

M 3690 3690 3690 000 0000 0000 0311 0000 M 3806 3681 3744 009 0062 -0062 1234 1848 0054

m 368334 m 368086 mediane D 00089

Cn(2p) 1867 Cn(2p) 1867 Cp 16101

Cn(p) 16101 Cn(p) 16101 Qn(D) 00325359

n=2p n 20 n=2p n 20 Qdiff 00523852

f 11 f 11 t 043

l 55 l 55

n=p n 10 n=p n 10

f 6 f 6

l 15 l 15

Qn(2p) 0014323 Qn(2p) 001811

Qn(p) 0013719 Qn(p) 003164

Qintra 002674 Qintra 003381

Qinter 002209 Qinter 005094

Sr(eacutecart-type de reacutepeacutetabiliteacute) 00378 Sr 00478

RSDr(coefficient de variation de la reacutepeacutetabiliteacute)103 RSDr 130

r(repeatability limit)) 0106 r 0134

SL(eacutecart-type interlaboratoire) -000023 SLlt0 SL 000145

SR(eacutecart-type de reproductibiliteacute) 00378 SR 00611

RSDR(coefficient de variation de la reproductibiliteacute)0010 RSDR 0017

R(limite de reproductibiliteacute)) 0105894 R(limite de reproductibiliteacute))017119

Ref Alt DLaboratoires

Niveau 3

Moyenne si Deviation h k Laboratoires

Niveau 3

Moyenne si Deviation h k

Page 14: ÞÀ¿¸7 ú Y5zñLjÜ< /u +æü t¬Á ... - NF Validation · NF VALIDATION Validation of alternative analytical methods Application in food microbiology ... 500 – 1 000 1 000 –

Thermo Fisher Scientific

ADRIA Deacuteveloppement 1438 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Figure 4 ndash Regression straight lines

Meat products

Fishery products

Dairy products

Vegetables

Thermo Fisher Scientific

ADRIA Deacuteveloppement 1538 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Environmental samples

Egg products

All products (estimated number)

Thermo Fisher Scientific

ADRIA Deacuteveloppement 1638 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

The hypotheses that the ordinates are close to 0 and the slopes close to 1

are approved in all cases except for

- Meat products (slope P = 1 ordinate P = 1)

- Environmental samples (ordinate P = 3)

- All products (ordinate P = 0)

The bias between the two methods vary from ndash 0133 to 0008 log CFU For

all categories the bias is ndash 0032 log CFU The repeatability limits of the

alternative and the reference method are similar For all products the values

are respectively 0215 and 0196 log CFU For all products these

interpretations are provided taking into account data with low contamination

level or not (estimated colonies number)

For all food products the regression straight line is the following

log (Alt) = 1040 log Ref ndash 0182

The Listeria PrecisTM method shows satisfying relative accuracy

All products

All products (Level 2)

Thermo Fisher Scientific

ADRIA Deacuteveloppement 1738 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

2113 Detection limit (LOD) and quantification limit (LOQ)

The critical level is the defined as the smallest amount which can be detected (not null) but not

quantified as an exact value Below this value it cannot be sure that the true value is not null

The detection limit is defined as being higher than the critical level because it involves a power the

probability 1- which has to be well over 50 for example 95

The quantification limit is defined as the smallest amount of analyte (that is the lowest actual number of

organisms) which can be measured and quantified with defined precision and accuracy under the

experimental conditions by the method under validation

Protocol

The detection limit of the alternative method was realised with pure cultures

Three different inoculation levels were tested with six replicates per level ie

18 analyses were done by the alternative method

Listeria monocytogenes 18 was used for the detection limit of the alternative

method

Quantification limit was calculated for six independent blank samples

determinations

Results

These data are intrinsic to the method used and are presented in the

following tables

Table 8 ndash Inoculation 1 ml on 3 plates

Level (CFUml) Positive samples number Standard deviation Bias

0 06

05 26 1211 0

1 36 0816 05

5 66 2483 85

Thermo Fisher Scientific

ADRIA Deacuteveloppement 1838 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Table 9 ndash Inoculation 01 ml on 1 plate

Level (CFUml) Positive samples number Standard deviation Bias

0 06

5 36 0548 05

10 46 1414 2

50 66 2258 55

Table 10

Formulas Obtained values

LC 165 S0 + X0 18

LOD 33 S0 + X0 32

LOQ 10 S0 + X0 87

2114 Relative Sensitivity

The relative sensitivity is defined as the ability of the alternative method to detect two different amounts

of analyte measured by the reference method within a given matrix at a specified average value or

over the whole measurement range that is it is the minimal quantity variation (increase of the analyte

concentration x) which gives a significant variation of the measured signal (response y)

These data are intrinsic to the used method and are obtained from the results

of the linearity study

Sensitivity patterns obtained for tested (matrixstrain) pairs are presented in

figure 5

Thermo Fisher Scientific

ADRIA Deacuteveloppement 1938 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Figure 5 ndash Sensitivity patterns for the tested (matrixstrain) pairs

2115 SpecificitySelectivity

The specificity is defined as the degree to which a method is affected (or not) by the other components

present in a multi-component sample That is the ability of a method to measure exactly a given

analyte or its amount within the sample without interference from non-target components such as a

matrix effect or background noise

The selectivity is defined as a measure of the degree of non-interference in the presence of non-target

analytes A method is selective if it can be used to detect the analyte under examination and that a

guarantee can be provided that the detected signal can only be a product by that specific analyte

The specificity and selectivity study was performed for the Listeria PrecisTM

detection method no additional test was realised for the enumeration

method The results were the following

- Among the Listeria monocytogenes strains tested 2 strains (Listeria

monocytogenes CIP 7834 and Listeria monocytogenes CIP 105459) gave

negative results These 2 strains were tested twice by running 48 h

incubation time of the compared agars Positive results were then

observed by the alternative method Pale characteristic colonies were

observed on the ISO agar by streaking the Half Fraser broth only

- No cross reaction was observed with the non-target strains except with

Listeria ivanovii which gave characteristic colonies on Brilliancetrade Listeria

Agar but the confirmatory tests provide a clear discrimination between

Listeria monocytogenes and Listeria ivanovi species

Rillettes

Lettuce

Raw milk

Smoked salmon

Raw egg

Process water

x (CFUg)

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2038 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

212 Practicability

1 Packaging Ready to use Brilliance Listeria agar plates are available

(x 10 plates)

2 Reagent volume

3 Storage conditionsndash

Shelf life of products

not opened

The plates are stored at 6 - 12degC in darkness conditions The

expiration date is mentioned on the package and on the plates It

is around 3 months

4 Modalities of use

after first use

5 Required

equipments No specific equipment is required

6 Reagents Ready to use plates

7 Training 05 day

8 Real time handling

Reference method

9 min for one sample

55 min for 10 samples

Alternative method

7 min for one sample

35 minutes for 10 samples

9 Time to result

Negative results

Reference method Day 2

Alternative method Day 2

Negative results after confirmation or positive results

Reference method Day 4 ndash Day 7

Alternative method Day 4 ndash Day 7

Positive results

Reference method Day 4 ndash Day 7

Alternative method Day 2 ndash Day 3 (with OBIS Mono Test)

or Day 4 ndash Day 7 (by the classical tests described in the

standardised method)

10 Technician skill Identical to that required for the reference method

11 Steps common to

the reference method Mother suspension revivification step and decimal dilutions

12 Traceability

13 Maintenance

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2138 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

213 Inter-laboratory study

2131 Organisation

12 laboratories participated to this study Pasteurised milk was inoculated by

Listeria monocytogenes 12a isolated from raw milk The targeted

inoculation levels were

- 0 CFUml

- 50 CFU ml

- 500 CFU ml

- 5 000 CFU ml

Each laboratory received eight flasks of 10 ml sample ie two flasks per

inoculation level Furthermore one non-inoculated sample has been added

to the package for total viable count microflora (NF ISO 4833 method)

Detailed instructions were transmitted to the collaborators by the expert

laboratory

Collaborative study laboratories and the expert laboratory carried out the

analyses with the alternative and reference methods

2132 Verification of experimental parameters

Strain stability during transport

In order to evaluate the Listeria monocytogenes strain stability during

transport bacterial count of all inoculated flasks were checked at different

time ie inoculation time after 24 h of conservation at 2deg - 8degC Results are

reported in table 11

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2238 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Table 11 ndash Listeria monocytogenes count

by ISO 11290-2A1 method (log CFUml)

Day of the analyses Level contamination Reference method

Day 0

(29052006)

1 45

2 440

3 4 500

Day 1

(30052006)

1 60

2 430

3 4 900

No evolution of the strain was observed after storage for 24 h at 2degC ndash 8degC

Contamination level

The real contamination levels are provided in table 12

Table 12 ndash Real contamination level (CFUml)

Target inoculation

(CFU ml) Samples Ndeg

Real contamination levels

(CFUml)

0 1 - 8 0

50 5 - 6 45

500 3 - 7 440

5000 2 - 4 4 500

Sample temperature at receipt

Measured temperatures at receipt are listed in table 13

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2338 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Table 13 - Sample temperature on receipt

Laboratories Receipt

day

Hour

receipt

Measured temperature

at receipt (degC)

Measured temperature by the

temperature probe (degC)

A

B

C

D

E

F

G

H

I

J

K

L

M

30052006

10h45

10h15

11h15

10h00

09h30

8h30

11h50

10h00

08h30

14h45

08h30

09h45

08h45

82

55

45

63

60

62

81

38

48

-

14

67

76

03

03

-13

03

04

-14

05

04

01

05

-13

04

Conclusion

The temperatures during the transport were correct for all the labs except for

Lab J which didnrsquot provide the information

All the labs received their packages with several broken flasks

Three labs (B D and L) received some damaged samples but proceeded to

analyses

All the labs started the analyses at Day 1 except Lab J

2133 Results

Aerobic mesophilic flora

The count of aerobic mesophilic flora varied from 94 102 CFUml to

33 103 CFUml

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2438 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Expert lab results

The results obtained by the expert lab are the following

Table 14

Target inoculation level (CFUml)

Reference method ISO 11290-2

Alternative method Listeria Precis Enumeration

Rep 1 Rep 2 Rep 1 Rep 2

lt 10 lt 10 lt 10 lt 10 lt10

50 50 (Ne) 60 (Ne) 50 (Ne) 30 (Ne)

500 470 390 490 500

5 000 4 700 5 100 5 900 3 400

Ne estimated number

The obtained results are in agreement with those expected

Collaborator Labs results

A synthesis of the results obtained by the collaborator labs is provided

Table 15 (CFUml) and Table 16 (log CFUml)

Table 15 ndash Synthesis of results obtained by the alternative

and the reference methods (CFUml)

Labs Level 1 Level 2 Level 2 bis Level 3

Reference method

Alternative method

Reference method

Alternative method

Reference method

Alternative method

Reference method

Alternative method

A 40 40 70 40 480 410 210 340 1000 300 400 300 4900 4600 4900 5100

B 40 15 40 20 350 300 360 450 350 650 800 600 4100 4700 5100 3700

C 50 20 20 70 440 430 480 450 300 650 300 500 4600 5400 4600 5000

D 40 60 40 40 490 460 600 540 250 450 600 1000 4900 4900 4400 4600

E 50 40 50 20 490 520 530 560 750 550 400 200 5400 4500 4100 5400

F 60 20 60 50 370 250 300 130 300 350 600 300 4400 4700 4900 5900

G 70 35 120 50 440 390 570 370 450 550 700 700 5000 4500 4000 4400

H 110 40 100 90 520 440 450 540 150 650 300 300 4200 4700 5600 4800

L 35 60 20 40 390 390 170 380 650 350 800 200 5300 4600 5600 5900

M 45 45 30 40 450 490 550 410 400 550 800 180 4900 4900 6400 4800

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2538 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Table 16 ndash Synthesis of results obtained by the alternative

and the reference methods (log CFUml)

Labs Level 1 Level 2 Level 2 bis Level 3

Reference method

Alternative method

Reference method

Alternative method

Reference method

Alternative method

Reference method

Alternative method

A 1602 1602 1845 1602 2681 2613 2322 2531 3000 2477 2602 2477 3690 3663 3690 3708

B 1602 1176 1602 1301 2544 2477 2556 2653 2544 2813 2903 2778 3613 3672 3708 3568

C 1699 1301 1301 1845 2643 2633 2681 2653 2477 2813 2477 2699 3663 3732 3663 3699

D 1602 1778 1602 1602 2690 2663 2778 2732 2398 2653 2778 3000 3690 3690 3643 3663

E 1699 1602 1699 1301 2690 2716 2724 2748 2875 2740 2602 2301 3732 3653 3613 3732

F 1778 1301 1778 1699 2568 2398 2477 2114 2477 2544 2778 2477 3643 3672 3690 3771

G 1845 1544 2079 1699 2643 2591 2756 2568 2653 2740 2845 2845 3699 3653 3602 3643

H 2041 1602 2000 1954 2716 2643 2653 2732 2176 2813 2477 2477 3623 3672 3748 3681

L 1544 1778 1301 1602 2591 2591 2230 2580 2813 2544 2903 2301 3724 3663 3748 3771

M 1653 1653 1477 1602 2653 2690 2740 2613 2602 2740 2903 2255 3690 3690 3806 3681

Level 2 results obtained taking account the 3 plates inoculated with the mother-suspension

Level 2 bis results obtained taking account the plates inoculated with 01 ml of the mother-suspension

Scrutiny of the measurement results for consistency

In order to identify the measurement results or laboratories that could be

inconsistent two graphical consistency techniques were realised Mandelrsquos

h-ank-statistics Mandel indicators h and k at 5 significance highlighted

some possible inconsistent data (See tables and graphics in Appendix 3 and

Appendix 4)

Mandelrsquos values Number of values observed over the threshold

Reference method Alternative method

h gt 1 Lab F Level 2 Lab F Level 2

h gt 5 Lab F Level 2

Lab B Levels 1 and 2

Lab A Level 2

Lab D Level 3

Lab F Level 2

Lab L Level 2

kgt 1 Lab F Level 3

k gt 5 Lab F Level 2 Lab C Level 2

Lab F Level 3

Lab L Levels 1 and 3

Lab M Level 1

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2638 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

The emphasized results appear (i) coherent with the targeted contamination

levels (ii) concordant between both methods

All the results were thus kept for the statistical analysis

Comparison of the trueness and precision characteristics of

the reference method and alternative methods

The statistical values are summarised hereafter

Level p

Reference method Alternative method

Repeatability ratio

Reproducibility ratio Median

Repeatability standard deviation

Reproducibility standard deviation

Median Repeatability

standard deviation

Reproducibility standard deviation

1 10 16519 02415 02425 16021 02045 02433 0847 1007

2 10 26551 00543 00659 26719 00914 01325 1682 2011

2 bis 10 26581 02607 02607 25951 01900 01953 0729 0749

3 10 18671 00378 00378 18671 00478 00611 1264 1617

Bias of the alternative method

In order to estimate the bias of the alternative method with regard to the reference method for every

level Dij and t are calculated according to the following equations

RefAlt ijYijYDij

Diffp

Dijit

)2(

)(median

If the t value is superior to 2 the alternative method is significantly biased

with regard to the reference method

The values are given in table 17

Table 17 ndash t (d) values obtained by level

Level Bias t (d) Conclusion

1 0068 114 Non significant bias

2 0021 082 Non significant bias

2 bis - 0037 035 Non significant bias

3 - 0037 043 Non significant bias

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2738 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

The statistical test concludes to a non significant bias for all the inoculation

levels The bias varies from ndash 0037 to 0068 log CFUml

Comparison of the repeatability standard deviations

If the ratio Srj Alt Srj Ref of the repeatability standard deviations of the

alternative method and the reference method is larger than 2 the precision

under repeatability conditions of the alternative method is considered to be

lower than that of the reference method If this ratio is smaller than 05 the

precision under repeatability conditions of the alternative method is

considered to be greater than that of the reference method

The ratio values are given in table 18

Table 18

Level

Repeatability standard deviation

Reference method Alternative method Alternative method

reference method ratio

1 0242 0205 0847

2 0054 0091 1682

2 bis 0261 0190 0729

3 0038 0048 1264

Level Repeatability limit

Reference method Alternative method

1 0676 0573

2 0152 0256

2 bis 0730 0532

3 0106 0134

The ratios of the repeatability standard deviations are below 2 in all cases

The precision under repeatability conditions of the alternative method is

equivalent to that of the reference method

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2838 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Comparison of the reproducibility standard deviations

If the ratio Srj Alt Srj Ref of the reproducibility standard deviations of the alternative method and the

reference method is larger than 2 the precision under reproducibility conditions of the alternative

method is considered to be lower than that of the reference method If this ratio is smaller than 05 the

precision under reproducibility conditions of the alternative method is considered to be greater than

that of the reference method

The ratio values are given in table 19

Table 19

Level

Reproducibility standard deviation

Reference method Alternative method Alternative method

reference method ratio

1 0242 0243 1007

2 0066 0133 2011

2 bis 0261 0195 0749

3 0038 0061 1617

Level Reproducibility limit

Reference method Alternative method

1 0676 0681

2 0185 0371

2 bis 0730 0547

3 0106 0171

The ratios of the reproducibility standard deviations are below or close to 2

The precision under reproducibility conditions of the alternative method is

equivalent to that of the reference method

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2938 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

214 Conclusion

Method comparison study

The Listeria PrecisTM enumeration method shows satisfying linearity and

relative accuracy

The alternative method shows satisfying inclusivity and exclusivity results

Two Listeria monocytogenes strains gave negative results with the

alternative method These strains grew weakly on the reference method

media (OampA Palcam and Oxford)

The limits of repeatability of the alternative method are similar to those of the

reference method

The bias between both methods are low between - 0133 Log CFUg and

- 0008 Log CFUg

The Listeria PrecisTM enumeration method offers the saving for confirmatory

tests

Positive results are obtained with the alternative method in 2-3 days (with

OBIS Mono test) or in 4-7 days (by the classical tests described in the

standardized method) against 4-7 days for the reference method

Inter-laboratory study

The bias between the reference and the alternative methods shows satisfying

and correct values

- level 1 (100 - 1 000 CFUg) bias = + 007 log CFUg

- level 2 (1 000 - 10 000 CFUg) bias = 002 log CFUg

- level 2 bis (1 000 - 10 000 CFUg) bias = - 004 log CFUg

- level 3 (10 000 - 100 000 CFUg) bias = - 004 log CFUg

The repeatability and the reproducibility of the alternative method look similar

to those of the reference method

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3038 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

22 Extension study (realised by ASEPT 2007)

An extension study was performed in 2007 for a new confirmatory test OBIS

MONO

150 Listeria monocytogenes strains and 100 non-target strains were isolated

onto TSAYE and BrillianceTM Listeria Agar plates All the Listeria

monocytogenes and the non-target strains gave the expected results

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3138 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Appendix 1 ndash Flow diagram of the Listeria PrecisTM method

x g sample + 9 x ml buffered peptone water

Revivification step for 1 h plusmn 5 min at 20degC plusmn 1degC

Streaking 1 ml onto 3 Brilliance Listeria Agar plates (mother suspension)

or 01 ml onto 1 Brilliance Listeria Agar plates (mother suspension and decimal dilutions)

Incubation for 48 h plusmn 3 h at 37degC plusmn 1degC

Confirmatory tests on at least one typical colony

- by the tests described in the ISO method

- by applying the OBIS mono test on an isolated colony

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3238 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Appendix 2 ndash ISO 11290-2 (1998) amp ISO 11290-2A1 (2004) Horizontal

method for the detection and enumeration of Listeria monocytogenes -

Part 2 enumeration of Listeria monocytogenes in foods

10 g cheese + 90 ml buffered peptone water or Half Fraser Broth (without supplements) (110 dilution)

Stomach 1 min 30 seconds

Keep at 20degC plusmn 2degC for 1 h plusmn 5 min

Decimal dilutions in 9 ml peptone salt tubes

Dilution 110

Dilution 1100

Dilution 11000

1 ml on 3 Chromogenic Listeria medium (ISO) plates (x 2) (-1)

01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-2)

01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-3)

01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-4)

Incubation at 37degC plusmn 1degC

24 h - 48 h

Numerate typical colonies at 24 h

(bluegreen surrounded by an opaque halo)

Numerate at 48 h

Streak typical colonies on TSYEA

(5 coloniesplate 2 successive dilutions)

Confirmation tests

Catalase

Gram stain

For confirmation of L monocytogenes

Haemolysis

Carbohydrate (rhamnose and xylose) utilisation

CAMP Test

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3338 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Appendix 3 ndash Mandelrsquos graphics

Laboratoire h Ref

Niveau 1 h Ref

Niveau 2 h Ref

Niveau 3 h Alt

Niveau 1 h Alt

Niveau 2 h Alt

Niveau 3 h5 h1 h5 h1

A -050 -015 083 062 -212 -039 198 299 -198 -299

B -261 -270 021 -077 -058 173 198 299 -198 -299

C -151 -031 -014 -015 -004 -005 198 299 -198 -299

D 038 040 -137 000 072 207 198 299 -198 -299

E -001 090 155 -052 056 -101 198 299 -198 -299

F -112 -321 -153 070 -325 023 198 299 -198 -299

G 042 -071 040 147 -009 176 198 299 -198 -299

H 169 046 -169 192 018 -083 198 299 -198 -299

L 009 -120 021 -077 -231 005 198 299 -198 -299

M 001 031 014 -032 004 -011 198 299 -198 -299

-300

-200

-100

000

100

200

300

A B C D E F G H L M

Vale

urs

h d

e M

an

del(

rob

uste

s)

Laboratoires

Meacutethode de reacutefeacuterence analyse robuste

h Ref Niveau 1

h Ref Niveau 2

h Ref Niveau 3

h 5=198

h 1=299

-300

-200

-100

000

100

200

300

A B C D E F G H L M

Vale

urs

de h

de M

an

del(

rob

uste

s)

Laboratoires

Meacutethode alternative analyse robuste

h Alt Niveau 1

h Alt Niveau 2

h Alt Niveau 2

h 1=299

h 5=198

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3438 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Laboratoire k Ref

Niveau 1 k Ref

Niveau 2 k Ref

Niveau 3 k Alt

Niveau 1 k Alt

Niveau 2 k Alt

Niveau 3 k1 k5

A 000 089 142 084 162 046 259 181

B 125 087 073 104 075 046 259 181

C 117 013 091 188 022 083 259 181

D 052 036 069 000 035 083 259 181

E 028 034 037 138 019 112 259 181

F 140 222 018 027 281 112 259 181

G 088 068 024 131 145 000 259 181

H 129 094 173 016 061 000 259 181

L 069 000 073 104 270 224 259 181

M 000 048 038 043 099 241 259 181

000

050

100

150

200

250

300

350

400

A B C D E F G H L M

Vale

urs

k d

e M

an

del(

rob

uste

s)

Laboratoires

Meacutethode de reacutefeacuterence analyse robuste

k Ref Niveau 1k Ref Niveau 2k Ref Niveau 3

k 1=259

k 5=181

000

050

100

150

200

250

300

A B C D E F G H L M

Vale

urs

k d

e M

an

del

(ro

bu

ste

s)

Laboratoires

Meacutethode alternative analyse robuste

k Alt Niveau 3

k Alt Niveau 1

k Alt Niveau 2

k 1=259

k 5=181

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3538 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Appendix 4 ndash Statistical results

Level 1

Nombre de laboratoires (p) 10

A 1602 1602 1602 000 0000 0000 -0495 0000 A 1845 1602 1724 017 0122 -0122 0621 0840 0122

B 1602 1176 1389 030 0213 -0213 -2613 1247 B 1602 1301 1452 021 0151 -0151 -0769 1041 0062

C 1699 1301 1500 028 0199 -0199 -1510 1165 C 1301 1845 1573 038 -0272 0272 -0148 1881 0073

D 1602 1778 1690 012 -0088 0088 0380 0516 D 1602 1602 1602 000 0000 0000 0000 0000 -0088

E 1699 1602 1651 007 0048 -0048 -0013 0284 E 1699 1301 1500 028 0199 -0199 -0522 1376 -0151

F 1778 1301 1540 034 0239 -0239 -1116 1397 F 1778 1699 1739 006 0040 -0040 0698 0274 0199

G 1845 1544 1695 021 0151 -0151 0425 0881 G 2079 1699 1889 027 0190 -0190 1467 1315 0194

H 2041 1602 1822 031 0220 -0220 1689 1286 H 2000 1954 1977 003 0023 -0023 1917 0158 0155

L 1544 1778 1661 017 -0117 0117 0092 0685 L 1301 1602 1452 021 -0151 0151 -0769 1041 -0210

M 1653 1653 1653 000 0000 0000 0013 0000 M 1477 1602 1540 009 -0062 0062 -0319 0432 -0114

m 165186 m 160206 mediane D 00678

Cn(2p) 1867 Cn(2p) 1867 Cp 16101

Cn(p) 16101 Cn(p) 16101 Qn(D) 0092926

n=2p n 20 n=2p n 20 Qdiff 01496169

f 11 f 11 t 114

l 55 l 55

n=p n 10 n=p n 10

f 6 f 6

l 15 l 15

Qn(2p) 0091465 Qn(2p) 007745

Qn(p) 0062469 Qn(p) 0121519

Qintra 017078 Qintra 014461

Qinter 010058 Qinter 019565

Sr(eacutecart-type de reacutepeacutetabiliteacute) 02415 Sr 02045

RSDr(coefficient de variation de la reacutepeacutetabiliteacute)1462 RSDr 1277

r(repeatability limit)) 0676 r 0573

SL(eacutecart-type interlaboratoire) -001905 SLlt0 SL 001737

SR(eacutecart-type de reproductibiliteacute) 02415 SR 02433

RSDR(coefficient de variation de la reproductibiliteacute)0146 RSDR 0152

R(limite de reproductibiliteacute)) 06762 R(limite de reproductibiliteacute))068123

h k h MoyenneLaboratoires

Niveau 1

si LaboratoiresDeviationMoyenne Dk DeviationsiAlt

Niveau 1

Ref

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3638 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Level 2

Nombre de laboratoires (p) 10

A 2681 2613 2647 005 0034 -0034 -0151 0891 A 2322 2531 2427 015 -0105 0105 -2119 1620 -0220

B 2544 2477 2511 005 0033 -0033 -2699 0872 B 2556 2653 2605 007 -0048 0048 -0581 0750 0094

C 2643 2633 2638 001 0005 -0005 -0311 0130 C 2681 2653 2667 002 0014 -0014 -0040 0217 0029

D 2690 2663 2676 002 0014 -0014 0400 0357 D 2778 2732 2755 003 0023 -0023 0721 0354 0079

E 2690 2716 2703 002 -0013 0013 0897 0336 E 2724 2748 2736 002 -0012 0012 0556 0185 0033

F 2568 2398 2483 012 0085 -0085 -3214 2217 F 2477 2114 2296 026 0182 -0182 -3255 2811 -0188

G 2643 2591 2617 004 0026 -0026 -0707 0682 G 2756 2568 2662 013 0094 -0094 -0085 1453 0045

H 2716 2643 2680 005 0036 -0036 0460 0945 H 2653 2732 2693 006 -0040 0040 0181 0613 0013

L 2591 2591 2591 000 0000 0000 -1196 0000 L 2230 2580 2405 025 -0175 0175 -2307 2704 -0186

M 2653 2690 2672 003 -0018 0018 0311 0481 M 2740 2613 2677 009 0064 -0064 0040 0987 0005

m 2655082 m 26719001 mediane D 00209

Cn(2p) 1867 Cn(2p) 1867 Cp 16101

Cn(p) 16101 Cn(p) 16101 Qn(D) 0039911

n=2p n 20 n=2p n 20 Qdiff 00642591

f 11 f 11 t 082

l 55 l 55

n=p n 10 n=p n 10

f 6 f 6

l 15 l 15

Qn(2p) 0020570 Qn(2p) 00345985

Qn(p) 00332437 Qn(p) 00718158

Qintra 003841 Qintra 006460

Qinter 005352 Qinter 011563

Sr(eacutecart-type de reacutepeacutetabiliteacute) 00543 Sr 00914

RSDr(coefficient de variation de la reacutepeacutetabiliteacute)205 RSDr 342

r(repeatability limit)) 0152 r 0256

SL(eacutecart-type interlaboratoire) 000139 SL 000920

SR(eacutecart-type de reproductibiliteacute) 00659 SR 01325

RSDR(coefficient de variation de la reproductibiliteacute)0025 RSDR 0050

R(limite de reproductibiliteacute)) 018446 R(limite de reproductibiliteacute))03708618

Laboratoires

Niveau 2

Moyenne si Laboratoires

Niveau 2

DRef Alt Moyenne si Deviation h Deviation h k k

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3738 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Level 2 bis

Nombre de laboratoires (p) 10

A 3000 2477 2739 037 0261 -0261 0832 1418 A 2602 2477 2540 009 0062 -0062 -0391 0465 -0199

B 2544 2813 2678 019 -0134 0134 0211 0729 B 2903 2778 2841 009 0062 -0062 1732 0465 0162

C 2477 2813 2645 024 -0168 0168 -0135 0911 C 2477 2699 2588 016 -0111 0111 -0049 0826 -0057

D 2398 2653 2526 018 -0128 0128 -1370 0692 D 2778 3000 2889 016 -0111 0111 2074 0826 0363

E 2875 2740 2808 010 0067 -0067 1547 0365 E 2602 2301 2452 021 0151 -0151 -1012 1120 -0356

F 2477 2544 2511 005 -0033 0033 -1525 0182 F 2778 2477 2628 021 0151 -0151 0230 1120 0117

G 2653 2740 2697 006 -0044 0044 0400 0236 G 2845 2845 2845 000 0000 0000 1764 0000 0148

H 2176 2813 2495 045 -0318 0318 -1692 1727 H 2477 2477 2477 000 0000 0000 -0832 0000 -0017

L 2813 2544 2678 019 0134 -0134 0211 0729 L 2903 2301 2602 043 0301 -0301 0049 2241 -0076

M 2602 2740 2671 010 -0069 0069 0135 0375 M 2903 2255 2579 046 0324 -0324 -0112 2411 -0092

m 2658114 m 259505 mediane D -00372

Cn(2p) 1867 Cn(2p) 1867 Cp 16101

Cn(p) 16101 Cn(p) 16101 Qn(D) 01656915

n=2p n 20 n=2p n 20 Qdiff 02667753

f 11 f 11 t 035

l 55 l 55

n=p n 10 n=p n 10

f 6 f 6

l 15 l 15

Qn(2p) 0098745 Qn(2p) 007195

Qn(p) 006007 Qn(p) 008805

Qintra 018437 Qintra 013435

Qinter 009672 Qinter 014176

Sr(eacutecart-type de reacutepeacutetabiliteacute) 02607 Sr 01900

RSDr(coefficient de variation de la reacutepeacutetabiliteacute)981 RSDr 732

r(repeatability limit)) 0730 r 0532

SL(eacutecart-type interlaboratoire) -002464 SLlt0 SL 000205

SR(eacutecart-type de reproductibiliteacute) 02607 SR 01953

RSDR(coefficient de variation de la reproductibiliteacute)0098 RSDR 0075

R(limite de reproductibiliteacute)) 072996 R(limite de reproductibiliteacute))054686

Laboratoires

Niveau 2 bis

Moyenne si Laboratoires

Niveau 2 bis

DRef Alt Moyenne si Deviation h Deviation h k k

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3838 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Level 3

Nombre de laboratoires (p) 10

A 3690 3663 3676 002 0014 -0014 -0311 0513 A 3690 3708 3699 001 -0009 0009 0354 0257 0022

B 3613 3672 3642 004 -0030 0030 -1851 1109 B 3708 3568 3638 010 0070 -0070 -0844 2061 -0005

C 3663 3732 3698 005 -0035 0035 0645 1302 C 3663 3699 3681 003 -0018 0018 0000 0536 -0017

D 3690 3690 3690 000 0000 0000 0311 0000 D 3643 3663 3653 001 -0010 0010 -0545 0286 -0037

E 3732 3653 3693 006 0040 -0040 0429 1480 E 3613 3732 3673 008 -0060 0060 -0162 1769 -0020

F 3643 3672 3658 002 -0014 0014 -1157 0536 F 3690 3771 3731 006 -0040 0040 0975 1193 0073

G 3699 3653 3676 003 0023 -0023 -0328 0856 G 3602 3643 3623 003 -0021 0021 -1141 0612 -0053

H 3623 3672 3648 003 -0024 0024 -1615 0913 H 3748 3681 3715 005 0033 -0033 0664 0990 0067

L 3724 3663 3694 004 0031 -0031 0461 1150 L 3748 3771 3760 002 -0011 0011 1544 0335 0066

M 3690 3690 3690 000 0000 0000 0311 0000 M 3806 3681 3744 009 0062 -0062 1234 1848 0054

m 368334 m 368086 mediane D 00089

Cn(2p) 1867 Cn(2p) 1867 Cp 16101

Cn(p) 16101 Cn(p) 16101 Qn(D) 00325359

n=2p n 20 n=2p n 20 Qdiff 00523852

f 11 f 11 t 043

l 55 l 55

n=p n 10 n=p n 10

f 6 f 6

l 15 l 15

Qn(2p) 0014323 Qn(2p) 001811

Qn(p) 0013719 Qn(p) 003164

Qintra 002674 Qintra 003381

Qinter 002209 Qinter 005094

Sr(eacutecart-type de reacutepeacutetabiliteacute) 00378 Sr 00478

RSDr(coefficient de variation de la reacutepeacutetabiliteacute)103 RSDr 130

r(repeatability limit)) 0106 r 0134

SL(eacutecart-type interlaboratoire) -000023 SLlt0 SL 000145

SR(eacutecart-type de reproductibiliteacute) 00378 SR 00611

RSDR(coefficient de variation de la reproductibiliteacute)0010 RSDR 0017

R(limite de reproductibiliteacute)) 0105894 R(limite de reproductibiliteacute))017119

Ref Alt DLaboratoires

Niveau 3

Moyenne si Deviation h k Laboratoires

Niveau 3

Moyenne si Deviation h k

Page 15: ÞÀ¿¸7 ú Y5zñLjÜ< /u +æü t¬Á ... - NF Validation · NF VALIDATION Validation of alternative analytical methods Application in food microbiology ... 500 – 1 000 1 000 –

Thermo Fisher Scientific

ADRIA Deacuteveloppement 1538 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Environmental samples

Egg products

All products (estimated number)

Thermo Fisher Scientific

ADRIA Deacuteveloppement 1638 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

The hypotheses that the ordinates are close to 0 and the slopes close to 1

are approved in all cases except for

- Meat products (slope P = 1 ordinate P = 1)

- Environmental samples (ordinate P = 3)

- All products (ordinate P = 0)

The bias between the two methods vary from ndash 0133 to 0008 log CFU For

all categories the bias is ndash 0032 log CFU The repeatability limits of the

alternative and the reference method are similar For all products the values

are respectively 0215 and 0196 log CFU For all products these

interpretations are provided taking into account data with low contamination

level or not (estimated colonies number)

For all food products the regression straight line is the following

log (Alt) = 1040 log Ref ndash 0182

The Listeria PrecisTM method shows satisfying relative accuracy

All products

All products (Level 2)

Thermo Fisher Scientific

ADRIA Deacuteveloppement 1738 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

2113 Detection limit (LOD) and quantification limit (LOQ)

The critical level is the defined as the smallest amount which can be detected (not null) but not

quantified as an exact value Below this value it cannot be sure that the true value is not null

The detection limit is defined as being higher than the critical level because it involves a power the

probability 1- which has to be well over 50 for example 95

The quantification limit is defined as the smallest amount of analyte (that is the lowest actual number of

organisms) which can be measured and quantified with defined precision and accuracy under the

experimental conditions by the method under validation

Protocol

The detection limit of the alternative method was realised with pure cultures

Three different inoculation levels were tested with six replicates per level ie

18 analyses were done by the alternative method

Listeria monocytogenes 18 was used for the detection limit of the alternative

method

Quantification limit was calculated for six independent blank samples

determinations

Results

These data are intrinsic to the method used and are presented in the

following tables

Table 8 ndash Inoculation 1 ml on 3 plates

Level (CFUml) Positive samples number Standard deviation Bias

0 06

05 26 1211 0

1 36 0816 05

5 66 2483 85

Thermo Fisher Scientific

ADRIA Deacuteveloppement 1838 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Table 9 ndash Inoculation 01 ml on 1 plate

Level (CFUml) Positive samples number Standard deviation Bias

0 06

5 36 0548 05

10 46 1414 2

50 66 2258 55

Table 10

Formulas Obtained values

LC 165 S0 + X0 18

LOD 33 S0 + X0 32

LOQ 10 S0 + X0 87

2114 Relative Sensitivity

The relative sensitivity is defined as the ability of the alternative method to detect two different amounts

of analyte measured by the reference method within a given matrix at a specified average value or

over the whole measurement range that is it is the minimal quantity variation (increase of the analyte

concentration x) which gives a significant variation of the measured signal (response y)

These data are intrinsic to the used method and are obtained from the results

of the linearity study

Sensitivity patterns obtained for tested (matrixstrain) pairs are presented in

figure 5

Thermo Fisher Scientific

ADRIA Deacuteveloppement 1938 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Figure 5 ndash Sensitivity patterns for the tested (matrixstrain) pairs

2115 SpecificitySelectivity

The specificity is defined as the degree to which a method is affected (or not) by the other components

present in a multi-component sample That is the ability of a method to measure exactly a given

analyte or its amount within the sample without interference from non-target components such as a

matrix effect or background noise

The selectivity is defined as a measure of the degree of non-interference in the presence of non-target

analytes A method is selective if it can be used to detect the analyte under examination and that a

guarantee can be provided that the detected signal can only be a product by that specific analyte

The specificity and selectivity study was performed for the Listeria PrecisTM

detection method no additional test was realised for the enumeration

method The results were the following

- Among the Listeria monocytogenes strains tested 2 strains (Listeria

monocytogenes CIP 7834 and Listeria monocytogenes CIP 105459) gave

negative results These 2 strains were tested twice by running 48 h

incubation time of the compared agars Positive results were then

observed by the alternative method Pale characteristic colonies were

observed on the ISO agar by streaking the Half Fraser broth only

- No cross reaction was observed with the non-target strains except with

Listeria ivanovii which gave characteristic colonies on Brilliancetrade Listeria

Agar but the confirmatory tests provide a clear discrimination between

Listeria monocytogenes and Listeria ivanovi species

Rillettes

Lettuce

Raw milk

Smoked salmon

Raw egg

Process water

x (CFUg)

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2038 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

212 Practicability

1 Packaging Ready to use Brilliance Listeria agar plates are available

(x 10 plates)

2 Reagent volume

3 Storage conditionsndash

Shelf life of products

not opened

The plates are stored at 6 - 12degC in darkness conditions The

expiration date is mentioned on the package and on the plates It

is around 3 months

4 Modalities of use

after first use

5 Required

equipments No specific equipment is required

6 Reagents Ready to use plates

7 Training 05 day

8 Real time handling

Reference method

9 min for one sample

55 min for 10 samples

Alternative method

7 min for one sample

35 minutes for 10 samples

9 Time to result

Negative results

Reference method Day 2

Alternative method Day 2

Negative results after confirmation or positive results

Reference method Day 4 ndash Day 7

Alternative method Day 4 ndash Day 7

Positive results

Reference method Day 4 ndash Day 7

Alternative method Day 2 ndash Day 3 (with OBIS Mono Test)

or Day 4 ndash Day 7 (by the classical tests described in the

standardised method)

10 Technician skill Identical to that required for the reference method

11 Steps common to

the reference method Mother suspension revivification step and decimal dilutions

12 Traceability

13 Maintenance

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2138 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

213 Inter-laboratory study

2131 Organisation

12 laboratories participated to this study Pasteurised milk was inoculated by

Listeria monocytogenes 12a isolated from raw milk The targeted

inoculation levels were

- 0 CFUml

- 50 CFU ml

- 500 CFU ml

- 5 000 CFU ml

Each laboratory received eight flasks of 10 ml sample ie two flasks per

inoculation level Furthermore one non-inoculated sample has been added

to the package for total viable count microflora (NF ISO 4833 method)

Detailed instructions were transmitted to the collaborators by the expert

laboratory

Collaborative study laboratories and the expert laboratory carried out the

analyses with the alternative and reference methods

2132 Verification of experimental parameters

Strain stability during transport

In order to evaluate the Listeria monocytogenes strain stability during

transport bacterial count of all inoculated flasks were checked at different

time ie inoculation time after 24 h of conservation at 2deg - 8degC Results are

reported in table 11

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2238 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Table 11 ndash Listeria monocytogenes count

by ISO 11290-2A1 method (log CFUml)

Day of the analyses Level contamination Reference method

Day 0

(29052006)

1 45

2 440

3 4 500

Day 1

(30052006)

1 60

2 430

3 4 900

No evolution of the strain was observed after storage for 24 h at 2degC ndash 8degC

Contamination level

The real contamination levels are provided in table 12

Table 12 ndash Real contamination level (CFUml)

Target inoculation

(CFU ml) Samples Ndeg

Real contamination levels

(CFUml)

0 1 - 8 0

50 5 - 6 45

500 3 - 7 440

5000 2 - 4 4 500

Sample temperature at receipt

Measured temperatures at receipt are listed in table 13

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2338 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Table 13 - Sample temperature on receipt

Laboratories Receipt

day

Hour

receipt

Measured temperature

at receipt (degC)

Measured temperature by the

temperature probe (degC)

A

B

C

D

E

F

G

H

I

J

K

L

M

30052006

10h45

10h15

11h15

10h00

09h30

8h30

11h50

10h00

08h30

14h45

08h30

09h45

08h45

82

55

45

63

60

62

81

38

48

-

14

67

76

03

03

-13

03

04

-14

05

04

01

05

-13

04

Conclusion

The temperatures during the transport were correct for all the labs except for

Lab J which didnrsquot provide the information

All the labs received their packages with several broken flasks

Three labs (B D and L) received some damaged samples but proceeded to

analyses

All the labs started the analyses at Day 1 except Lab J

2133 Results

Aerobic mesophilic flora

The count of aerobic mesophilic flora varied from 94 102 CFUml to

33 103 CFUml

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2438 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Expert lab results

The results obtained by the expert lab are the following

Table 14

Target inoculation level (CFUml)

Reference method ISO 11290-2

Alternative method Listeria Precis Enumeration

Rep 1 Rep 2 Rep 1 Rep 2

lt 10 lt 10 lt 10 lt 10 lt10

50 50 (Ne) 60 (Ne) 50 (Ne) 30 (Ne)

500 470 390 490 500

5 000 4 700 5 100 5 900 3 400

Ne estimated number

The obtained results are in agreement with those expected

Collaborator Labs results

A synthesis of the results obtained by the collaborator labs is provided

Table 15 (CFUml) and Table 16 (log CFUml)

Table 15 ndash Synthesis of results obtained by the alternative

and the reference methods (CFUml)

Labs Level 1 Level 2 Level 2 bis Level 3

Reference method

Alternative method

Reference method

Alternative method

Reference method

Alternative method

Reference method

Alternative method

A 40 40 70 40 480 410 210 340 1000 300 400 300 4900 4600 4900 5100

B 40 15 40 20 350 300 360 450 350 650 800 600 4100 4700 5100 3700

C 50 20 20 70 440 430 480 450 300 650 300 500 4600 5400 4600 5000

D 40 60 40 40 490 460 600 540 250 450 600 1000 4900 4900 4400 4600

E 50 40 50 20 490 520 530 560 750 550 400 200 5400 4500 4100 5400

F 60 20 60 50 370 250 300 130 300 350 600 300 4400 4700 4900 5900

G 70 35 120 50 440 390 570 370 450 550 700 700 5000 4500 4000 4400

H 110 40 100 90 520 440 450 540 150 650 300 300 4200 4700 5600 4800

L 35 60 20 40 390 390 170 380 650 350 800 200 5300 4600 5600 5900

M 45 45 30 40 450 490 550 410 400 550 800 180 4900 4900 6400 4800

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2538 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Table 16 ndash Synthesis of results obtained by the alternative

and the reference methods (log CFUml)

Labs Level 1 Level 2 Level 2 bis Level 3

Reference method

Alternative method

Reference method

Alternative method

Reference method

Alternative method

Reference method

Alternative method

A 1602 1602 1845 1602 2681 2613 2322 2531 3000 2477 2602 2477 3690 3663 3690 3708

B 1602 1176 1602 1301 2544 2477 2556 2653 2544 2813 2903 2778 3613 3672 3708 3568

C 1699 1301 1301 1845 2643 2633 2681 2653 2477 2813 2477 2699 3663 3732 3663 3699

D 1602 1778 1602 1602 2690 2663 2778 2732 2398 2653 2778 3000 3690 3690 3643 3663

E 1699 1602 1699 1301 2690 2716 2724 2748 2875 2740 2602 2301 3732 3653 3613 3732

F 1778 1301 1778 1699 2568 2398 2477 2114 2477 2544 2778 2477 3643 3672 3690 3771

G 1845 1544 2079 1699 2643 2591 2756 2568 2653 2740 2845 2845 3699 3653 3602 3643

H 2041 1602 2000 1954 2716 2643 2653 2732 2176 2813 2477 2477 3623 3672 3748 3681

L 1544 1778 1301 1602 2591 2591 2230 2580 2813 2544 2903 2301 3724 3663 3748 3771

M 1653 1653 1477 1602 2653 2690 2740 2613 2602 2740 2903 2255 3690 3690 3806 3681

Level 2 results obtained taking account the 3 plates inoculated with the mother-suspension

Level 2 bis results obtained taking account the plates inoculated with 01 ml of the mother-suspension

Scrutiny of the measurement results for consistency

In order to identify the measurement results or laboratories that could be

inconsistent two graphical consistency techniques were realised Mandelrsquos

h-ank-statistics Mandel indicators h and k at 5 significance highlighted

some possible inconsistent data (See tables and graphics in Appendix 3 and

Appendix 4)

Mandelrsquos values Number of values observed over the threshold

Reference method Alternative method

h gt 1 Lab F Level 2 Lab F Level 2

h gt 5 Lab F Level 2

Lab B Levels 1 and 2

Lab A Level 2

Lab D Level 3

Lab F Level 2

Lab L Level 2

kgt 1 Lab F Level 3

k gt 5 Lab F Level 2 Lab C Level 2

Lab F Level 3

Lab L Levels 1 and 3

Lab M Level 1

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2638 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

The emphasized results appear (i) coherent with the targeted contamination

levels (ii) concordant between both methods

All the results were thus kept for the statistical analysis

Comparison of the trueness and precision characteristics of

the reference method and alternative methods

The statistical values are summarised hereafter

Level p

Reference method Alternative method

Repeatability ratio

Reproducibility ratio Median

Repeatability standard deviation

Reproducibility standard deviation

Median Repeatability

standard deviation

Reproducibility standard deviation

1 10 16519 02415 02425 16021 02045 02433 0847 1007

2 10 26551 00543 00659 26719 00914 01325 1682 2011

2 bis 10 26581 02607 02607 25951 01900 01953 0729 0749

3 10 18671 00378 00378 18671 00478 00611 1264 1617

Bias of the alternative method

In order to estimate the bias of the alternative method with regard to the reference method for every

level Dij and t are calculated according to the following equations

RefAlt ijYijYDij

Diffp

Dijit

)2(

)(median

If the t value is superior to 2 the alternative method is significantly biased

with regard to the reference method

The values are given in table 17

Table 17 ndash t (d) values obtained by level

Level Bias t (d) Conclusion

1 0068 114 Non significant bias

2 0021 082 Non significant bias

2 bis - 0037 035 Non significant bias

3 - 0037 043 Non significant bias

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2738 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

The statistical test concludes to a non significant bias for all the inoculation

levels The bias varies from ndash 0037 to 0068 log CFUml

Comparison of the repeatability standard deviations

If the ratio Srj Alt Srj Ref of the repeatability standard deviations of the

alternative method and the reference method is larger than 2 the precision

under repeatability conditions of the alternative method is considered to be

lower than that of the reference method If this ratio is smaller than 05 the

precision under repeatability conditions of the alternative method is

considered to be greater than that of the reference method

The ratio values are given in table 18

Table 18

Level

Repeatability standard deviation

Reference method Alternative method Alternative method

reference method ratio

1 0242 0205 0847

2 0054 0091 1682

2 bis 0261 0190 0729

3 0038 0048 1264

Level Repeatability limit

Reference method Alternative method

1 0676 0573

2 0152 0256

2 bis 0730 0532

3 0106 0134

The ratios of the repeatability standard deviations are below 2 in all cases

The precision under repeatability conditions of the alternative method is

equivalent to that of the reference method

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2838 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Comparison of the reproducibility standard deviations

If the ratio Srj Alt Srj Ref of the reproducibility standard deviations of the alternative method and the

reference method is larger than 2 the precision under reproducibility conditions of the alternative

method is considered to be lower than that of the reference method If this ratio is smaller than 05 the

precision under reproducibility conditions of the alternative method is considered to be greater than

that of the reference method

The ratio values are given in table 19

Table 19

Level

Reproducibility standard deviation

Reference method Alternative method Alternative method

reference method ratio

1 0242 0243 1007

2 0066 0133 2011

2 bis 0261 0195 0749

3 0038 0061 1617

Level Reproducibility limit

Reference method Alternative method

1 0676 0681

2 0185 0371

2 bis 0730 0547

3 0106 0171

The ratios of the reproducibility standard deviations are below or close to 2

The precision under reproducibility conditions of the alternative method is

equivalent to that of the reference method

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2938 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

214 Conclusion

Method comparison study

The Listeria PrecisTM enumeration method shows satisfying linearity and

relative accuracy

The alternative method shows satisfying inclusivity and exclusivity results

Two Listeria monocytogenes strains gave negative results with the

alternative method These strains grew weakly on the reference method

media (OampA Palcam and Oxford)

The limits of repeatability of the alternative method are similar to those of the

reference method

The bias between both methods are low between - 0133 Log CFUg and

- 0008 Log CFUg

The Listeria PrecisTM enumeration method offers the saving for confirmatory

tests

Positive results are obtained with the alternative method in 2-3 days (with

OBIS Mono test) or in 4-7 days (by the classical tests described in the

standardized method) against 4-7 days for the reference method

Inter-laboratory study

The bias between the reference and the alternative methods shows satisfying

and correct values

- level 1 (100 - 1 000 CFUg) bias = + 007 log CFUg

- level 2 (1 000 - 10 000 CFUg) bias = 002 log CFUg

- level 2 bis (1 000 - 10 000 CFUg) bias = - 004 log CFUg

- level 3 (10 000 - 100 000 CFUg) bias = - 004 log CFUg

The repeatability and the reproducibility of the alternative method look similar

to those of the reference method

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3038 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

22 Extension study (realised by ASEPT 2007)

An extension study was performed in 2007 for a new confirmatory test OBIS

MONO

150 Listeria monocytogenes strains and 100 non-target strains were isolated

onto TSAYE and BrillianceTM Listeria Agar plates All the Listeria

monocytogenes and the non-target strains gave the expected results

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3138 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Appendix 1 ndash Flow diagram of the Listeria PrecisTM method

x g sample + 9 x ml buffered peptone water

Revivification step for 1 h plusmn 5 min at 20degC plusmn 1degC

Streaking 1 ml onto 3 Brilliance Listeria Agar plates (mother suspension)

or 01 ml onto 1 Brilliance Listeria Agar plates (mother suspension and decimal dilutions)

Incubation for 48 h plusmn 3 h at 37degC plusmn 1degC

Confirmatory tests on at least one typical colony

- by the tests described in the ISO method

- by applying the OBIS mono test on an isolated colony

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3238 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Appendix 2 ndash ISO 11290-2 (1998) amp ISO 11290-2A1 (2004) Horizontal

method for the detection and enumeration of Listeria monocytogenes -

Part 2 enumeration of Listeria monocytogenes in foods

10 g cheese + 90 ml buffered peptone water or Half Fraser Broth (without supplements) (110 dilution)

Stomach 1 min 30 seconds

Keep at 20degC plusmn 2degC for 1 h plusmn 5 min

Decimal dilutions in 9 ml peptone salt tubes

Dilution 110

Dilution 1100

Dilution 11000

1 ml on 3 Chromogenic Listeria medium (ISO) plates (x 2) (-1)

01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-2)

01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-3)

01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-4)

Incubation at 37degC plusmn 1degC

24 h - 48 h

Numerate typical colonies at 24 h

(bluegreen surrounded by an opaque halo)

Numerate at 48 h

Streak typical colonies on TSYEA

(5 coloniesplate 2 successive dilutions)

Confirmation tests

Catalase

Gram stain

For confirmation of L monocytogenes

Haemolysis

Carbohydrate (rhamnose and xylose) utilisation

CAMP Test

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3338 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Appendix 3 ndash Mandelrsquos graphics

Laboratoire h Ref

Niveau 1 h Ref

Niveau 2 h Ref

Niveau 3 h Alt

Niveau 1 h Alt

Niveau 2 h Alt

Niveau 3 h5 h1 h5 h1

A -050 -015 083 062 -212 -039 198 299 -198 -299

B -261 -270 021 -077 -058 173 198 299 -198 -299

C -151 -031 -014 -015 -004 -005 198 299 -198 -299

D 038 040 -137 000 072 207 198 299 -198 -299

E -001 090 155 -052 056 -101 198 299 -198 -299

F -112 -321 -153 070 -325 023 198 299 -198 -299

G 042 -071 040 147 -009 176 198 299 -198 -299

H 169 046 -169 192 018 -083 198 299 -198 -299

L 009 -120 021 -077 -231 005 198 299 -198 -299

M 001 031 014 -032 004 -011 198 299 -198 -299

-300

-200

-100

000

100

200

300

A B C D E F G H L M

Vale

urs

h d

e M

an

del(

rob

uste

s)

Laboratoires

Meacutethode de reacutefeacuterence analyse robuste

h Ref Niveau 1

h Ref Niveau 2

h Ref Niveau 3

h 5=198

h 1=299

-300

-200

-100

000

100

200

300

A B C D E F G H L M

Vale

urs

de h

de M

an

del(

rob

uste

s)

Laboratoires

Meacutethode alternative analyse robuste

h Alt Niveau 1

h Alt Niveau 2

h Alt Niveau 2

h 1=299

h 5=198

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3438 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Laboratoire k Ref

Niveau 1 k Ref

Niveau 2 k Ref

Niveau 3 k Alt

Niveau 1 k Alt

Niveau 2 k Alt

Niveau 3 k1 k5

A 000 089 142 084 162 046 259 181

B 125 087 073 104 075 046 259 181

C 117 013 091 188 022 083 259 181

D 052 036 069 000 035 083 259 181

E 028 034 037 138 019 112 259 181

F 140 222 018 027 281 112 259 181

G 088 068 024 131 145 000 259 181

H 129 094 173 016 061 000 259 181

L 069 000 073 104 270 224 259 181

M 000 048 038 043 099 241 259 181

000

050

100

150

200

250

300

350

400

A B C D E F G H L M

Vale

urs

k d

e M

an

del(

rob

uste

s)

Laboratoires

Meacutethode de reacutefeacuterence analyse robuste

k Ref Niveau 1k Ref Niveau 2k Ref Niveau 3

k 1=259

k 5=181

000

050

100

150

200

250

300

A B C D E F G H L M

Vale

urs

k d

e M

an

del

(ro

bu

ste

s)

Laboratoires

Meacutethode alternative analyse robuste

k Alt Niveau 3

k Alt Niveau 1

k Alt Niveau 2

k 1=259

k 5=181

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3538 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Appendix 4 ndash Statistical results

Level 1

Nombre de laboratoires (p) 10

A 1602 1602 1602 000 0000 0000 -0495 0000 A 1845 1602 1724 017 0122 -0122 0621 0840 0122

B 1602 1176 1389 030 0213 -0213 -2613 1247 B 1602 1301 1452 021 0151 -0151 -0769 1041 0062

C 1699 1301 1500 028 0199 -0199 -1510 1165 C 1301 1845 1573 038 -0272 0272 -0148 1881 0073

D 1602 1778 1690 012 -0088 0088 0380 0516 D 1602 1602 1602 000 0000 0000 0000 0000 -0088

E 1699 1602 1651 007 0048 -0048 -0013 0284 E 1699 1301 1500 028 0199 -0199 -0522 1376 -0151

F 1778 1301 1540 034 0239 -0239 -1116 1397 F 1778 1699 1739 006 0040 -0040 0698 0274 0199

G 1845 1544 1695 021 0151 -0151 0425 0881 G 2079 1699 1889 027 0190 -0190 1467 1315 0194

H 2041 1602 1822 031 0220 -0220 1689 1286 H 2000 1954 1977 003 0023 -0023 1917 0158 0155

L 1544 1778 1661 017 -0117 0117 0092 0685 L 1301 1602 1452 021 -0151 0151 -0769 1041 -0210

M 1653 1653 1653 000 0000 0000 0013 0000 M 1477 1602 1540 009 -0062 0062 -0319 0432 -0114

m 165186 m 160206 mediane D 00678

Cn(2p) 1867 Cn(2p) 1867 Cp 16101

Cn(p) 16101 Cn(p) 16101 Qn(D) 0092926

n=2p n 20 n=2p n 20 Qdiff 01496169

f 11 f 11 t 114

l 55 l 55

n=p n 10 n=p n 10

f 6 f 6

l 15 l 15

Qn(2p) 0091465 Qn(2p) 007745

Qn(p) 0062469 Qn(p) 0121519

Qintra 017078 Qintra 014461

Qinter 010058 Qinter 019565

Sr(eacutecart-type de reacutepeacutetabiliteacute) 02415 Sr 02045

RSDr(coefficient de variation de la reacutepeacutetabiliteacute)1462 RSDr 1277

r(repeatability limit)) 0676 r 0573

SL(eacutecart-type interlaboratoire) -001905 SLlt0 SL 001737

SR(eacutecart-type de reproductibiliteacute) 02415 SR 02433

RSDR(coefficient de variation de la reproductibiliteacute)0146 RSDR 0152

R(limite de reproductibiliteacute)) 06762 R(limite de reproductibiliteacute))068123

h k h MoyenneLaboratoires

Niveau 1

si LaboratoiresDeviationMoyenne Dk DeviationsiAlt

Niveau 1

Ref

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3638 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Level 2

Nombre de laboratoires (p) 10

A 2681 2613 2647 005 0034 -0034 -0151 0891 A 2322 2531 2427 015 -0105 0105 -2119 1620 -0220

B 2544 2477 2511 005 0033 -0033 -2699 0872 B 2556 2653 2605 007 -0048 0048 -0581 0750 0094

C 2643 2633 2638 001 0005 -0005 -0311 0130 C 2681 2653 2667 002 0014 -0014 -0040 0217 0029

D 2690 2663 2676 002 0014 -0014 0400 0357 D 2778 2732 2755 003 0023 -0023 0721 0354 0079

E 2690 2716 2703 002 -0013 0013 0897 0336 E 2724 2748 2736 002 -0012 0012 0556 0185 0033

F 2568 2398 2483 012 0085 -0085 -3214 2217 F 2477 2114 2296 026 0182 -0182 -3255 2811 -0188

G 2643 2591 2617 004 0026 -0026 -0707 0682 G 2756 2568 2662 013 0094 -0094 -0085 1453 0045

H 2716 2643 2680 005 0036 -0036 0460 0945 H 2653 2732 2693 006 -0040 0040 0181 0613 0013

L 2591 2591 2591 000 0000 0000 -1196 0000 L 2230 2580 2405 025 -0175 0175 -2307 2704 -0186

M 2653 2690 2672 003 -0018 0018 0311 0481 M 2740 2613 2677 009 0064 -0064 0040 0987 0005

m 2655082 m 26719001 mediane D 00209

Cn(2p) 1867 Cn(2p) 1867 Cp 16101

Cn(p) 16101 Cn(p) 16101 Qn(D) 0039911

n=2p n 20 n=2p n 20 Qdiff 00642591

f 11 f 11 t 082

l 55 l 55

n=p n 10 n=p n 10

f 6 f 6

l 15 l 15

Qn(2p) 0020570 Qn(2p) 00345985

Qn(p) 00332437 Qn(p) 00718158

Qintra 003841 Qintra 006460

Qinter 005352 Qinter 011563

Sr(eacutecart-type de reacutepeacutetabiliteacute) 00543 Sr 00914

RSDr(coefficient de variation de la reacutepeacutetabiliteacute)205 RSDr 342

r(repeatability limit)) 0152 r 0256

SL(eacutecart-type interlaboratoire) 000139 SL 000920

SR(eacutecart-type de reproductibiliteacute) 00659 SR 01325

RSDR(coefficient de variation de la reproductibiliteacute)0025 RSDR 0050

R(limite de reproductibiliteacute)) 018446 R(limite de reproductibiliteacute))03708618

Laboratoires

Niveau 2

Moyenne si Laboratoires

Niveau 2

DRef Alt Moyenne si Deviation h Deviation h k k

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3738 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Level 2 bis

Nombre de laboratoires (p) 10

A 3000 2477 2739 037 0261 -0261 0832 1418 A 2602 2477 2540 009 0062 -0062 -0391 0465 -0199

B 2544 2813 2678 019 -0134 0134 0211 0729 B 2903 2778 2841 009 0062 -0062 1732 0465 0162

C 2477 2813 2645 024 -0168 0168 -0135 0911 C 2477 2699 2588 016 -0111 0111 -0049 0826 -0057

D 2398 2653 2526 018 -0128 0128 -1370 0692 D 2778 3000 2889 016 -0111 0111 2074 0826 0363

E 2875 2740 2808 010 0067 -0067 1547 0365 E 2602 2301 2452 021 0151 -0151 -1012 1120 -0356

F 2477 2544 2511 005 -0033 0033 -1525 0182 F 2778 2477 2628 021 0151 -0151 0230 1120 0117

G 2653 2740 2697 006 -0044 0044 0400 0236 G 2845 2845 2845 000 0000 0000 1764 0000 0148

H 2176 2813 2495 045 -0318 0318 -1692 1727 H 2477 2477 2477 000 0000 0000 -0832 0000 -0017

L 2813 2544 2678 019 0134 -0134 0211 0729 L 2903 2301 2602 043 0301 -0301 0049 2241 -0076

M 2602 2740 2671 010 -0069 0069 0135 0375 M 2903 2255 2579 046 0324 -0324 -0112 2411 -0092

m 2658114 m 259505 mediane D -00372

Cn(2p) 1867 Cn(2p) 1867 Cp 16101

Cn(p) 16101 Cn(p) 16101 Qn(D) 01656915

n=2p n 20 n=2p n 20 Qdiff 02667753

f 11 f 11 t 035

l 55 l 55

n=p n 10 n=p n 10

f 6 f 6

l 15 l 15

Qn(2p) 0098745 Qn(2p) 007195

Qn(p) 006007 Qn(p) 008805

Qintra 018437 Qintra 013435

Qinter 009672 Qinter 014176

Sr(eacutecart-type de reacutepeacutetabiliteacute) 02607 Sr 01900

RSDr(coefficient de variation de la reacutepeacutetabiliteacute)981 RSDr 732

r(repeatability limit)) 0730 r 0532

SL(eacutecart-type interlaboratoire) -002464 SLlt0 SL 000205

SR(eacutecart-type de reproductibiliteacute) 02607 SR 01953

RSDR(coefficient de variation de la reproductibiliteacute)0098 RSDR 0075

R(limite de reproductibiliteacute)) 072996 R(limite de reproductibiliteacute))054686

Laboratoires

Niveau 2 bis

Moyenne si Laboratoires

Niveau 2 bis

DRef Alt Moyenne si Deviation h Deviation h k k

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3838 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Level 3

Nombre de laboratoires (p) 10

A 3690 3663 3676 002 0014 -0014 -0311 0513 A 3690 3708 3699 001 -0009 0009 0354 0257 0022

B 3613 3672 3642 004 -0030 0030 -1851 1109 B 3708 3568 3638 010 0070 -0070 -0844 2061 -0005

C 3663 3732 3698 005 -0035 0035 0645 1302 C 3663 3699 3681 003 -0018 0018 0000 0536 -0017

D 3690 3690 3690 000 0000 0000 0311 0000 D 3643 3663 3653 001 -0010 0010 -0545 0286 -0037

E 3732 3653 3693 006 0040 -0040 0429 1480 E 3613 3732 3673 008 -0060 0060 -0162 1769 -0020

F 3643 3672 3658 002 -0014 0014 -1157 0536 F 3690 3771 3731 006 -0040 0040 0975 1193 0073

G 3699 3653 3676 003 0023 -0023 -0328 0856 G 3602 3643 3623 003 -0021 0021 -1141 0612 -0053

H 3623 3672 3648 003 -0024 0024 -1615 0913 H 3748 3681 3715 005 0033 -0033 0664 0990 0067

L 3724 3663 3694 004 0031 -0031 0461 1150 L 3748 3771 3760 002 -0011 0011 1544 0335 0066

M 3690 3690 3690 000 0000 0000 0311 0000 M 3806 3681 3744 009 0062 -0062 1234 1848 0054

m 368334 m 368086 mediane D 00089

Cn(2p) 1867 Cn(2p) 1867 Cp 16101

Cn(p) 16101 Cn(p) 16101 Qn(D) 00325359

n=2p n 20 n=2p n 20 Qdiff 00523852

f 11 f 11 t 043

l 55 l 55

n=p n 10 n=p n 10

f 6 f 6

l 15 l 15

Qn(2p) 0014323 Qn(2p) 001811

Qn(p) 0013719 Qn(p) 003164

Qintra 002674 Qintra 003381

Qinter 002209 Qinter 005094

Sr(eacutecart-type de reacutepeacutetabiliteacute) 00378 Sr 00478

RSDr(coefficient de variation de la reacutepeacutetabiliteacute)103 RSDr 130

r(repeatability limit)) 0106 r 0134

SL(eacutecart-type interlaboratoire) -000023 SLlt0 SL 000145

SR(eacutecart-type de reproductibiliteacute) 00378 SR 00611

RSDR(coefficient de variation de la reproductibiliteacute)0010 RSDR 0017

R(limite de reproductibiliteacute)) 0105894 R(limite de reproductibiliteacute))017119

Ref Alt DLaboratoires

Niveau 3

Moyenne si Deviation h k Laboratoires

Niveau 3

Moyenne si Deviation h k

Page 16: ÞÀ¿¸7 ú Y5zñLjÜ< /u +æü t¬Á ... - NF Validation · NF VALIDATION Validation of alternative analytical methods Application in food microbiology ... 500 – 1 000 1 000 –

Thermo Fisher Scientific

ADRIA Deacuteveloppement 1638 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

The hypotheses that the ordinates are close to 0 and the slopes close to 1

are approved in all cases except for

- Meat products (slope P = 1 ordinate P = 1)

- Environmental samples (ordinate P = 3)

- All products (ordinate P = 0)

The bias between the two methods vary from ndash 0133 to 0008 log CFU For

all categories the bias is ndash 0032 log CFU The repeatability limits of the

alternative and the reference method are similar For all products the values

are respectively 0215 and 0196 log CFU For all products these

interpretations are provided taking into account data with low contamination

level or not (estimated colonies number)

For all food products the regression straight line is the following

log (Alt) = 1040 log Ref ndash 0182

The Listeria PrecisTM method shows satisfying relative accuracy

All products

All products (Level 2)

Thermo Fisher Scientific

ADRIA Deacuteveloppement 1738 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

2113 Detection limit (LOD) and quantification limit (LOQ)

The critical level is the defined as the smallest amount which can be detected (not null) but not

quantified as an exact value Below this value it cannot be sure that the true value is not null

The detection limit is defined as being higher than the critical level because it involves a power the

probability 1- which has to be well over 50 for example 95

The quantification limit is defined as the smallest amount of analyte (that is the lowest actual number of

organisms) which can be measured and quantified with defined precision and accuracy under the

experimental conditions by the method under validation

Protocol

The detection limit of the alternative method was realised with pure cultures

Three different inoculation levels were tested with six replicates per level ie

18 analyses were done by the alternative method

Listeria monocytogenes 18 was used for the detection limit of the alternative

method

Quantification limit was calculated for six independent blank samples

determinations

Results

These data are intrinsic to the method used and are presented in the

following tables

Table 8 ndash Inoculation 1 ml on 3 plates

Level (CFUml) Positive samples number Standard deviation Bias

0 06

05 26 1211 0

1 36 0816 05

5 66 2483 85

Thermo Fisher Scientific

ADRIA Deacuteveloppement 1838 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Table 9 ndash Inoculation 01 ml on 1 plate

Level (CFUml) Positive samples number Standard deviation Bias

0 06

5 36 0548 05

10 46 1414 2

50 66 2258 55

Table 10

Formulas Obtained values

LC 165 S0 + X0 18

LOD 33 S0 + X0 32

LOQ 10 S0 + X0 87

2114 Relative Sensitivity

The relative sensitivity is defined as the ability of the alternative method to detect two different amounts

of analyte measured by the reference method within a given matrix at a specified average value or

over the whole measurement range that is it is the minimal quantity variation (increase of the analyte

concentration x) which gives a significant variation of the measured signal (response y)

These data are intrinsic to the used method and are obtained from the results

of the linearity study

Sensitivity patterns obtained for tested (matrixstrain) pairs are presented in

figure 5

Thermo Fisher Scientific

ADRIA Deacuteveloppement 1938 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Figure 5 ndash Sensitivity patterns for the tested (matrixstrain) pairs

2115 SpecificitySelectivity

The specificity is defined as the degree to which a method is affected (or not) by the other components

present in a multi-component sample That is the ability of a method to measure exactly a given

analyte or its amount within the sample without interference from non-target components such as a

matrix effect or background noise

The selectivity is defined as a measure of the degree of non-interference in the presence of non-target

analytes A method is selective if it can be used to detect the analyte under examination and that a

guarantee can be provided that the detected signal can only be a product by that specific analyte

The specificity and selectivity study was performed for the Listeria PrecisTM

detection method no additional test was realised for the enumeration

method The results were the following

- Among the Listeria monocytogenes strains tested 2 strains (Listeria

monocytogenes CIP 7834 and Listeria monocytogenes CIP 105459) gave

negative results These 2 strains were tested twice by running 48 h

incubation time of the compared agars Positive results were then

observed by the alternative method Pale characteristic colonies were

observed on the ISO agar by streaking the Half Fraser broth only

- No cross reaction was observed with the non-target strains except with

Listeria ivanovii which gave characteristic colonies on Brilliancetrade Listeria

Agar but the confirmatory tests provide a clear discrimination between

Listeria monocytogenes and Listeria ivanovi species

Rillettes

Lettuce

Raw milk

Smoked salmon

Raw egg

Process water

x (CFUg)

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2038 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

212 Practicability

1 Packaging Ready to use Brilliance Listeria agar plates are available

(x 10 plates)

2 Reagent volume

3 Storage conditionsndash

Shelf life of products

not opened

The plates are stored at 6 - 12degC in darkness conditions The

expiration date is mentioned on the package and on the plates It

is around 3 months

4 Modalities of use

after first use

5 Required

equipments No specific equipment is required

6 Reagents Ready to use plates

7 Training 05 day

8 Real time handling

Reference method

9 min for one sample

55 min for 10 samples

Alternative method

7 min for one sample

35 minutes for 10 samples

9 Time to result

Negative results

Reference method Day 2

Alternative method Day 2

Negative results after confirmation or positive results

Reference method Day 4 ndash Day 7

Alternative method Day 4 ndash Day 7

Positive results

Reference method Day 4 ndash Day 7

Alternative method Day 2 ndash Day 3 (with OBIS Mono Test)

or Day 4 ndash Day 7 (by the classical tests described in the

standardised method)

10 Technician skill Identical to that required for the reference method

11 Steps common to

the reference method Mother suspension revivification step and decimal dilutions

12 Traceability

13 Maintenance

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2138 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

213 Inter-laboratory study

2131 Organisation

12 laboratories participated to this study Pasteurised milk was inoculated by

Listeria monocytogenes 12a isolated from raw milk The targeted

inoculation levels were

- 0 CFUml

- 50 CFU ml

- 500 CFU ml

- 5 000 CFU ml

Each laboratory received eight flasks of 10 ml sample ie two flasks per

inoculation level Furthermore one non-inoculated sample has been added

to the package for total viable count microflora (NF ISO 4833 method)

Detailed instructions were transmitted to the collaborators by the expert

laboratory

Collaborative study laboratories and the expert laboratory carried out the

analyses with the alternative and reference methods

2132 Verification of experimental parameters

Strain stability during transport

In order to evaluate the Listeria monocytogenes strain stability during

transport bacterial count of all inoculated flasks were checked at different

time ie inoculation time after 24 h of conservation at 2deg - 8degC Results are

reported in table 11

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2238 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Table 11 ndash Listeria monocytogenes count

by ISO 11290-2A1 method (log CFUml)

Day of the analyses Level contamination Reference method

Day 0

(29052006)

1 45

2 440

3 4 500

Day 1

(30052006)

1 60

2 430

3 4 900

No evolution of the strain was observed after storage for 24 h at 2degC ndash 8degC

Contamination level

The real contamination levels are provided in table 12

Table 12 ndash Real contamination level (CFUml)

Target inoculation

(CFU ml) Samples Ndeg

Real contamination levels

(CFUml)

0 1 - 8 0

50 5 - 6 45

500 3 - 7 440

5000 2 - 4 4 500

Sample temperature at receipt

Measured temperatures at receipt are listed in table 13

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2338 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Table 13 - Sample temperature on receipt

Laboratories Receipt

day

Hour

receipt

Measured temperature

at receipt (degC)

Measured temperature by the

temperature probe (degC)

A

B

C

D

E

F

G

H

I

J

K

L

M

30052006

10h45

10h15

11h15

10h00

09h30

8h30

11h50

10h00

08h30

14h45

08h30

09h45

08h45

82

55

45

63

60

62

81

38

48

-

14

67

76

03

03

-13

03

04

-14

05

04

01

05

-13

04

Conclusion

The temperatures during the transport were correct for all the labs except for

Lab J which didnrsquot provide the information

All the labs received their packages with several broken flasks

Three labs (B D and L) received some damaged samples but proceeded to

analyses

All the labs started the analyses at Day 1 except Lab J

2133 Results

Aerobic mesophilic flora

The count of aerobic mesophilic flora varied from 94 102 CFUml to

33 103 CFUml

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2438 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Expert lab results

The results obtained by the expert lab are the following

Table 14

Target inoculation level (CFUml)

Reference method ISO 11290-2

Alternative method Listeria Precis Enumeration

Rep 1 Rep 2 Rep 1 Rep 2

lt 10 lt 10 lt 10 lt 10 lt10

50 50 (Ne) 60 (Ne) 50 (Ne) 30 (Ne)

500 470 390 490 500

5 000 4 700 5 100 5 900 3 400

Ne estimated number

The obtained results are in agreement with those expected

Collaborator Labs results

A synthesis of the results obtained by the collaborator labs is provided

Table 15 (CFUml) and Table 16 (log CFUml)

Table 15 ndash Synthesis of results obtained by the alternative

and the reference methods (CFUml)

Labs Level 1 Level 2 Level 2 bis Level 3

Reference method

Alternative method

Reference method

Alternative method

Reference method

Alternative method

Reference method

Alternative method

A 40 40 70 40 480 410 210 340 1000 300 400 300 4900 4600 4900 5100

B 40 15 40 20 350 300 360 450 350 650 800 600 4100 4700 5100 3700

C 50 20 20 70 440 430 480 450 300 650 300 500 4600 5400 4600 5000

D 40 60 40 40 490 460 600 540 250 450 600 1000 4900 4900 4400 4600

E 50 40 50 20 490 520 530 560 750 550 400 200 5400 4500 4100 5400

F 60 20 60 50 370 250 300 130 300 350 600 300 4400 4700 4900 5900

G 70 35 120 50 440 390 570 370 450 550 700 700 5000 4500 4000 4400

H 110 40 100 90 520 440 450 540 150 650 300 300 4200 4700 5600 4800

L 35 60 20 40 390 390 170 380 650 350 800 200 5300 4600 5600 5900

M 45 45 30 40 450 490 550 410 400 550 800 180 4900 4900 6400 4800

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2538 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Table 16 ndash Synthesis of results obtained by the alternative

and the reference methods (log CFUml)

Labs Level 1 Level 2 Level 2 bis Level 3

Reference method

Alternative method

Reference method

Alternative method

Reference method

Alternative method

Reference method

Alternative method

A 1602 1602 1845 1602 2681 2613 2322 2531 3000 2477 2602 2477 3690 3663 3690 3708

B 1602 1176 1602 1301 2544 2477 2556 2653 2544 2813 2903 2778 3613 3672 3708 3568

C 1699 1301 1301 1845 2643 2633 2681 2653 2477 2813 2477 2699 3663 3732 3663 3699

D 1602 1778 1602 1602 2690 2663 2778 2732 2398 2653 2778 3000 3690 3690 3643 3663

E 1699 1602 1699 1301 2690 2716 2724 2748 2875 2740 2602 2301 3732 3653 3613 3732

F 1778 1301 1778 1699 2568 2398 2477 2114 2477 2544 2778 2477 3643 3672 3690 3771

G 1845 1544 2079 1699 2643 2591 2756 2568 2653 2740 2845 2845 3699 3653 3602 3643

H 2041 1602 2000 1954 2716 2643 2653 2732 2176 2813 2477 2477 3623 3672 3748 3681

L 1544 1778 1301 1602 2591 2591 2230 2580 2813 2544 2903 2301 3724 3663 3748 3771

M 1653 1653 1477 1602 2653 2690 2740 2613 2602 2740 2903 2255 3690 3690 3806 3681

Level 2 results obtained taking account the 3 plates inoculated with the mother-suspension

Level 2 bis results obtained taking account the plates inoculated with 01 ml of the mother-suspension

Scrutiny of the measurement results for consistency

In order to identify the measurement results or laboratories that could be

inconsistent two graphical consistency techniques were realised Mandelrsquos

h-ank-statistics Mandel indicators h and k at 5 significance highlighted

some possible inconsistent data (See tables and graphics in Appendix 3 and

Appendix 4)

Mandelrsquos values Number of values observed over the threshold

Reference method Alternative method

h gt 1 Lab F Level 2 Lab F Level 2

h gt 5 Lab F Level 2

Lab B Levels 1 and 2

Lab A Level 2

Lab D Level 3

Lab F Level 2

Lab L Level 2

kgt 1 Lab F Level 3

k gt 5 Lab F Level 2 Lab C Level 2

Lab F Level 3

Lab L Levels 1 and 3

Lab M Level 1

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2638 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

The emphasized results appear (i) coherent with the targeted contamination

levels (ii) concordant between both methods

All the results were thus kept for the statistical analysis

Comparison of the trueness and precision characteristics of

the reference method and alternative methods

The statistical values are summarised hereafter

Level p

Reference method Alternative method

Repeatability ratio

Reproducibility ratio Median

Repeatability standard deviation

Reproducibility standard deviation

Median Repeatability

standard deviation

Reproducibility standard deviation

1 10 16519 02415 02425 16021 02045 02433 0847 1007

2 10 26551 00543 00659 26719 00914 01325 1682 2011

2 bis 10 26581 02607 02607 25951 01900 01953 0729 0749

3 10 18671 00378 00378 18671 00478 00611 1264 1617

Bias of the alternative method

In order to estimate the bias of the alternative method with regard to the reference method for every

level Dij and t are calculated according to the following equations

RefAlt ijYijYDij

Diffp

Dijit

)2(

)(median

If the t value is superior to 2 the alternative method is significantly biased

with regard to the reference method

The values are given in table 17

Table 17 ndash t (d) values obtained by level

Level Bias t (d) Conclusion

1 0068 114 Non significant bias

2 0021 082 Non significant bias

2 bis - 0037 035 Non significant bias

3 - 0037 043 Non significant bias

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2738 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

The statistical test concludes to a non significant bias for all the inoculation

levels The bias varies from ndash 0037 to 0068 log CFUml

Comparison of the repeatability standard deviations

If the ratio Srj Alt Srj Ref of the repeatability standard deviations of the

alternative method and the reference method is larger than 2 the precision

under repeatability conditions of the alternative method is considered to be

lower than that of the reference method If this ratio is smaller than 05 the

precision under repeatability conditions of the alternative method is

considered to be greater than that of the reference method

The ratio values are given in table 18

Table 18

Level

Repeatability standard deviation

Reference method Alternative method Alternative method

reference method ratio

1 0242 0205 0847

2 0054 0091 1682

2 bis 0261 0190 0729

3 0038 0048 1264

Level Repeatability limit

Reference method Alternative method

1 0676 0573

2 0152 0256

2 bis 0730 0532

3 0106 0134

The ratios of the repeatability standard deviations are below 2 in all cases

The precision under repeatability conditions of the alternative method is

equivalent to that of the reference method

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2838 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Comparison of the reproducibility standard deviations

If the ratio Srj Alt Srj Ref of the reproducibility standard deviations of the alternative method and the

reference method is larger than 2 the precision under reproducibility conditions of the alternative

method is considered to be lower than that of the reference method If this ratio is smaller than 05 the

precision under reproducibility conditions of the alternative method is considered to be greater than

that of the reference method

The ratio values are given in table 19

Table 19

Level

Reproducibility standard deviation

Reference method Alternative method Alternative method

reference method ratio

1 0242 0243 1007

2 0066 0133 2011

2 bis 0261 0195 0749

3 0038 0061 1617

Level Reproducibility limit

Reference method Alternative method

1 0676 0681

2 0185 0371

2 bis 0730 0547

3 0106 0171

The ratios of the reproducibility standard deviations are below or close to 2

The precision under reproducibility conditions of the alternative method is

equivalent to that of the reference method

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2938 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

214 Conclusion

Method comparison study

The Listeria PrecisTM enumeration method shows satisfying linearity and

relative accuracy

The alternative method shows satisfying inclusivity and exclusivity results

Two Listeria monocytogenes strains gave negative results with the

alternative method These strains grew weakly on the reference method

media (OampA Palcam and Oxford)

The limits of repeatability of the alternative method are similar to those of the

reference method

The bias between both methods are low between - 0133 Log CFUg and

- 0008 Log CFUg

The Listeria PrecisTM enumeration method offers the saving for confirmatory

tests

Positive results are obtained with the alternative method in 2-3 days (with

OBIS Mono test) or in 4-7 days (by the classical tests described in the

standardized method) against 4-7 days for the reference method

Inter-laboratory study

The bias between the reference and the alternative methods shows satisfying

and correct values

- level 1 (100 - 1 000 CFUg) bias = + 007 log CFUg

- level 2 (1 000 - 10 000 CFUg) bias = 002 log CFUg

- level 2 bis (1 000 - 10 000 CFUg) bias = - 004 log CFUg

- level 3 (10 000 - 100 000 CFUg) bias = - 004 log CFUg

The repeatability and the reproducibility of the alternative method look similar

to those of the reference method

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3038 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

22 Extension study (realised by ASEPT 2007)

An extension study was performed in 2007 for a new confirmatory test OBIS

MONO

150 Listeria monocytogenes strains and 100 non-target strains were isolated

onto TSAYE and BrillianceTM Listeria Agar plates All the Listeria

monocytogenes and the non-target strains gave the expected results

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3138 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Appendix 1 ndash Flow diagram of the Listeria PrecisTM method

x g sample + 9 x ml buffered peptone water

Revivification step for 1 h plusmn 5 min at 20degC plusmn 1degC

Streaking 1 ml onto 3 Brilliance Listeria Agar plates (mother suspension)

or 01 ml onto 1 Brilliance Listeria Agar plates (mother suspension and decimal dilutions)

Incubation for 48 h plusmn 3 h at 37degC plusmn 1degC

Confirmatory tests on at least one typical colony

- by the tests described in the ISO method

- by applying the OBIS mono test on an isolated colony

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3238 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Appendix 2 ndash ISO 11290-2 (1998) amp ISO 11290-2A1 (2004) Horizontal

method for the detection and enumeration of Listeria monocytogenes -

Part 2 enumeration of Listeria monocytogenes in foods

10 g cheese + 90 ml buffered peptone water or Half Fraser Broth (without supplements) (110 dilution)

Stomach 1 min 30 seconds

Keep at 20degC plusmn 2degC for 1 h plusmn 5 min

Decimal dilutions in 9 ml peptone salt tubes

Dilution 110

Dilution 1100

Dilution 11000

1 ml on 3 Chromogenic Listeria medium (ISO) plates (x 2) (-1)

01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-2)

01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-3)

01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-4)

Incubation at 37degC plusmn 1degC

24 h - 48 h

Numerate typical colonies at 24 h

(bluegreen surrounded by an opaque halo)

Numerate at 48 h

Streak typical colonies on TSYEA

(5 coloniesplate 2 successive dilutions)

Confirmation tests

Catalase

Gram stain

For confirmation of L monocytogenes

Haemolysis

Carbohydrate (rhamnose and xylose) utilisation

CAMP Test

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3338 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Appendix 3 ndash Mandelrsquos graphics

Laboratoire h Ref

Niveau 1 h Ref

Niveau 2 h Ref

Niveau 3 h Alt

Niveau 1 h Alt

Niveau 2 h Alt

Niveau 3 h5 h1 h5 h1

A -050 -015 083 062 -212 -039 198 299 -198 -299

B -261 -270 021 -077 -058 173 198 299 -198 -299

C -151 -031 -014 -015 -004 -005 198 299 -198 -299

D 038 040 -137 000 072 207 198 299 -198 -299

E -001 090 155 -052 056 -101 198 299 -198 -299

F -112 -321 -153 070 -325 023 198 299 -198 -299

G 042 -071 040 147 -009 176 198 299 -198 -299

H 169 046 -169 192 018 -083 198 299 -198 -299

L 009 -120 021 -077 -231 005 198 299 -198 -299

M 001 031 014 -032 004 -011 198 299 -198 -299

-300

-200

-100

000

100

200

300

A B C D E F G H L M

Vale

urs

h d

e M

an

del(

rob

uste

s)

Laboratoires

Meacutethode de reacutefeacuterence analyse robuste

h Ref Niveau 1

h Ref Niveau 2

h Ref Niveau 3

h 5=198

h 1=299

-300

-200

-100

000

100

200

300

A B C D E F G H L M

Vale

urs

de h

de M

an

del(

rob

uste

s)

Laboratoires

Meacutethode alternative analyse robuste

h Alt Niveau 1

h Alt Niveau 2

h Alt Niveau 2

h 1=299

h 5=198

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3438 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Laboratoire k Ref

Niveau 1 k Ref

Niveau 2 k Ref

Niveau 3 k Alt

Niveau 1 k Alt

Niveau 2 k Alt

Niveau 3 k1 k5

A 000 089 142 084 162 046 259 181

B 125 087 073 104 075 046 259 181

C 117 013 091 188 022 083 259 181

D 052 036 069 000 035 083 259 181

E 028 034 037 138 019 112 259 181

F 140 222 018 027 281 112 259 181

G 088 068 024 131 145 000 259 181

H 129 094 173 016 061 000 259 181

L 069 000 073 104 270 224 259 181

M 000 048 038 043 099 241 259 181

000

050

100

150

200

250

300

350

400

A B C D E F G H L M

Vale

urs

k d

e M

an

del(

rob

uste

s)

Laboratoires

Meacutethode de reacutefeacuterence analyse robuste

k Ref Niveau 1k Ref Niveau 2k Ref Niveau 3

k 1=259

k 5=181

000

050

100

150

200

250

300

A B C D E F G H L M

Vale

urs

k d

e M

an

del

(ro

bu

ste

s)

Laboratoires

Meacutethode alternative analyse robuste

k Alt Niveau 3

k Alt Niveau 1

k Alt Niveau 2

k 1=259

k 5=181

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3538 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Appendix 4 ndash Statistical results

Level 1

Nombre de laboratoires (p) 10

A 1602 1602 1602 000 0000 0000 -0495 0000 A 1845 1602 1724 017 0122 -0122 0621 0840 0122

B 1602 1176 1389 030 0213 -0213 -2613 1247 B 1602 1301 1452 021 0151 -0151 -0769 1041 0062

C 1699 1301 1500 028 0199 -0199 -1510 1165 C 1301 1845 1573 038 -0272 0272 -0148 1881 0073

D 1602 1778 1690 012 -0088 0088 0380 0516 D 1602 1602 1602 000 0000 0000 0000 0000 -0088

E 1699 1602 1651 007 0048 -0048 -0013 0284 E 1699 1301 1500 028 0199 -0199 -0522 1376 -0151

F 1778 1301 1540 034 0239 -0239 -1116 1397 F 1778 1699 1739 006 0040 -0040 0698 0274 0199

G 1845 1544 1695 021 0151 -0151 0425 0881 G 2079 1699 1889 027 0190 -0190 1467 1315 0194

H 2041 1602 1822 031 0220 -0220 1689 1286 H 2000 1954 1977 003 0023 -0023 1917 0158 0155

L 1544 1778 1661 017 -0117 0117 0092 0685 L 1301 1602 1452 021 -0151 0151 -0769 1041 -0210

M 1653 1653 1653 000 0000 0000 0013 0000 M 1477 1602 1540 009 -0062 0062 -0319 0432 -0114

m 165186 m 160206 mediane D 00678

Cn(2p) 1867 Cn(2p) 1867 Cp 16101

Cn(p) 16101 Cn(p) 16101 Qn(D) 0092926

n=2p n 20 n=2p n 20 Qdiff 01496169

f 11 f 11 t 114

l 55 l 55

n=p n 10 n=p n 10

f 6 f 6

l 15 l 15

Qn(2p) 0091465 Qn(2p) 007745

Qn(p) 0062469 Qn(p) 0121519

Qintra 017078 Qintra 014461

Qinter 010058 Qinter 019565

Sr(eacutecart-type de reacutepeacutetabiliteacute) 02415 Sr 02045

RSDr(coefficient de variation de la reacutepeacutetabiliteacute)1462 RSDr 1277

r(repeatability limit)) 0676 r 0573

SL(eacutecart-type interlaboratoire) -001905 SLlt0 SL 001737

SR(eacutecart-type de reproductibiliteacute) 02415 SR 02433

RSDR(coefficient de variation de la reproductibiliteacute)0146 RSDR 0152

R(limite de reproductibiliteacute)) 06762 R(limite de reproductibiliteacute))068123

h k h MoyenneLaboratoires

Niveau 1

si LaboratoiresDeviationMoyenne Dk DeviationsiAlt

Niveau 1

Ref

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3638 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Level 2

Nombre de laboratoires (p) 10

A 2681 2613 2647 005 0034 -0034 -0151 0891 A 2322 2531 2427 015 -0105 0105 -2119 1620 -0220

B 2544 2477 2511 005 0033 -0033 -2699 0872 B 2556 2653 2605 007 -0048 0048 -0581 0750 0094

C 2643 2633 2638 001 0005 -0005 -0311 0130 C 2681 2653 2667 002 0014 -0014 -0040 0217 0029

D 2690 2663 2676 002 0014 -0014 0400 0357 D 2778 2732 2755 003 0023 -0023 0721 0354 0079

E 2690 2716 2703 002 -0013 0013 0897 0336 E 2724 2748 2736 002 -0012 0012 0556 0185 0033

F 2568 2398 2483 012 0085 -0085 -3214 2217 F 2477 2114 2296 026 0182 -0182 -3255 2811 -0188

G 2643 2591 2617 004 0026 -0026 -0707 0682 G 2756 2568 2662 013 0094 -0094 -0085 1453 0045

H 2716 2643 2680 005 0036 -0036 0460 0945 H 2653 2732 2693 006 -0040 0040 0181 0613 0013

L 2591 2591 2591 000 0000 0000 -1196 0000 L 2230 2580 2405 025 -0175 0175 -2307 2704 -0186

M 2653 2690 2672 003 -0018 0018 0311 0481 M 2740 2613 2677 009 0064 -0064 0040 0987 0005

m 2655082 m 26719001 mediane D 00209

Cn(2p) 1867 Cn(2p) 1867 Cp 16101

Cn(p) 16101 Cn(p) 16101 Qn(D) 0039911

n=2p n 20 n=2p n 20 Qdiff 00642591

f 11 f 11 t 082

l 55 l 55

n=p n 10 n=p n 10

f 6 f 6

l 15 l 15

Qn(2p) 0020570 Qn(2p) 00345985

Qn(p) 00332437 Qn(p) 00718158

Qintra 003841 Qintra 006460

Qinter 005352 Qinter 011563

Sr(eacutecart-type de reacutepeacutetabiliteacute) 00543 Sr 00914

RSDr(coefficient de variation de la reacutepeacutetabiliteacute)205 RSDr 342

r(repeatability limit)) 0152 r 0256

SL(eacutecart-type interlaboratoire) 000139 SL 000920

SR(eacutecart-type de reproductibiliteacute) 00659 SR 01325

RSDR(coefficient de variation de la reproductibiliteacute)0025 RSDR 0050

R(limite de reproductibiliteacute)) 018446 R(limite de reproductibiliteacute))03708618

Laboratoires

Niveau 2

Moyenne si Laboratoires

Niveau 2

DRef Alt Moyenne si Deviation h Deviation h k k

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3738 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Level 2 bis

Nombre de laboratoires (p) 10

A 3000 2477 2739 037 0261 -0261 0832 1418 A 2602 2477 2540 009 0062 -0062 -0391 0465 -0199

B 2544 2813 2678 019 -0134 0134 0211 0729 B 2903 2778 2841 009 0062 -0062 1732 0465 0162

C 2477 2813 2645 024 -0168 0168 -0135 0911 C 2477 2699 2588 016 -0111 0111 -0049 0826 -0057

D 2398 2653 2526 018 -0128 0128 -1370 0692 D 2778 3000 2889 016 -0111 0111 2074 0826 0363

E 2875 2740 2808 010 0067 -0067 1547 0365 E 2602 2301 2452 021 0151 -0151 -1012 1120 -0356

F 2477 2544 2511 005 -0033 0033 -1525 0182 F 2778 2477 2628 021 0151 -0151 0230 1120 0117

G 2653 2740 2697 006 -0044 0044 0400 0236 G 2845 2845 2845 000 0000 0000 1764 0000 0148

H 2176 2813 2495 045 -0318 0318 -1692 1727 H 2477 2477 2477 000 0000 0000 -0832 0000 -0017

L 2813 2544 2678 019 0134 -0134 0211 0729 L 2903 2301 2602 043 0301 -0301 0049 2241 -0076

M 2602 2740 2671 010 -0069 0069 0135 0375 M 2903 2255 2579 046 0324 -0324 -0112 2411 -0092

m 2658114 m 259505 mediane D -00372

Cn(2p) 1867 Cn(2p) 1867 Cp 16101

Cn(p) 16101 Cn(p) 16101 Qn(D) 01656915

n=2p n 20 n=2p n 20 Qdiff 02667753

f 11 f 11 t 035

l 55 l 55

n=p n 10 n=p n 10

f 6 f 6

l 15 l 15

Qn(2p) 0098745 Qn(2p) 007195

Qn(p) 006007 Qn(p) 008805

Qintra 018437 Qintra 013435

Qinter 009672 Qinter 014176

Sr(eacutecart-type de reacutepeacutetabiliteacute) 02607 Sr 01900

RSDr(coefficient de variation de la reacutepeacutetabiliteacute)981 RSDr 732

r(repeatability limit)) 0730 r 0532

SL(eacutecart-type interlaboratoire) -002464 SLlt0 SL 000205

SR(eacutecart-type de reproductibiliteacute) 02607 SR 01953

RSDR(coefficient de variation de la reproductibiliteacute)0098 RSDR 0075

R(limite de reproductibiliteacute)) 072996 R(limite de reproductibiliteacute))054686

Laboratoires

Niveau 2 bis

Moyenne si Laboratoires

Niveau 2 bis

DRef Alt Moyenne si Deviation h Deviation h k k

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3838 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Level 3

Nombre de laboratoires (p) 10

A 3690 3663 3676 002 0014 -0014 -0311 0513 A 3690 3708 3699 001 -0009 0009 0354 0257 0022

B 3613 3672 3642 004 -0030 0030 -1851 1109 B 3708 3568 3638 010 0070 -0070 -0844 2061 -0005

C 3663 3732 3698 005 -0035 0035 0645 1302 C 3663 3699 3681 003 -0018 0018 0000 0536 -0017

D 3690 3690 3690 000 0000 0000 0311 0000 D 3643 3663 3653 001 -0010 0010 -0545 0286 -0037

E 3732 3653 3693 006 0040 -0040 0429 1480 E 3613 3732 3673 008 -0060 0060 -0162 1769 -0020

F 3643 3672 3658 002 -0014 0014 -1157 0536 F 3690 3771 3731 006 -0040 0040 0975 1193 0073

G 3699 3653 3676 003 0023 -0023 -0328 0856 G 3602 3643 3623 003 -0021 0021 -1141 0612 -0053

H 3623 3672 3648 003 -0024 0024 -1615 0913 H 3748 3681 3715 005 0033 -0033 0664 0990 0067

L 3724 3663 3694 004 0031 -0031 0461 1150 L 3748 3771 3760 002 -0011 0011 1544 0335 0066

M 3690 3690 3690 000 0000 0000 0311 0000 M 3806 3681 3744 009 0062 -0062 1234 1848 0054

m 368334 m 368086 mediane D 00089

Cn(2p) 1867 Cn(2p) 1867 Cp 16101

Cn(p) 16101 Cn(p) 16101 Qn(D) 00325359

n=2p n 20 n=2p n 20 Qdiff 00523852

f 11 f 11 t 043

l 55 l 55

n=p n 10 n=p n 10

f 6 f 6

l 15 l 15

Qn(2p) 0014323 Qn(2p) 001811

Qn(p) 0013719 Qn(p) 003164

Qintra 002674 Qintra 003381

Qinter 002209 Qinter 005094

Sr(eacutecart-type de reacutepeacutetabiliteacute) 00378 Sr 00478

RSDr(coefficient de variation de la reacutepeacutetabiliteacute)103 RSDr 130

r(repeatability limit)) 0106 r 0134

SL(eacutecart-type interlaboratoire) -000023 SLlt0 SL 000145

SR(eacutecart-type de reproductibiliteacute) 00378 SR 00611

RSDR(coefficient de variation de la reproductibiliteacute)0010 RSDR 0017

R(limite de reproductibiliteacute)) 0105894 R(limite de reproductibiliteacute))017119

Ref Alt DLaboratoires

Niveau 3

Moyenne si Deviation h k Laboratoires

Niveau 3

Moyenne si Deviation h k

Page 17: ÞÀ¿¸7 ú Y5zñLjÜ< /u +æü t¬Á ... - NF Validation · NF VALIDATION Validation of alternative analytical methods Application in food microbiology ... 500 – 1 000 1 000 –

Thermo Fisher Scientific

ADRIA Deacuteveloppement 1738 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

2113 Detection limit (LOD) and quantification limit (LOQ)

The critical level is the defined as the smallest amount which can be detected (not null) but not

quantified as an exact value Below this value it cannot be sure that the true value is not null

The detection limit is defined as being higher than the critical level because it involves a power the

probability 1- which has to be well over 50 for example 95

The quantification limit is defined as the smallest amount of analyte (that is the lowest actual number of

organisms) which can be measured and quantified with defined precision and accuracy under the

experimental conditions by the method under validation

Protocol

The detection limit of the alternative method was realised with pure cultures

Three different inoculation levels were tested with six replicates per level ie

18 analyses were done by the alternative method

Listeria monocytogenes 18 was used for the detection limit of the alternative

method

Quantification limit was calculated for six independent blank samples

determinations

Results

These data are intrinsic to the method used and are presented in the

following tables

Table 8 ndash Inoculation 1 ml on 3 plates

Level (CFUml) Positive samples number Standard deviation Bias

0 06

05 26 1211 0

1 36 0816 05

5 66 2483 85

Thermo Fisher Scientific

ADRIA Deacuteveloppement 1838 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Table 9 ndash Inoculation 01 ml on 1 plate

Level (CFUml) Positive samples number Standard deviation Bias

0 06

5 36 0548 05

10 46 1414 2

50 66 2258 55

Table 10

Formulas Obtained values

LC 165 S0 + X0 18

LOD 33 S0 + X0 32

LOQ 10 S0 + X0 87

2114 Relative Sensitivity

The relative sensitivity is defined as the ability of the alternative method to detect two different amounts

of analyte measured by the reference method within a given matrix at a specified average value or

over the whole measurement range that is it is the minimal quantity variation (increase of the analyte

concentration x) which gives a significant variation of the measured signal (response y)

These data are intrinsic to the used method and are obtained from the results

of the linearity study

Sensitivity patterns obtained for tested (matrixstrain) pairs are presented in

figure 5

Thermo Fisher Scientific

ADRIA Deacuteveloppement 1938 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Figure 5 ndash Sensitivity patterns for the tested (matrixstrain) pairs

2115 SpecificitySelectivity

The specificity is defined as the degree to which a method is affected (or not) by the other components

present in a multi-component sample That is the ability of a method to measure exactly a given

analyte or its amount within the sample without interference from non-target components such as a

matrix effect or background noise

The selectivity is defined as a measure of the degree of non-interference in the presence of non-target

analytes A method is selective if it can be used to detect the analyte under examination and that a

guarantee can be provided that the detected signal can only be a product by that specific analyte

The specificity and selectivity study was performed for the Listeria PrecisTM

detection method no additional test was realised for the enumeration

method The results were the following

- Among the Listeria monocytogenes strains tested 2 strains (Listeria

monocytogenes CIP 7834 and Listeria monocytogenes CIP 105459) gave

negative results These 2 strains were tested twice by running 48 h

incubation time of the compared agars Positive results were then

observed by the alternative method Pale characteristic colonies were

observed on the ISO agar by streaking the Half Fraser broth only

- No cross reaction was observed with the non-target strains except with

Listeria ivanovii which gave characteristic colonies on Brilliancetrade Listeria

Agar but the confirmatory tests provide a clear discrimination between

Listeria monocytogenes and Listeria ivanovi species

Rillettes

Lettuce

Raw milk

Smoked salmon

Raw egg

Process water

x (CFUg)

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2038 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

212 Practicability

1 Packaging Ready to use Brilliance Listeria agar plates are available

(x 10 plates)

2 Reagent volume

3 Storage conditionsndash

Shelf life of products

not opened

The plates are stored at 6 - 12degC in darkness conditions The

expiration date is mentioned on the package and on the plates It

is around 3 months

4 Modalities of use

after first use

5 Required

equipments No specific equipment is required

6 Reagents Ready to use plates

7 Training 05 day

8 Real time handling

Reference method

9 min for one sample

55 min for 10 samples

Alternative method

7 min for one sample

35 minutes for 10 samples

9 Time to result

Negative results

Reference method Day 2

Alternative method Day 2

Negative results after confirmation or positive results

Reference method Day 4 ndash Day 7

Alternative method Day 4 ndash Day 7

Positive results

Reference method Day 4 ndash Day 7

Alternative method Day 2 ndash Day 3 (with OBIS Mono Test)

or Day 4 ndash Day 7 (by the classical tests described in the

standardised method)

10 Technician skill Identical to that required for the reference method

11 Steps common to

the reference method Mother suspension revivification step and decimal dilutions

12 Traceability

13 Maintenance

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2138 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

213 Inter-laboratory study

2131 Organisation

12 laboratories participated to this study Pasteurised milk was inoculated by

Listeria monocytogenes 12a isolated from raw milk The targeted

inoculation levels were

- 0 CFUml

- 50 CFU ml

- 500 CFU ml

- 5 000 CFU ml

Each laboratory received eight flasks of 10 ml sample ie two flasks per

inoculation level Furthermore one non-inoculated sample has been added

to the package for total viable count microflora (NF ISO 4833 method)

Detailed instructions were transmitted to the collaborators by the expert

laboratory

Collaborative study laboratories and the expert laboratory carried out the

analyses with the alternative and reference methods

2132 Verification of experimental parameters

Strain stability during transport

In order to evaluate the Listeria monocytogenes strain stability during

transport bacterial count of all inoculated flasks were checked at different

time ie inoculation time after 24 h of conservation at 2deg - 8degC Results are

reported in table 11

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2238 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Table 11 ndash Listeria monocytogenes count

by ISO 11290-2A1 method (log CFUml)

Day of the analyses Level contamination Reference method

Day 0

(29052006)

1 45

2 440

3 4 500

Day 1

(30052006)

1 60

2 430

3 4 900

No evolution of the strain was observed after storage for 24 h at 2degC ndash 8degC

Contamination level

The real contamination levels are provided in table 12

Table 12 ndash Real contamination level (CFUml)

Target inoculation

(CFU ml) Samples Ndeg

Real contamination levels

(CFUml)

0 1 - 8 0

50 5 - 6 45

500 3 - 7 440

5000 2 - 4 4 500

Sample temperature at receipt

Measured temperatures at receipt are listed in table 13

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2338 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Table 13 - Sample temperature on receipt

Laboratories Receipt

day

Hour

receipt

Measured temperature

at receipt (degC)

Measured temperature by the

temperature probe (degC)

A

B

C

D

E

F

G

H

I

J

K

L

M

30052006

10h45

10h15

11h15

10h00

09h30

8h30

11h50

10h00

08h30

14h45

08h30

09h45

08h45

82

55

45

63

60

62

81

38

48

-

14

67

76

03

03

-13

03

04

-14

05

04

01

05

-13

04

Conclusion

The temperatures during the transport were correct for all the labs except for

Lab J which didnrsquot provide the information

All the labs received their packages with several broken flasks

Three labs (B D and L) received some damaged samples but proceeded to

analyses

All the labs started the analyses at Day 1 except Lab J

2133 Results

Aerobic mesophilic flora

The count of aerobic mesophilic flora varied from 94 102 CFUml to

33 103 CFUml

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2438 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Expert lab results

The results obtained by the expert lab are the following

Table 14

Target inoculation level (CFUml)

Reference method ISO 11290-2

Alternative method Listeria Precis Enumeration

Rep 1 Rep 2 Rep 1 Rep 2

lt 10 lt 10 lt 10 lt 10 lt10

50 50 (Ne) 60 (Ne) 50 (Ne) 30 (Ne)

500 470 390 490 500

5 000 4 700 5 100 5 900 3 400

Ne estimated number

The obtained results are in agreement with those expected

Collaborator Labs results

A synthesis of the results obtained by the collaborator labs is provided

Table 15 (CFUml) and Table 16 (log CFUml)

Table 15 ndash Synthesis of results obtained by the alternative

and the reference methods (CFUml)

Labs Level 1 Level 2 Level 2 bis Level 3

Reference method

Alternative method

Reference method

Alternative method

Reference method

Alternative method

Reference method

Alternative method

A 40 40 70 40 480 410 210 340 1000 300 400 300 4900 4600 4900 5100

B 40 15 40 20 350 300 360 450 350 650 800 600 4100 4700 5100 3700

C 50 20 20 70 440 430 480 450 300 650 300 500 4600 5400 4600 5000

D 40 60 40 40 490 460 600 540 250 450 600 1000 4900 4900 4400 4600

E 50 40 50 20 490 520 530 560 750 550 400 200 5400 4500 4100 5400

F 60 20 60 50 370 250 300 130 300 350 600 300 4400 4700 4900 5900

G 70 35 120 50 440 390 570 370 450 550 700 700 5000 4500 4000 4400

H 110 40 100 90 520 440 450 540 150 650 300 300 4200 4700 5600 4800

L 35 60 20 40 390 390 170 380 650 350 800 200 5300 4600 5600 5900

M 45 45 30 40 450 490 550 410 400 550 800 180 4900 4900 6400 4800

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2538 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Table 16 ndash Synthesis of results obtained by the alternative

and the reference methods (log CFUml)

Labs Level 1 Level 2 Level 2 bis Level 3

Reference method

Alternative method

Reference method

Alternative method

Reference method

Alternative method

Reference method

Alternative method

A 1602 1602 1845 1602 2681 2613 2322 2531 3000 2477 2602 2477 3690 3663 3690 3708

B 1602 1176 1602 1301 2544 2477 2556 2653 2544 2813 2903 2778 3613 3672 3708 3568

C 1699 1301 1301 1845 2643 2633 2681 2653 2477 2813 2477 2699 3663 3732 3663 3699

D 1602 1778 1602 1602 2690 2663 2778 2732 2398 2653 2778 3000 3690 3690 3643 3663

E 1699 1602 1699 1301 2690 2716 2724 2748 2875 2740 2602 2301 3732 3653 3613 3732

F 1778 1301 1778 1699 2568 2398 2477 2114 2477 2544 2778 2477 3643 3672 3690 3771

G 1845 1544 2079 1699 2643 2591 2756 2568 2653 2740 2845 2845 3699 3653 3602 3643

H 2041 1602 2000 1954 2716 2643 2653 2732 2176 2813 2477 2477 3623 3672 3748 3681

L 1544 1778 1301 1602 2591 2591 2230 2580 2813 2544 2903 2301 3724 3663 3748 3771

M 1653 1653 1477 1602 2653 2690 2740 2613 2602 2740 2903 2255 3690 3690 3806 3681

Level 2 results obtained taking account the 3 plates inoculated with the mother-suspension

Level 2 bis results obtained taking account the plates inoculated with 01 ml of the mother-suspension

Scrutiny of the measurement results for consistency

In order to identify the measurement results or laboratories that could be

inconsistent two graphical consistency techniques were realised Mandelrsquos

h-ank-statistics Mandel indicators h and k at 5 significance highlighted

some possible inconsistent data (See tables and graphics in Appendix 3 and

Appendix 4)

Mandelrsquos values Number of values observed over the threshold

Reference method Alternative method

h gt 1 Lab F Level 2 Lab F Level 2

h gt 5 Lab F Level 2

Lab B Levels 1 and 2

Lab A Level 2

Lab D Level 3

Lab F Level 2

Lab L Level 2

kgt 1 Lab F Level 3

k gt 5 Lab F Level 2 Lab C Level 2

Lab F Level 3

Lab L Levels 1 and 3

Lab M Level 1

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2638 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

The emphasized results appear (i) coherent with the targeted contamination

levels (ii) concordant between both methods

All the results were thus kept for the statistical analysis

Comparison of the trueness and precision characteristics of

the reference method and alternative methods

The statistical values are summarised hereafter

Level p

Reference method Alternative method

Repeatability ratio

Reproducibility ratio Median

Repeatability standard deviation

Reproducibility standard deviation

Median Repeatability

standard deviation

Reproducibility standard deviation

1 10 16519 02415 02425 16021 02045 02433 0847 1007

2 10 26551 00543 00659 26719 00914 01325 1682 2011

2 bis 10 26581 02607 02607 25951 01900 01953 0729 0749

3 10 18671 00378 00378 18671 00478 00611 1264 1617

Bias of the alternative method

In order to estimate the bias of the alternative method with regard to the reference method for every

level Dij and t are calculated according to the following equations

RefAlt ijYijYDij

Diffp

Dijit

)2(

)(median

If the t value is superior to 2 the alternative method is significantly biased

with regard to the reference method

The values are given in table 17

Table 17 ndash t (d) values obtained by level

Level Bias t (d) Conclusion

1 0068 114 Non significant bias

2 0021 082 Non significant bias

2 bis - 0037 035 Non significant bias

3 - 0037 043 Non significant bias

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2738 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

The statistical test concludes to a non significant bias for all the inoculation

levels The bias varies from ndash 0037 to 0068 log CFUml

Comparison of the repeatability standard deviations

If the ratio Srj Alt Srj Ref of the repeatability standard deviations of the

alternative method and the reference method is larger than 2 the precision

under repeatability conditions of the alternative method is considered to be

lower than that of the reference method If this ratio is smaller than 05 the

precision under repeatability conditions of the alternative method is

considered to be greater than that of the reference method

The ratio values are given in table 18

Table 18

Level

Repeatability standard deviation

Reference method Alternative method Alternative method

reference method ratio

1 0242 0205 0847

2 0054 0091 1682

2 bis 0261 0190 0729

3 0038 0048 1264

Level Repeatability limit

Reference method Alternative method

1 0676 0573

2 0152 0256

2 bis 0730 0532

3 0106 0134

The ratios of the repeatability standard deviations are below 2 in all cases

The precision under repeatability conditions of the alternative method is

equivalent to that of the reference method

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2838 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Comparison of the reproducibility standard deviations

If the ratio Srj Alt Srj Ref of the reproducibility standard deviations of the alternative method and the

reference method is larger than 2 the precision under reproducibility conditions of the alternative

method is considered to be lower than that of the reference method If this ratio is smaller than 05 the

precision under reproducibility conditions of the alternative method is considered to be greater than

that of the reference method

The ratio values are given in table 19

Table 19

Level

Reproducibility standard deviation

Reference method Alternative method Alternative method

reference method ratio

1 0242 0243 1007

2 0066 0133 2011

2 bis 0261 0195 0749

3 0038 0061 1617

Level Reproducibility limit

Reference method Alternative method

1 0676 0681

2 0185 0371

2 bis 0730 0547

3 0106 0171

The ratios of the reproducibility standard deviations are below or close to 2

The precision under reproducibility conditions of the alternative method is

equivalent to that of the reference method

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2938 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

214 Conclusion

Method comparison study

The Listeria PrecisTM enumeration method shows satisfying linearity and

relative accuracy

The alternative method shows satisfying inclusivity and exclusivity results

Two Listeria monocytogenes strains gave negative results with the

alternative method These strains grew weakly on the reference method

media (OampA Palcam and Oxford)

The limits of repeatability of the alternative method are similar to those of the

reference method

The bias between both methods are low between - 0133 Log CFUg and

- 0008 Log CFUg

The Listeria PrecisTM enumeration method offers the saving for confirmatory

tests

Positive results are obtained with the alternative method in 2-3 days (with

OBIS Mono test) or in 4-7 days (by the classical tests described in the

standardized method) against 4-7 days for the reference method

Inter-laboratory study

The bias between the reference and the alternative methods shows satisfying

and correct values

- level 1 (100 - 1 000 CFUg) bias = + 007 log CFUg

- level 2 (1 000 - 10 000 CFUg) bias = 002 log CFUg

- level 2 bis (1 000 - 10 000 CFUg) bias = - 004 log CFUg

- level 3 (10 000 - 100 000 CFUg) bias = - 004 log CFUg

The repeatability and the reproducibility of the alternative method look similar

to those of the reference method

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3038 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

22 Extension study (realised by ASEPT 2007)

An extension study was performed in 2007 for a new confirmatory test OBIS

MONO

150 Listeria monocytogenes strains and 100 non-target strains were isolated

onto TSAYE and BrillianceTM Listeria Agar plates All the Listeria

monocytogenes and the non-target strains gave the expected results

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3138 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Appendix 1 ndash Flow diagram of the Listeria PrecisTM method

x g sample + 9 x ml buffered peptone water

Revivification step for 1 h plusmn 5 min at 20degC plusmn 1degC

Streaking 1 ml onto 3 Brilliance Listeria Agar plates (mother suspension)

or 01 ml onto 1 Brilliance Listeria Agar plates (mother suspension and decimal dilutions)

Incubation for 48 h plusmn 3 h at 37degC plusmn 1degC

Confirmatory tests on at least one typical colony

- by the tests described in the ISO method

- by applying the OBIS mono test on an isolated colony

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3238 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Appendix 2 ndash ISO 11290-2 (1998) amp ISO 11290-2A1 (2004) Horizontal

method for the detection and enumeration of Listeria monocytogenes -

Part 2 enumeration of Listeria monocytogenes in foods

10 g cheese + 90 ml buffered peptone water or Half Fraser Broth (without supplements) (110 dilution)

Stomach 1 min 30 seconds

Keep at 20degC plusmn 2degC for 1 h plusmn 5 min

Decimal dilutions in 9 ml peptone salt tubes

Dilution 110

Dilution 1100

Dilution 11000

1 ml on 3 Chromogenic Listeria medium (ISO) plates (x 2) (-1)

01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-2)

01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-3)

01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-4)

Incubation at 37degC plusmn 1degC

24 h - 48 h

Numerate typical colonies at 24 h

(bluegreen surrounded by an opaque halo)

Numerate at 48 h

Streak typical colonies on TSYEA

(5 coloniesplate 2 successive dilutions)

Confirmation tests

Catalase

Gram stain

For confirmation of L monocytogenes

Haemolysis

Carbohydrate (rhamnose and xylose) utilisation

CAMP Test

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3338 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Appendix 3 ndash Mandelrsquos graphics

Laboratoire h Ref

Niveau 1 h Ref

Niveau 2 h Ref

Niveau 3 h Alt

Niveau 1 h Alt

Niveau 2 h Alt

Niveau 3 h5 h1 h5 h1

A -050 -015 083 062 -212 -039 198 299 -198 -299

B -261 -270 021 -077 -058 173 198 299 -198 -299

C -151 -031 -014 -015 -004 -005 198 299 -198 -299

D 038 040 -137 000 072 207 198 299 -198 -299

E -001 090 155 -052 056 -101 198 299 -198 -299

F -112 -321 -153 070 -325 023 198 299 -198 -299

G 042 -071 040 147 -009 176 198 299 -198 -299

H 169 046 -169 192 018 -083 198 299 -198 -299

L 009 -120 021 -077 -231 005 198 299 -198 -299

M 001 031 014 -032 004 -011 198 299 -198 -299

-300

-200

-100

000

100

200

300

A B C D E F G H L M

Vale

urs

h d

e M

an

del(

rob

uste

s)

Laboratoires

Meacutethode de reacutefeacuterence analyse robuste

h Ref Niveau 1

h Ref Niveau 2

h Ref Niveau 3

h 5=198

h 1=299

-300

-200

-100

000

100

200

300

A B C D E F G H L M

Vale

urs

de h

de M

an

del(

rob

uste

s)

Laboratoires

Meacutethode alternative analyse robuste

h Alt Niveau 1

h Alt Niveau 2

h Alt Niveau 2

h 1=299

h 5=198

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3438 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Laboratoire k Ref

Niveau 1 k Ref

Niveau 2 k Ref

Niveau 3 k Alt

Niveau 1 k Alt

Niveau 2 k Alt

Niveau 3 k1 k5

A 000 089 142 084 162 046 259 181

B 125 087 073 104 075 046 259 181

C 117 013 091 188 022 083 259 181

D 052 036 069 000 035 083 259 181

E 028 034 037 138 019 112 259 181

F 140 222 018 027 281 112 259 181

G 088 068 024 131 145 000 259 181

H 129 094 173 016 061 000 259 181

L 069 000 073 104 270 224 259 181

M 000 048 038 043 099 241 259 181

000

050

100

150

200

250

300

350

400

A B C D E F G H L M

Vale

urs

k d

e M

an

del(

rob

uste

s)

Laboratoires

Meacutethode de reacutefeacuterence analyse robuste

k Ref Niveau 1k Ref Niveau 2k Ref Niveau 3

k 1=259

k 5=181

000

050

100

150

200

250

300

A B C D E F G H L M

Vale

urs

k d

e M

an

del

(ro

bu

ste

s)

Laboratoires

Meacutethode alternative analyse robuste

k Alt Niveau 3

k Alt Niveau 1

k Alt Niveau 2

k 1=259

k 5=181

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3538 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Appendix 4 ndash Statistical results

Level 1

Nombre de laboratoires (p) 10

A 1602 1602 1602 000 0000 0000 -0495 0000 A 1845 1602 1724 017 0122 -0122 0621 0840 0122

B 1602 1176 1389 030 0213 -0213 -2613 1247 B 1602 1301 1452 021 0151 -0151 -0769 1041 0062

C 1699 1301 1500 028 0199 -0199 -1510 1165 C 1301 1845 1573 038 -0272 0272 -0148 1881 0073

D 1602 1778 1690 012 -0088 0088 0380 0516 D 1602 1602 1602 000 0000 0000 0000 0000 -0088

E 1699 1602 1651 007 0048 -0048 -0013 0284 E 1699 1301 1500 028 0199 -0199 -0522 1376 -0151

F 1778 1301 1540 034 0239 -0239 -1116 1397 F 1778 1699 1739 006 0040 -0040 0698 0274 0199

G 1845 1544 1695 021 0151 -0151 0425 0881 G 2079 1699 1889 027 0190 -0190 1467 1315 0194

H 2041 1602 1822 031 0220 -0220 1689 1286 H 2000 1954 1977 003 0023 -0023 1917 0158 0155

L 1544 1778 1661 017 -0117 0117 0092 0685 L 1301 1602 1452 021 -0151 0151 -0769 1041 -0210

M 1653 1653 1653 000 0000 0000 0013 0000 M 1477 1602 1540 009 -0062 0062 -0319 0432 -0114

m 165186 m 160206 mediane D 00678

Cn(2p) 1867 Cn(2p) 1867 Cp 16101

Cn(p) 16101 Cn(p) 16101 Qn(D) 0092926

n=2p n 20 n=2p n 20 Qdiff 01496169

f 11 f 11 t 114

l 55 l 55

n=p n 10 n=p n 10

f 6 f 6

l 15 l 15

Qn(2p) 0091465 Qn(2p) 007745

Qn(p) 0062469 Qn(p) 0121519

Qintra 017078 Qintra 014461

Qinter 010058 Qinter 019565

Sr(eacutecart-type de reacutepeacutetabiliteacute) 02415 Sr 02045

RSDr(coefficient de variation de la reacutepeacutetabiliteacute)1462 RSDr 1277

r(repeatability limit)) 0676 r 0573

SL(eacutecart-type interlaboratoire) -001905 SLlt0 SL 001737

SR(eacutecart-type de reproductibiliteacute) 02415 SR 02433

RSDR(coefficient de variation de la reproductibiliteacute)0146 RSDR 0152

R(limite de reproductibiliteacute)) 06762 R(limite de reproductibiliteacute))068123

h k h MoyenneLaboratoires

Niveau 1

si LaboratoiresDeviationMoyenne Dk DeviationsiAlt

Niveau 1

Ref

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3638 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Level 2

Nombre de laboratoires (p) 10

A 2681 2613 2647 005 0034 -0034 -0151 0891 A 2322 2531 2427 015 -0105 0105 -2119 1620 -0220

B 2544 2477 2511 005 0033 -0033 -2699 0872 B 2556 2653 2605 007 -0048 0048 -0581 0750 0094

C 2643 2633 2638 001 0005 -0005 -0311 0130 C 2681 2653 2667 002 0014 -0014 -0040 0217 0029

D 2690 2663 2676 002 0014 -0014 0400 0357 D 2778 2732 2755 003 0023 -0023 0721 0354 0079

E 2690 2716 2703 002 -0013 0013 0897 0336 E 2724 2748 2736 002 -0012 0012 0556 0185 0033

F 2568 2398 2483 012 0085 -0085 -3214 2217 F 2477 2114 2296 026 0182 -0182 -3255 2811 -0188

G 2643 2591 2617 004 0026 -0026 -0707 0682 G 2756 2568 2662 013 0094 -0094 -0085 1453 0045

H 2716 2643 2680 005 0036 -0036 0460 0945 H 2653 2732 2693 006 -0040 0040 0181 0613 0013

L 2591 2591 2591 000 0000 0000 -1196 0000 L 2230 2580 2405 025 -0175 0175 -2307 2704 -0186

M 2653 2690 2672 003 -0018 0018 0311 0481 M 2740 2613 2677 009 0064 -0064 0040 0987 0005

m 2655082 m 26719001 mediane D 00209

Cn(2p) 1867 Cn(2p) 1867 Cp 16101

Cn(p) 16101 Cn(p) 16101 Qn(D) 0039911

n=2p n 20 n=2p n 20 Qdiff 00642591

f 11 f 11 t 082

l 55 l 55

n=p n 10 n=p n 10

f 6 f 6

l 15 l 15

Qn(2p) 0020570 Qn(2p) 00345985

Qn(p) 00332437 Qn(p) 00718158

Qintra 003841 Qintra 006460

Qinter 005352 Qinter 011563

Sr(eacutecart-type de reacutepeacutetabiliteacute) 00543 Sr 00914

RSDr(coefficient de variation de la reacutepeacutetabiliteacute)205 RSDr 342

r(repeatability limit)) 0152 r 0256

SL(eacutecart-type interlaboratoire) 000139 SL 000920

SR(eacutecart-type de reproductibiliteacute) 00659 SR 01325

RSDR(coefficient de variation de la reproductibiliteacute)0025 RSDR 0050

R(limite de reproductibiliteacute)) 018446 R(limite de reproductibiliteacute))03708618

Laboratoires

Niveau 2

Moyenne si Laboratoires

Niveau 2

DRef Alt Moyenne si Deviation h Deviation h k k

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3738 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Level 2 bis

Nombre de laboratoires (p) 10

A 3000 2477 2739 037 0261 -0261 0832 1418 A 2602 2477 2540 009 0062 -0062 -0391 0465 -0199

B 2544 2813 2678 019 -0134 0134 0211 0729 B 2903 2778 2841 009 0062 -0062 1732 0465 0162

C 2477 2813 2645 024 -0168 0168 -0135 0911 C 2477 2699 2588 016 -0111 0111 -0049 0826 -0057

D 2398 2653 2526 018 -0128 0128 -1370 0692 D 2778 3000 2889 016 -0111 0111 2074 0826 0363

E 2875 2740 2808 010 0067 -0067 1547 0365 E 2602 2301 2452 021 0151 -0151 -1012 1120 -0356

F 2477 2544 2511 005 -0033 0033 -1525 0182 F 2778 2477 2628 021 0151 -0151 0230 1120 0117

G 2653 2740 2697 006 -0044 0044 0400 0236 G 2845 2845 2845 000 0000 0000 1764 0000 0148

H 2176 2813 2495 045 -0318 0318 -1692 1727 H 2477 2477 2477 000 0000 0000 -0832 0000 -0017

L 2813 2544 2678 019 0134 -0134 0211 0729 L 2903 2301 2602 043 0301 -0301 0049 2241 -0076

M 2602 2740 2671 010 -0069 0069 0135 0375 M 2903 2255 2579 046 0324 -0324 -0112 2411 -0092

m 2658114 m 259505 mediane D -00372

Cn(2p) 1867 Cn(2p) 1867 Cp 16101

Cn(p) 16101 Cn(p) 16101 Qn(D) 01656915

n=2p n 20 n=2p n 20 Qdiff 02667753

f 11 f 11 t 035

l 55 l 55

n=p n 10 n=p n 10

f 6 f 6

l 15 l 15

Qn(2p) 0098745 Qn(2p) 007195

Qn(p) 006007 Qn(p) 008805

Qintra 018437 Qintra 013435

Qinter 009672 Qinter 014176

Sr(eacutecart-type de reacutepeacutetabiliteacute) 02607 Sr 01900

RSDr(coefficient de variation de la reacutepeacutetabiliteacute)981 RSDr 732

r(repeatability limit)) 0730 r 0532

SL(eacutecart-type interlaboratoire) -002464 SLlt0 SL 000205

SR(eacutecart-type de reproductibiliteacute) 02607 SR 01953

RSDR(coefficient de variation de la reproductibiliteacute)0098 RSDR 0075

R(limite de reproductibiliteacute)) 072996 R(limite de reproductibiliteacute))054686

Laboratoires

Niveau 2 bis

Moyenne si Laboratoires

Niveau 2 bis

DRef Alt Moyenne si Deviation h Deviation h k k

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3838 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Level 3

Nombre de laboratoires (p) 10

A 3690 3663 3676 002 0014 -0014 -0311 0513 A 3690 3708 3699 001 -0009 0009 0354 0257 0022

B 3613 3672 3642 004 -0030 0030 -1851 1109 B 3708 3568 3638 010 0070 -0070 -0844 2061 -0005

C 3663 3732 3698 005 -0035 0035 0645 1302 C 3663 3699 3681 003 -0018 0018 0000 0536 -0017

D 3690 3690 3690 000 0000 0000 0311 0000 D 3643 3663 3653 001 -0010 0010 -0545 0286 -0037

E 3732 3653 3693 006 0040 -0040 0429 1480 E 3613 3732 3673 008 -0060 0060 -0162 1769 -0020

F 3643 3672 3658 002 -0014 0014 -1157 0536 F 3690 3771 3731 006 -0040 0040 0975 1193 0073

G 3699 3653 3676 003 0023 -0023 -0328 0856 G 3602 3643 3623 003 -0021 0021 -1141 0612 -0053

H 3623 3672 3648 003 -0024 0024 -1615 0913 H 3748 3681 3715 005 0033 -0033 0664 0990 0067

L 3724 3663 3694 004 0031 -0031 0461 1150 L 3748 3771 3760 002 -0011 0011 1544 0335 0066

M 3690 3690 3690 000 0000 0000 0311 0000 M 3806 3681 3744 009 0062 -0062 1234 1848 0054

m 368334 m 368086 mediane D 00089

Cn(2p) 1867 Cn(2p) 1867 Cp 16101

Cn(p) 16101 Cn(p) 16101 Qn(D) 00325359

n=2p n 20 n=2p n 20 Qdiff 00523852

f 11 f 11 t 043

l 55 l 55

n=p n 10 n=p n 10

f 6 f 6

l 15 l 15

Qn(2p) 0014323 Qn(2p) 001811

Qn(p) 0013719 Qn(p) 003164

Qintra 002674 Qintra 003381

Qinter 002209 Qinter 005094

Sr(eacutecart-type de reacutepeacutetabiliteacute) 00378 Sr 00478

RSDr(coefficient de variation de la reacutepeacutetabiliteacute)103 RSDr 130

r(repeatability limit)) 0106 r 0134

SL(eacutecart-type interlaboratoire) -000023 SLlt0 SL 000145

SR(eacutecart-type de reproductibiliteacute) 00378 SR 00611

RSDR(coefficient de variation de la reproductibiliteacute)0010 RSDR 0017

R(limite de reproductibiliteacute)) 0105894 R(limite de reproductibiliteacute))017119

Ref Alt DLaboratoires

Niveau 3

Moyenne si Deviation h k Laboratoires

Niveau 3

Moyenne si Deviation h k

Page 18: ÞÀ¿¸7 ú Y5zñLjÜ< /u +æü t¬Á ... - NF Validation · NF VALIDATION Validation of alternative analytical methods Application in food microbiology ... 500 – 1 000 1 000 –

Thermo Fisher Scientific

ADRIA Deacuteveloppement 1838 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Table 9 ndash Inoculation 01 ml on 1 plate

Level (CFUml) Positive samples number Standard deviation Bias

0 06

5 36 0548 05

10 46 1414 2

50 66 2258 55

Table 10

Formulas Obtained values

LC 165 S0 + X0 18

LOD 33 S0 + X0 32

LOQ 10 S0 + X0 87

2114 Relative Sensitivity

The relative sensitivity is defined as the ability of the alternative method to detect two different amounts

of analyte measured by the reference method within a given matrix at a specified average value or

over the whole measurement range that is it is the minimal quantity variation (increase of the analyte

concentration x) which gives a significant variation of the measured signal (response y)

These data are intrinsic to the used method and are obtained from the results

of the linearity study

Sensitivity patterns obtained for tested (matrixstrain) pairs are presented in

figure 5

Thermo Fisher Scientific

ADRIA Deacuteveloppement 1938 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Figure 5 ndash Sensitivity patterns for the tested (matrixstrain) pairs

2115 SpecificitySelectivity

The specificity is defined as the degree to which a method is affected (or not) by the other components

present in a multi-component sample That is the ability of a method to measure exactly a given

analyte or its amount within the sample without interference from non-target components such as a

matrix effect or background noise

The selectivity is defined as a measure of the degree of non-interference in the presence of non-target

analytes A method is selective if it can be used to detect the analyte under examination and that a

guarantee can be provided that the detected signal can only be a product by that specific analyte

The specificity and selectivity study was performed for the Listeria PrecisTM

detection method no additional test was realised for the enumeration

method The results were the following

- Among the Listeria monocytogenes strains tested 2 strains (Listeria

monocytogenes CIP 7834 and Listeria monocytogenes CIP 105459) gave

negative results These 2 strains were tested twice by running 48 h

incubation time of the compared agars Positive results were then

observed by the alternative method Pale characteristic colonies were

observed on the ISO agar by streaking the Half Fraser broth only

- No cross reaction was observed with the non-target strains except with

Listeria ivanovii which gave characteristic colonies on Brilliancetrade Listeria

Agar but the confirmatory tests provide a clear discrimination between

Listeria monocytogenes and Listeria ivanovi species

Rillettes

Lettuce

Raw milk

Smoked salmon

Raw egg

Process water

x (CFUg)

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2038 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

212 Practicability

1 Packaging Ready to use Brilliance Listeria agar plates are available

(x 10 plates)

2 Reagent volume

3 Storage conditionsndash

Shelf life of products

not opened

The plates are stored at 6 - 12degC in darkness conditions The

expiration date is mentioned on the package and on the plates It

is around 3 months

4 Modalities of use

after first use

5 Required

equipments No specific equipment is required

6 Reagents Ready to use plates

7 Training 05 day

8 Real time handling

Reference method

9 min for one sample

55 min for 10 samples

Alternative method

7 min for one sample

35 minutes for 10 samples

9 Time to result

Negative results

Reference method Day 2

Alternative method Day 2

Negative results after confirmation or positive results

Reference method Day 4 ndash Day 7

Alternative method Day 4 ndash Day 7

Positive results

Reference method Day 4 ndash Day 7

Alternative method Day 2 ndash Day 3 (with OBIS Mono Test)

or Day 4 ndash Day 7 (by the classical tests described in the

standardised method)

10 Technician skill Identical to that required for the reference method

11 Steps common to

the reference method Mother suspension revivification step and decimal dilutions

12 Traceability

13 Maintenance

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2138 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

213 Inter-laboratory study

2131 Organisation

12 laboratories participated to this study Pasteurised milk was inoculated by

Listeria monocytogenes 12a isolated from raw milk The targeted

inoculation levels were

- 0 CFUml

- 50 CFU ml

- 500 CFU ml

- 5 000 CFU ml

Each laboratory received eight flasks of 10 ml sample ie two flasks per

inoculation level Furthermore one non-inoculated sample has been added

to the package for total viable count microflora (NF ISO 4833 method)

Detailed instructions were transmitted to the collaborators by the expert

laboratory

Collaborative study laboratories and the expert laboratory carried out the

analyses with the alternative and reference methods

2132 Verification of experimental parameters

Strain stability during transport

In order to evaluate the Listeria monocytogenes strain stability during

transport bacterial count of all inoculated flasks were checked at different

time ie inoculation time after 24 h of conservation at 2deg - 8degC Results are

reported in table 11

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2238 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Table 11 ndash Listeria monocytogenes count

by ISO 11290-2A1 method (log CFUml)

Day of the analyses Level contamination Reference method

Day 0

(29052006)

1 45

2 440

3 4 500

Day 1

(30052006)

1 60

2 430

3 4 900

No evolution of the strain was observed after storage for 24 h at 2degC ndash 8degC

Contamination level

The real contamination levels are provided in table 12

Table 12 ndash Real contamination level (CFUml)

Target inoculation

(CFU ml) Samples Ndeg

Real contamination levels

(CFUml)

0 1 - 8 0

50 5 - 6 45

500 3 - 7 440

5000 2 - 4 4 500

Sample temperature at receipt

Measured temperatures at receipt are listed in table 13

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2338 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Table 13 - Sample temperature on receipt

Laboratories Receipt

day

Hour

receipt

Measured temperature

at receipt (degC)

Measured temperature by the

temperature probe (degC)

A

B

C

D

E

F

G

H

I

J

K

L

M

30052006

10h45

10h15

11h15

10h00

09h30

8h30

11h50

10h00

08h30

14h45

08h30

09h45

08h45

82

55

45

63

60

62

81

38

48

-

14

67

76

03

03

-13

03

04

-14

05

04

01

05

-13

04

Conclusion

The temperatures during the transport were correct for all the labs except for

Lab J which didnrsquot provide the information

All the labs received their packages with several broken flasks

Three labs (B D and L) received some damaged samples but proceeded to

analyses

All the labs started the analyses at Day 1 except Lab J

2133 Results

Aerobic mesophilic flora

The count of aerobic mesophilic flora varied from 94 102 CFUml to

33 103 CFUml

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2438 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Expert lab results

The results obtained by the expert lab are the following

Table 14

Target inoculation level (CFUml)

Reference method ISO 11290-2

Alternative method Listeria Precis Enumeration

Rep 1 Rep 2 Rep 1 Rep 2

lt 10 lt 10 lt 10 lt 10 lt10

50 50 (Ne) 60 (Ne) 50 (Ne) 30 (Ne)

500 470 390 490 500

5 000 4 700 5 100 5 900 3 400

Ne estimated number

The obtained results are in agreement with those expected

Collaborator Labs results

A synthesis of the results obtained by the collaborator labs is provided

Table 15 (CFUml) and Table 16 (log CFUml)

Table 15 ndash Synthesis of results obtained by the alternative

and the reference methods (CFUml)

Labs Level 1 Level 2 Level 2 bis Level 3

Reference method

Alternative method

Reference method

Alternative method

Reference method

Alternative method

Reference method

Alternative method

A 40 40 70 40 480 410 210 340 1000 300 400 300 4900 4600 4900 5100

B 40 15 40 20 350 300 360 450 350 650 800 600 4100 4700 5100 3700

C 50 20 20 70 440 430 480 450 300 650 300 500 4600 5400 4600 5000

D 40 60 40 40 490 460 600 540 250 450 600 1000 4900 4900 4400 4600

E 50 40 50 20 490 520 530 560 750 550 400 200 5400 4500 4100 5400

F 60 20 60 50 370 250 300 130 300 350 600 300 4400 4700 4900 5900

G 70 35 120 50 440 390 570 370 450 550 700 700 5000 4500 4000 4400

H 110 40 100 90 520 440 450 540 150 650 300 300 4200 4700 5600 4800

L 35 60 20 40 390 390 170 380 650 350 800 200 5300 4600 5600 5900

M 45 45 30 40 450 490 550 410 400 550 800 180 4900 4900 6400 4800

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2538 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Table 16 ndash Synthesis of results obtained by the alternative

and the reference methods (log CFUml)

Labs Level 1 Level 2 Level 2 bis Level 3

Reference method

Alternative method

Reference method

Alternative method

Reference method

Alternative method

Reference method

Alternative method

A 1602 1602 1845 1602 2681 2613 2322 2531 3000 2477 2602 2477 3690 3663 3690 3708

B 1602 1176 1602 1301 2544 2477 2556 2653 2544 2813 2903 2778 3613 3672 3708 3568

C 1699 1301 1301 1845 2643 2633 2681 2653 2477 2813 2477 2699 3663 3732 3663 3699

D 1602 1778 1602 1602 2690 2663 2778 2732 2398 2653 2778 3000 3690 3690 3643 3663

E 1699 1602 1699 1301 2690 2716 2724 2748 2875 2740 2602 2301 3732 3653 3613 3732

F 1778 1301 1778 1699 2568 2398 2477 2114 2477 2544 2778 2477 3643 3672 3690 3771

G 1845 1544 2079 1699 2643 2591 2756 2568 2653 2740 2845 2845 3699 3653 3602 3643

H 2041 1602 2000 1954 2716 2643 2653 2732 2176 2813 2477 2477 3623 3672 3748 3681

L 1544 1778 1301 1602 2591 2591 2230 2580 2813 2544 2903 2301 3724 3663 3748 3771

M 1653 1653 1477 1602 2653 2690 2740 2613 2602 2740 2903 2255 3690 3690 3806 3681

Level 2 results obtained taking account the 3 plates inoculated with the mother-suspension

Level 2 bis results obtained taking account the plates inoculated with 01 ml of the mother-suspension

Scrutiny of the measurement results for consistency

In order to identify the measurement results or laboratories that could be

inconsistent two graphical consistency techniques were realised Mandelrsquos

h-ank-statistics Mandel indicators h and k at 5 significance highlighted

some possible inconsistent data (See tables and graphics in Appendix 3 and

Appendix 4)

Mandelrsquos values Number of values observed over the threshold

Reference method Alternative method

h gt 1 Lab F Level 2 Lab F Level 2

h gt 5 Lab F Level 2

Lab B Levels 1 and 2

Lab A Level 2

Lab D Level 3

Lab F Level 2

Lab L Level 2

kgt 1 Lab F Level 3

k gt 5 Lab F Level 2 Lab C Level 2

Lab F Level 3

Lab L Levels 1 and 3

Lab M Level 1

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2638 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

The emphasized results appear (i) coherent with the targeted contamination

levels (ii) concordant between both methods

All the results were thus kept for the statistical analysis

Comparison of the trueness and precision characteristics of

the reference method and alternative methods

The statistical values are summarised hereafter

Level p

Reference method Alternative method

Repeatability ratio

Reproducibility ratio Median

Repeatability standard deviation

Reproducibility standard deviation

Median Repeatability

standard deviation

Reproducibility standard deviation

1 10 16519 02415 02425 16021 02045 02433 0847 1007

2 10 26551 00543 00659 26719 00914 01325 1682 2011

2 bis 10 26581 02607 02607 25951 01900 01953 0729 0749

3 10 18671 00378 00378 18671 00478 00611 1264 1617

Bias of the alternative method

In order to estimate the bias of the alternative method with regard to the reference method for every

level Dij and t are calculated according to the following equations

RefAlt ijYijYDij

Diffp

Dijit

)2(

)(median

If the t value is superior to 2 the alternative method is significantly biased

with regard to the reference method

The values are given in table 17

Table 17 ndash t (d) values obtained by level

Level Bias t (d) Conclusion

1 0068 114 Non significant bias

2 0021 082 Non significant bias

2 bis - 0037 035 Non significant bias

3 - 0037 043 Non significant bias

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2738 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

The statistical test concludes to a non significant bias for all the inoculation

levels The bias varies from ndash 0037 to 0068 log CFUml

Comparison of the repeatability standard deviations

If the ratio Srj Alt Srj Ref of the repeatability standard deviations of the

alternative method and the reference method is larger than 2 the precision

under repeatability conditions of the alternative method is considered to be

lower than that of the reference method If this ratio is smaller than 05 the

precision under repeatability conditions of the alternative method is

considered to be greater than that of the reference method

The ratio values are given in table 18

Table 18

Level

Repeatability standard deviation

Reference method Alternative method Alternative method

reference method ratio

1 0242 0205 0847

2 0054 0091 1682

2 bis 0261 0190 0729

3 0038 0048 1264

Level Repeatability limit

Reference method Alternative method

1 0676 0573

2 0152 0256

2 bis 0730 0532

3 0106 0134

The ratios of the repeatability standard deviations are below 2 in all cases

The precision under repeatability conditions of the alternative method is

equivalent to that of the reference method

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2838 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Comparison of the reproducibility standard deviations

If the ratio Srj Alt Srj Ref of the reproducibility standard deviations of the alternative method and the

reference method is larger than 2 the precision under reproducibility conditions of the alternative

method is considered to be lower than that of the reference method If this ratio is smaller than 05 the

precision under reproducibility conditions of the alternative method is considered to be greater than

that of the reference method

The ratio values are given in table 19

Table 19

Level

Reproducibility standard deviation

Reference method Alternative method Alternative method

reference method ratio

1 0242 0243 1007

2 0066 0133 2011

2 bis 0261 0195 0749

3 0038 0061 1617

Level Reproducibility limit

Reference method Alternative method

1 0676 0681

2 0185 0371

2 bis 0730 0547

3 0106 0171

The ratios of the reproducibility standard deviations are below or close to 2

The precision under reproducibility conditions of the alternative method is

equivalent to that of the reference method

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2938 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

214 Conclusion

Method comparison study

The Listeria PrecisTM enumeration method shows satisfying linearity and

relative accuracy

The alternative method shows satisfying inclusivity and exclusivity results

Two Listeria monocytogenes strains gave negative results with the

alternative method These strains grew weakly on the reference method

media (OampA Palcam and Oxford)

The limits of repeatability of the alternative method are similar to those of the

reference method

The bias between both methods are low between - 0133 Log CFUg and

- 0008 Log CFUg

The Listeria PrecisTM enumeration method offers the saving for confirmatory

tests

Positive results are obtained with the alternative method in 2-3 days (with

OBIS Mono test) or in 4-7 days (by the classical tests described in the

standardized method) against 4-7 days for the reference method

Inter-laboratory study

The bias between the reference and the alternative methods shows satisfying

and correct values

- level 1 (100 - 1 000 CFUg) bias = + 007 log CFUg

- level 2 (1 000 - 10 000 CFUg) bias = 002 log CFUg

- level 2 bis (1 000 - 10 000 CFUg) bias = - 004 log CFUg

- level 3 (10 000 - 100 000 CFUg) bias = - 004 log CFUg

The repeatability and the reproducibility of the alternative method look similar

to those of the reference method

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3038 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

22 Extension study (realised by ASEPT 2007)

An extension study was performed in 2007 for a new confirmatory test OBIS

MONO

150 Listeria monocytogenes strains and 100 non-target strains were isolated

onto TSAYE and BrillianceTM Listeria Agar plates All the Listeria

monocytogenes and the non-target strains gave the expected results

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3138 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Appendix 1 ndash Flow diagram of the Listeria PrecisTM method

x g sample + 9 x ml buffered peptone water

Revivification step for 1 h plusmn 5 min at 20degC plusmn 1degC

Streaking 1 ml onto 3 Brilliance Listeria Agar plates (mother suspension)

or 01 ml onto 1 Brilliance Listeria Agar plates (mother suspension and decimal dilutions)

Incubation for 48 h plusmn 3 h at 37degC plusmn 1degC

Confirmatory tests on at least one typical colony

- by the tests described in the ISO method

- by applying the OBIS mono test on an isolated colony

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3238 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Appendix 2 ndash ISO 11290-2 (1998) amp ISO 11290-2A1 (2004) Horizontal

method for the detection and enumeration of Listeria monocytogenes -

Part 2 enumeration of Listeria monocytogenes in foods

10 g cheese + 90 ml buffered peptone water or Half Fraser Broth (without supplements) (110 dilution)

Stomach 1 min 30 seconds

Keep at 20degC plusmn 2degC for 1 h plusmn 5 min

Decimal dilutions in 9 ml peptone salt tubes

Dilution 110

Dilution 1100

Dilution 11000

1 ml on 3 Chromogenic Listeria medium (ISO) plates (x 2) (-1)

01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-2)

01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-3)

01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-4)

Incubation at 37degC plusmn 1degC

24 h - 48 h

Numerate typical colonies at 24 h

(bluegreen surrounded by an opaque halo)

Numerate at 48 h

Streak typical colonies on TSYEA

(5 coloniesplate 2 successive dilutions)

Confirmation tests

Catalase

Gram stain

For confirmation of L monocytogenes

Haemolysis

Carbohydrate (rhamnose and xylose) utilisation

CAMP Test

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3338 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Appendix 3 ndash Mandelrsquos graphics

Laboratoire h Ref

Niveau 1 h Ref

Niveau 2 h Ref

Niveau 3 h Alt

Niveau 1 h Alt

Niveau 2 h Alt

Niveau 3 h5 h1 h5 h1

A -050 -015 083 062 -212 -039 198 299 -198 -299

B -261 -270 021 -077 -058 173 198 299 -198 -299

C -151 -031 -014 -015 -004 -005 198 299 -198 -299

D 038 040 -137 000 072 207 198 299 -198 -299

E -001 090 155 -052 056 -101 198 299 -198 -299

F -112 -321 -153 070 -325 023 198 299 -198 -299

G 042 -071 040 147 -009 176 198 299 -198 -299

H 169 046 -169 192 018 -083 198 299 -198 -299

L 009 -120 021 -077 -231 005 198 299 -198 -299

M 001 031 014 -032 004 -011 198 299 -198 -299

-300

-200

-100

000

100

200

300

A B C D E F G H L M

Vale

urs

h d

e M

an

del(

rob

uste

s)

Laboratoires

Meacutethode de reacutefeacuterence analyse robuste

h Ref Niveau 1

h Ref Niveau 2

h Ref Niveau 3

h 5=198

h 1=299

-300

-200

-100

000

100

200

300

A B C D E F G H L M

Vale

urs

de h

de M

an

del(

rob

uste

s)

Laboratoires

Meacutethode alternative analyse robuste

h Alt Niveau 1

h Alt Niveau 2

h Alt Niveau 2

h 1=299

h 5=198

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3438 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Laboratoire k Ref

Niveau 1 k Ref

Niveau 2 k Ref

Niveau 3 k Alt

Niveau 1 k Alt

Niveau 2 k Alt

Niveau 3 k1 k5

A 000 089 142 084 162 046 259 181

B 125 087 073 104 075 046 259 181

C 117 013 091 188 022 083 259 181

D 052 036 069 000 035 083 259 181

E 028 034 037 138 019 112 259 181

F 140 222 018 027 281 112 259 181

G 088 068 024 131 145 000 259 181

H 129 094 173 016 061 000 259 181

L 069 000 073 104 270 224 259 181

M 000 048 038 043 099 241 259 181

000

050

100

150

200

250

300

350

400

A B C D E F G H L M

Vale

urs

k d

e M

an

del(

rob

uste

s)

Laboratoires

Meacutethode de reacutefeacuterence analyse robuste

k Ref Niveau 1k Ref Niveau 2k Ref Niveau 3

k 1=259

k 5=181

000

050

100

150

200

250

300

A B C D E F G H L M

Vale

urs

k d

e M

an

del

(ro

bu

ste

s)

Laboratoires

Meacutethode alternative analyse robuste

k Alt Niveau 3

k Alt Niveau 1

k Alt Niveau 2

k 1=259

k 5=181

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3538 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Appendix 4 ndash Statistical results

Level 1

Nombre de laboratoires (p) 10

A 1602 1602 1602 000 0000 0000 -0495 0000 A 1845 1602 1724 017 0122 -0122 0621 0840 0122

B 1602 1176 1389 030 0213 -0213 -2613 1247 B 1602 1301 1452 021 0151 -0151 -0769 1041 0062

C 1699 1301 1500 028 0199 -0199 -1510 1165 C 1301 1845 1573 038 -0272 0272 -0148 1881 0073

D 1602 1778 1690 012 -0088 0088 0380 0516 D 1602 1602 1602 000 0000 0000 0000 0000 -0088

E 1699 1602 1651 007 0048 -0048 -0013 0284 E 1699 1301 1500 028 0199 -0199 -0522 1376 -0151

F 1778 1301 1540 034 0239 -0239 -1116 1397 F 1778 1699 1739 006 0040 -0040 0698 0274 0199

G 1845 1544 1695 021 0151 -0151 0425 0881 G 2079 1699 1889 027 0190 -0190 1467 1315 0194

H 2041 1602 1822 031 0220 -0220 1689 1286 H 2000 1954 1977 003 0023 -0023 1917 0158 0155

L 1544 1778 1661 017 -0117 0117 0092 0685 L 1301 1602 1452 021 -0151 0151 -0769 1041 -0210

M 1653 1653 1653 000 0000 0000 0013 0000 M 1477 1602 1540 009 -0062 0062 -0319 0432 -0114

m 165186 m 160206 mediane D 00678

Cn(2p) 1867 Cn(2p) 1867 Cp 16101

Cn(p) 16101 Cn(p) 16101 Qn(D) 0092926

n=2p n 20 n=2p n 20 Qdiff 01496169

f 11 f 11 t 114

l 55 l 55

n=p n 10 n=p n 10

f 6 f 6

l 15 l 15

Qn(2p) 0091465 Qn(2p) 007745

Qn(p) 0062469 Qn(p) 0121519

Qintra 017078 Qintra 014461

Qinter 010058 Qinter 019565

Sr(eacutecart-type de reacutepeacutetabiliteacute) 02415 Sr 02045

RSDr(coefficient de variation de la reacutepeacutetabiliteacute)1462 RSDr 1277

r(repeatability limit)) 0676 r 0573

SL(eacutecart-type interlaboratoire) -001905 SLlt0 SL 001737

SR(eacutecart-type de reproductibiliteacute) 02415 SR 02433

RSDR(coefficient de variation de la reproductibiliteacute)0146 RSDR 0152

R(limite de reproductibiliteacute)) 06762 R(limite de reproductibiliteacute))068123

h k h MoyenneLaboratoires

Niveau 1

si LaboratoiresDeviationMoyenne Dk DeviationsiAlt

Niveau 1

Ref

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3638 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Level 2

Nombre de laboratoires (p) 10

A 2681 2613 2647 005 0034 -0034 -0151 0891 A 2322 2531 2427 015 -0105 0105 -2119 1620 -0220

B 2544 2477 2511 005 0033 -0033 -2699 0872 B 2556 2653 2605 007 -0048 0048 -0581 0750 0094

C 2643 2633 2638 001 0005 -0005 -0311 0130 C 2681 2653 2667 002 0014 -0014 -0040 0217 0029

D 2690 2663 2676 002 0014 -0014 0400 0357 D 2778 2732 2755 003 0023 -0023 0721 0354 0079

E 2690 2716 2703 002 -0013 0013 0897 0336 E 2724 2748 2736 002 -0012 0012 0556 0185 0033

F 2568 2398 2483 012 0085 -0085 -3214 2217 F 2477 2114 2296 026 0182 -0182 -3255 2811 -0188

G 2643 2591 2617 004 0026 -0026 -0707 0682 G 2756 2568 2662 013 0094 -0094 -0085 1453 0045

H 2716 2643 2680 005 0036 -0036 0460 0945 H 2653 2732 2693 006 -0040 0040 0181 0613 0013

L 2591 2591 2591 000 0000 0000 -1196 0000 L 2230 2580 2405 025 -0175 0175 -2307 2704 -0186

M 2653 2690 2672 003 -0018 0018 0311 0481 M 2740 2613 2677 009 0064 -0064 0040 0987 0005

m 2655082 m 26719001 mediane D 00209

Cn(2p) 1867 Cn(2p) 1867 Cp 16101

Cn(p) 16101 Cn(p) 16101 Qn(D) 0039911

n=2p n 20 n=2p n 20 Qdiff 00642591

f 11 f 11 t 082

l 55 l 55

n=p n 10 n=p n 10

f 6 f 6

l 15 l 15

Qn(2p) 0020570 Qn(2p) 00345985

Qn(p) 00332437 Qn(p) 00718158

Qintra 003841 Qintra 006460

Qinter 005352 Qinter 011563

Sr(eacutecart-type de reacutepeacutetabiliteacute) 00543 Sr 00914

RSDr(coefficient de variation de la reacutepeacutetabiliteacute)205 RSDr 342

r(repeatability limit)) 0152 r 0256

SL(eacutecart-type interlaboratoire) 000139 SL 000920

SR(eacutecart-type de reproductibiliteacute) 00659 SR 01325

RSDR(coefficient de variation de la reproductibiliteacute)0025 RSDR 0050

R(limite de reproductibiliteacute)) 018446 R(limite de reproductibiliteacute))03708618

Laboratoires

Niveau 2

Moyenne si Laboratoires

Niveau 2

DRef Alt Moyenne si Deviation h Deviation h k k

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3738 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Level 2 bis

Nombre de laboratoires (p) 10

A 3000 2477 2739 037 0261 -0261 0832 1418 A 2602 2477 2540 009 0062 -0062 -0391 0465 -0199

B 2544 2813 2678 019 -0134 0134 0211 0729 B 2903 2778 2841 009 0062 -0062 1732 0465 0162

C 2477 2813 2645 024 -0168 0168 -0135 0911 C 2477 2699 2588 016 -0111 0111 -0049 0826 -0057

D 2398 2653 2526 018 -0128 0128 -1370 0692 D 2778 3000 2889 016 -0111 0111 2074 0826 0363

E 2875 2740 2808 010 0067 -0067 1547 0365 E 2602 2301 2452 021 0151 -0151 -1012 1120 -0356

F 2477 2544 2511 005 -0033 0033 -1525 0182 F 2778 2477 2628 021 0151 -0151 0230 1120 0117

G 2653 2740 2697 006 -0044 0044 0400 0236 G 2845 2845 2845 000 0000 0000 1764 0000 0148

H 2176 2813 2495 045 -0318 0318 -1692 1727 H 2477 2477 2477 000 0000 0000 -0832 0000 -0017

L 2813 2544 2678 019 0134 -0134 0211 0729 L 2903 2301 2602 043 0301 -0301 0049 2241 -0076

M 2602 2740 2671 010 -0069 0069 0135 0375 M 2903 2255 2579 046 0324 -0324 -0112 2411 -0092

m 2658114 m 259505 mediane D -00372

Cn(2p) 1867 Cn(2p) 1867 Cp 16101

Cn(p) 16101 Cn(p) 16101 Qn(D) 01656915

n=2p n 20 n=2p n 20 Qdiff 02667753

f 11 f 11 t 035

l 55 l 55

n=p n 10 n=p n 10

f 6 f 6

l 15 l 15

Qn(2p) 0098745 Qn(2p) 007195

Qn(p) 006007 Qn(p) 008805

Qintra 018437 Qintra 013435

Qinter 009672 Qinter 014176

Sr(eacutecart-type de reacutepeacutetabiliteacute) 02607 Sr 01900

RSDr(coefficient de variation de la reacutepeacutetabiliteacute)981 RSDr 732

r(repeatability limit)) 0730 r 0532

SL(eacutecart-type interlaboratoire) -002464 SLlt0 SL 000205

SR(eacutecart-type de reproductibiliteacute) 02607 SR 01953

RSDR(coefficient de variation de la reproductibiliteacute)0098 RSDR 0075

R(limite de reproductibiliteacute)) 072996 R(limite de reproductibiliteacute))054686

Laboratoires

Niveau 2 bis

Moyenne si Laboratoires

Niveau 2 bis

DRef Alt Moyenne si Deviation h Deviation h k k

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3838 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Level 3

Nombre de laboratoires (p) 10

A 3690 3663 3676 002 0014 -0014 -0311 0513 A 3690 3708 3699 001 -0009 0009 0354 0257 0022

B 3613 3672 3642 004 -0030 0030 -1851 1109 B 3708 3568 3638 010 0070 -0070 -0844 2061 -0005

C 3663 3732 3698 005 -0035 0035 0645 1302 C 3663 3699 3681 003 -0018 0018 0000 0536 -0017

D 3690 3690 3690 000 0000 0000 0311 0000 D 3643 3663 3653 001 -0010 0010 -0545 0286 -0037

E 3732 3653 3693 006 0040 -0040 0429 1480 E 3613 3732 3673 008 -0060 0060 -0162 1769 -0020

F 3643 3672 3658 002 -0014 0014 -1157 0536 F 3690 3771 3731 006 -0040 0040 0975 1193 0073

G 3699 3653 3676 003 0023 -0023 -0328 0856 G 3602 3643 3623 003 -0021 0021 -1141 0612 -0053

H 3623 3672 3648 003 -0024 0024 -1615 0913 H 3748 3681 3715 005 0033 -0033 0664 0990 0067

L 3724 3663 3694 004 0031 -0031 0461 1150 L 3748 3771 3760 002 -0011 0011 1544 0335 0066

M 3690 3690 3690 000 0000 0000 0311 0000 M 3806 3681 3744 009 0062 -0062 1234 1848 0054

m 368334 m 368086 mediane D 00089

Cn(2p) 1867 Cn(2p) 1867 Cp 16101

Cn(p) 16101 Cn(p) 16101 Qn(D) 00325359

n=2p n 20 n=2p n 20 Qdiff 00523852

f 11 f 11 t 043

l 55 l 55

n=p n 10 n=p n 10

f 6 f 6

l 15 l 15

Qn(2p) 0014323 Qn(2p) 001811

Qn(p) 0013719 Qn(p) 003164

Qintra 002674 Qintra 003381

Qinter 002209 Qinter 005094

Sr(eacutecart-type de reacutepeacutetabiliteacute) 00378 Sr 00478

RSDr(coefficient de variation de la reacutepeacutetabiliteacute)103 RSDr 130

r(repeatability limit)) 0106 r 0134

SL(eacutecart-type interlaboratoire) -000023 SLlt0 SL 000145

SR(eacutecart-type de reproductibiliteacute) 00378 SR 00611

RSDR(coefficient de variation de la reproductibiliteacute)0010 RSDR 0017

R(limite de reproductibiliteacute)) 0105894 R(limite de reproductibiliteacute))017119

Ref Alt DLaboratoires

Niveau 3

Moyenne si Deviation h k Laboratoires

Niveau 3

Moyenne si Deviation h k

Page 19: ÞÀ¿¸7 ú Y5zñLjÜ< /u +æü t¬Á ... - NF Validation · NF VALIDATION Validation of alternative analytical methods Application in food microbiology ... 500 – 1 000 1 000 –

Thermo Fisher Scientific

ADRIA Deacuteveloppement 1938 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Figure 5 ndash Sensitivity patterns for the tested (matrixstrain) pairs

2115 SpecificitySelectivity

The specificity is defined as the degree to which a method is affected (or not) by the other components

present in a multi-component sample That is the ability of a method to measure exactly a given

analyte or its amount within the sample without interference from non-target components such as a

matrix effect or background noise

The selectivity is defined as a measure of the degree of non-interference in the presence of non-target

analytes A method is selective if it can be used to detect the analyte under examination and that a

guarantee can be provided that the detected signal can only be a product by that specific analyte

The specificity and selectivity study was performed for the Listeria PrecisTM

detection method no additional test was realised for the enumeration

method The results were the following

- Among the Listeria monocytogenes strains tested 2 strains (Listeria

monocytogenes CIP 7834 and Listeria monocytogenes CIP 105459) gave

negative results These 2 strains were tested twice by running 48 h

incubation time of the compared agars Positive results were then

observed by the alternative method Pale characteristic colonies were

observed on the ISO agar by streaking the Half Fraser broth only

- No cross reaction was observed with the non-target strains except with

Listeria ivanovii which gave characteristic colonies on Brilliancetrade Listeria

Agar but the confirmatory tests provide a clear discrimination between

Listeria monocytogenes and Listeria ivanovi species

Rillettes

Lettuce

Raw milk

Smoked salmon

Raw egg

Process water

x (CFUg)

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2038 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

212 Practicability

1 Packaging Ready to use Brilliance Listeria agar plates are available

(x 10 plates)

2 Reagent volume

3 Storage conditionsndash

Shelf life of products

not opened

The plates are stored at 6 - 12degC in darkness conditions The

expiration date is mentioned on the package and on the plates It

is around 3 months

4 Modalities of use

after first use

5 Required

equipments No specific equipment is required

6 Reagents Ready to use plates

7 Training 05 day

8 Real time handling

Reference method

9 min for one sample

55 min for 10 samples

Alternative method

7 min for one sample

35 minutes for 10 samples

9 Time to result

Negative results

Reference method Day 2

Alternative method Day 2

Negative results after confirmation or positive results

Reference method Day 4 ndash Day 7

Alternative method Day 4 ndash Day 7

Positive results

Reference method Day 4 ndash Day 7

Alternative method Day 2 ndash Day 3 (with OBIS Mono Test)

or Day 4 ndash Day 7 (by the classical tests described in the

standardised method)

10 Technician skill Identical to that required for the reference method

11 Steps common to

the reference method Mother suspension revivification step and decimal dilutions

12 Traceability

13 Maintenance

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2138 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

213 Inter-laboratory study

2131 Organisation

12 laboratories participated to this study Pasteurised milk was inoculated by

Listeria monocytogenes 12a isolated from raw milk The targeted

inoculation levels were

- 0 CFUml

- 50 CFU ml

- 500 CFU ml

- 5 000 CFU ml

Each laboratory received eight flasks of 10 ml sample ie two flasks per

inoculation level Furthermore one non-inoculated sample has been added

to the package for total viable count microflora (NF ISO 4833 method)

Detailed instructions were transmitted to the collaborators by the expert

laboratory

Collaborative study laboratories and the expert laboratory carried out the

analyses with the alternative and reference methods

2132 Verification of experimental parameters

Strain stability during transport

In order to evaluate the Listeria monocytogenes strain stability during

transport bacterial count of all inoculated flasks were checked at different

time ie inoculation time after 24 h of conservation at 2deg - 8degC Results are

reported in table 11

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2238 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Table 11 ndash Listeria monocytogenes count

by ISO 11290-2A1 method (log CFUml)

Day of the analyses Level contamination Reference method

Day 0

(29052006)

1 45

2 440

3 4 500

Day 1

(30052006)

1 60

2 430

3 4 900

No evolution of the strain was observed after storage for 24 h at 2degC ndash 8degC

Contamination level

The real contamination levels are provided in table 12

Table 12 ndash Real contamination level (CFUml)

Target inoculation

(CFU ml) Samples Ndeg

Real contamination levels

(CFUml)

0 1 - 8 0

50 5 - 6 45

500 3 - 7 440

5000 2 - 4 4 500

Sample temperature at receipt

Measured temperatures at receipt are listed in table 13

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2338 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Table 13 - Sample temperature on receipt

Laboratories Receipt

day

Hour

receipt

Measured temperature

at receipt (degC)

Measured temperature by the

temperature probe (degC)

A

B

C

D

E

F

G

H

I

J

K

L

M

30052006

10h45

10h15

11h15

10h00

09h30

8h30

11h50

10h00

08h30

14h45

08h30

09h45

08h45

82

55

45

63

60

62

81

38

48

-

14

67

76

03

03

-13

03

04

-14

05

04

01

05

-13

04

Conclusion

The temperatures during the transport were correct for all the labs except for

Lab J which didnrsquot provide the information

All the labs received their packages with several broken flasks

Three labs (B D and L) received some damaged samples but proceeded to

analyses

All the labs started the analyses at Day 1 except Lab J

2133 Results

Aerobic mesophilic flora

The count of aerobic mesophilic flora varied from 94 102 CFUml to

33 103 CFUml

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2438 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Expert lab results

The results obtained by the expert lab are the following

Table 14

Target inoculation level (CFUml)

Reference method ISO 11290-2

Alternative method Listeria Precis Enumeration

Rep 1 Rep 2 Rep 1 Rep 2

lt 10 lt 10 lt 10 lt 10 lt10

50 50 (Ne) 60 (Ne) 50 (Ne) 30 (Ne)

500 470 390 490 500

5 000 4 700 5 100 5 900 3 400

Ne estimated number

The obtained results are in agreement with those expected

Collaborator Labs results

A synthesis of the results obtained by the collaborator labs is provided

Table 15 (CFUml) and Table 16 (log CFUml)

Table 15 ndash Synthesis of results obtained by the alternative

and the reference methods (CFUml)

Labs Level 1 Level 2 Level 2 bis Level 3

Reference method

Alternative method

Reference method

Alternative method

Reference method

Alternative method

Reference method

Alternative method

A 40 40 70 40 480 410 210 340 1000 300 400 300 4900 4600 4900 5100

B 40 15 40 20 350 300 360 450 350 650 800 600 4100 4700 5100 3700

C 50 20 20 70 440 430 480 450 300 650 300 500 4600 5400 4600 5000

D 40 60 40 40 490 460 600 540 250 450 600 1000 4900 4900 4400 4600

E 50 40 50 20 490 520 530 560 750 550 400 200 5400 4500 4100 5400

F 60 20 60 50 370 250 300 130 300 350 600 300 4400 4700 4900 5900

G 70 35 120 50 440 390 570 370 450 550 700 700 5000 4500 4000 4400

H 110 40 100 90 520 440 450 540 150 650 300 300 4200 4700 5600 4800

L 35 60 20 40 390 390 170 380 650 350 800 200 5300 4600 5600 5900

M 45 45 30 40 450 490 550 410 400 550 800 180 4900 4900 6400 4800

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2538 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Table 16 ndash Synthesis of results obtained by the alternative

and the reference methods (log CFUml)

Labs Level 1 Level 2 Level 2 bis Level 3

Reference method

Alternative method

Reference method

Alternative method

Reference method

Alternative method

Reference method

Alternative method

A 1602 1602 1845 1602 2681 2613 2322 2531 3000 2477 2602 2477 3690 3663 3690 3708

B 1602 1176 1602 1301 2544 2477 2556 2653 2544 2813 2903 2778 3613 3672 3708 3568

C 1699 1301 1301 1845 2643 2633 2681 2653 2477 2813 2477 2699 3663 3732 3663 3699

D 1602 1778 1602 1602 2690 2663 2778 2732 2398 2653 2778 3000 3690 3690 3643 3663

E 1699 1602 1699 1301 2690 2716 2724 2748 2875 2740 2602 2301 3732 3653 3613 3732

F 1778 1301 1778 1699 2568 2398 2477 2114 2477 2544 2778 2477 3643 3672 3690 3771

G 1845 1544 2079 1699 2643 2591 2756 2568 2653 2740 2845 2845 3699 3653 3602 3643

H 2041 1602 2000 1954 2716 2643 2653 2732 2176 2813 2477 2477 3623 3672 3748 3681

L 1544 1778 1301 1602 2591 2591 2230 2580 2813 2544 2903 2301 3724 3663 3748 3771

M 1653 1653 1477 1602 2653 2690 2740 2613 2602 2740 2903 2255 3690 3690 3806 3681

Level 2 results obtained taking account the 3 plates inoculated with the mother-suspension

Level 2 bis results obtained taking account the plates inoculated with 01 ml of the mother-suspension

Scrutiny of the measurement results for consistency

In order to identify the measurement results or laboratories that could be

inconsistent two graphical consistency techniques were realised Mandelrsquos

h-ank-statistics Mandel indicators h and k at 5 significance highlighted

some possible inconsistent data (See tables and graphics in Appendix 3 and

Appendix 4)

Mandelrsquos values Number of values observed over the threshold

Reference method Alternative method

h gt 1 Lab F Level 2 Lab F Level 2

h gt 5 Lab F Level 2

Lab B Levels 1 and 2

Lab A Level 2

Lab D Level 3

Lab F Level 2

Lab L Level 2

kgt 1 Lab F Level 3

k gt 5 Lab F Level 2 Lab C Level 2

Lab F Level 3

Lab L Levels 1 and 3

Lab M Level 1

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2638 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

The emphasized results appear (i) coherent with the targeted contamination

levels (ii) concordant between both methods

All the results were thus kept for the statistical analysis

Comparison of the trueness and precision characteristics of

the reference method and alternative methods

The statistical values are summarised hereafter

Level p

Reference method Alternative method

Repeatability ratio

Reproducibility ratio Median

Repeatability standard deviation

Reproducibility standard deviation

Median Repeatability

standard deviation

Reproducibility standard deviation

1 10 16519 02415 02425 16021 02045 02433 0847 1007

2 10 26551 00543 00659 26719 00914 01325 1682 2011

2 bis 10 26581 02607 02607 25951 01900 01953 0729 0749

3 10 18671 00378 00378 18671 00478 00611 1264 1617

Bias of the alternative method

In order to estimate the bias of the alternative method with regard to the reference method for every

level Dij and t are calculated according to the following equations

RefAlt ijYijYDij

Diffp

Dijit

)2(

)(median

If the t value is superior to 2 the alternative method is significantly biased

with regard to the reference method

The values are given in table 17

Table 17 ndash t (d) values obtained by level

Level Bias t (d) Conclusion

1 0068 114 Non significant bias

2 0021 082 Non significant bias

2 bis - 0037 035 Non significant bias

3 - 0037 043 Non significant bias

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2738 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

The statistical test concludes to a non significant bias for all the inoculation

levels The bias varies from ndash 0037 to 0068 log CFUml

Comparison of the repeatability standard deviations

If the ratio Srj Alt Srj Ref of the repeatability standard deviations of the

alternative method and the reference method is larger than 2 the precision

under repeatability conditions of the alternative method is considered to be

lower than that of the reference method If this ratio is smaller than 05 the

precision under repeatability conditions of the alternative method is

considered to be greater than that of the reference method

The ratio values are given in table 18

Table 18

Level

Repeatability standard deviation

Reference method Alternative method Alternative method

reference method ratio

1 0242 0205 0847

2 0054 0091 1682

2 bis 0261 0190 0729

3 0038 0048 1264

Level Repeatability limit

Reference method Alternative method

1 0676 0573

2 0152 0256

2 bis 0730 0532

3 0106 0134

The ratios of the repeatability standard deviations are below 2 in all cases

The precision under repeatability conditions of the alternative method is

equivalent to that of the reference method

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2838 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Comparison of the reproducibility standard deviations

If the ratio Srj Alt Srj Ref of the reproducibility standard deviations of the alternative method and the

reference method is larger than 2 the precision under reproducibility conditions of the alternative

method is considered to be lower than that of the reference method If this ratio is smaller than 05 the

precision under reproducibility conditions of the alternative method is considered to be greater than

that of the reference method

The ratio values are given in table 19

Table 19

Level

Reproducibility standard deviation

Reference method Alternative method Alternative method

reference method ratio

1 0242 0243 1007

2 0066 0133 2011

2 bis 0261 0195 0749

3 0038 0061 1617

Level Reproducibility limit

Reference method Alternative method

1 0676 0681

2 0185 0371

2 bis 0730 0547

3 0106 0171

The ratios of the reproducibility standard deviations are below or close to 2

The precision under reproducibility conditions of the alternative method is

equivalent to that of the reference method

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2938 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

214 Conclusion

Method comparison study

The Listeria PrecisTM enumeration method shows satisfying linearity and

relative accuracy

The alternative method shows satisfying inclusivity and exclusivity results

Two Listeria monocytogenes strains gave negative results with the

alternative method These strains grew weakly on the reference method

media (OampA Palcam and Oxford)

The limits of repeatability of the alternative method are similar to those of the

reference method

The bias between both methods are low between - 0133 Log CFUg and

- 0008 Log CFUg

The Listeria PrecisTM enumeration method offers the saving for confirmatory

tests

Positive results are obtained with the alternative method in 2-3 days (with

OBIS Mono test) or in 4-7 days (by the classical tests described in the

standardized method) against 4-7 days for the reference method

Inter-laboratory study

The bias between the reference and the alternative methods shows satisfying

and correct values

- level 1 (100 - 1 000 CFUg) bias = + 007 log CFUg

- level 2 (1 000 - 10 000 CFUg) bias = 002 log CFUg

- level 2 bis (1 000 - 10 000 CFUg) bias = - 004 log CFUg

- level 3 (10 000 - 100 000 CFUg) bias = - 004 log CFUg

The repeatability and the reproducibility of the alternative method look similar

to those of the reference method

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3038 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

22 Extension study (realised by ASEPT 2007)

An extension study was performed in 2007 for a new confirmatory test OBIS

MONO

150 Listeria monocytogenes strains and 100 non-target strains were isolated

onto TSAYE and BrillianceTM Listeria Agar plates All the Listeria

monocytogenes and the non-target strains gave the expected results

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3138 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Appendix 1 ndash Flow diagram of the Listeria PrecisTM method

x g sample + 9 x ml buffered peptone water

Revivification step for 1 h plusmn 5 min at 20degC plusmn 1degC

Streaking 1 ml onto 3 Brilliance Listeria Agar plates (mother suspension)

or 01 ml onto 1 Brilliance Listeria Agar plates (mother suspension and decimal dilutions)

Incubation for 48 h plusmn 3 h at 37degC plusmn 1degC

Confirmatory tests on at least one typical colony

- by the tests described in the ISO method

- by applying the OBIS mono test on an isolated colony

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3238 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Appendix 2 ndash ISO 11290-2 (1998) amp ISO 11290-2A1 (2004) Horizontal

method for the detection and enumeration of Listeria monocytogenes -

Part 2 enumeration of Listeria monocytogenes in foods

10 g cheese + 90 ml buffered peptone water or Half Fraser Broth (without supplements) (110 dilution)

Stomach 1 min 30 seconds

Keep at 20degC plusmn 2degC for 1 h plusmn 5 min

Decimal dilutions in 9 ml peptone salt tubes

Dilution 110

Dilution 1100

Dilution 11000

1 ml on 3 Chromogenic Listeria medium (ISO) plates (x 2) (-1)

01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-2)

01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-3)

01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-4)

Incubation at 37degC plusmn 1degC

24 h - 48 h

Numerate typical colonies at 24 h

(bluegreen surrounded by an opaque halo)

Numerate at 48 h

Streak typical colonies on TSYEA

(5 coloniesplate 2 successive dilutions)

Confirmation tests

Catalase

Gram stain

For confirmation of L monocytogenes

Haemolysis

Carbohydrate (rhamnose and xylose) utilisation

CAMP Test

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3338 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Appendix 3 ndash Mandelrsquos graphics

Laboratoire h Ref

Niveau 1 h Ref

Niveau 2 h Ref

Niveau 3 h Alt

Niveau 1 h Alt

Niveau 2 h Alt

Niveau 3 h5 h1 h5 h1

A -050 -015 083 062 -212 -039 198 299 -198 -299

B -261 -270 021 -077 -058 173 198 299 -198 -299

C -151 -031 -014 -015 -004 -005 198 299 -198 -299

D 038 040 -137 000 072 207 198 299 -198 -299

E -001 090 155 -052 056 -101 198 299 -198 -299

F -112 -321 -153 070 -325 023 198 299 -198 -299

G 042 -071 040 147 -009 176 198 299 -198 -299

H 169 046 -169 192 018 -083 198 299 -198 -299

L 009 -120 021 -077 -231 005 198 299 -198 -299

M 001 031 014 -032 004 -011 198 299 -198 -299

-300

-200

-100

000

100

200

300

A B C D E F G H L M

Vale

urs

h d

e M

an

del(

rob

uste

s)

Laboratoires

Meacutethode de reacutefeacuterence analyse robuste

h Ref Niveau 1

h Ref Niveau 2

h Ref Niveau 3

h 5=198

h 1=299

-300

-200

-100

000

100

200

300

A B C D E F G H L M

Vale

urs

de h

de M

an

del(

rob

uste

s)

Laboratoires

Meacutethode alternative analyse robuste

h Alt Niveau 1

h Alt Niveau 2

h Alt Niveau 2

h 1=299

h 5=198

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3438 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Laboratoire k Ref

Niveau 1 k Ref

Niveau 2 k Ref

Niveau 3 k Alt

Niveau 1 k Alt

Niveau 2 k Alt

Niveau 3 k1 k5

A 000 089 142 084 162 046 259 181

B 125 087 073 104 075 046 259 181

C 117 013 091 188 022 083 259 181

D 052 036 069 000 035 083 259 181

E 028 034 037 138 019 112 259 181

F 140 222 018 027 281 112 259 181

G 088 068 024 131 145 000 259 181

H 129 094 173 016 061 000 259 181

L 069 000 073 104 270 224 259 181

M 000 048 038 043 099 241 259 181

000

050

100

150

200

250

300

350

400

A B C D E F G H L M

Vale

urs

k d

e M

an

del(

rob

uste

s)

Laboratoires

Meacutethode de reacutefeacuterence analyse robuste

k Ref Niveau 1k Ref Niveau 2k Ref Niveau 3

k 1=259

k 5=181

000

050

100

150

200

250

300

A B C D E F G H L M

Vale

urs

k d

e M

an

del

(ro

bu

ste

s)

Laboratoires

Meacutethode alternative analyse robuste

k Alt Niveau 3

k Alt Niveau 1

k Alt Niveau 2

k 1=259

k 5=181

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3538 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Appendix 4 ndash Statistical results

Level 1

Nombre de laboratoires (p) 10

A 1602 1602 1602 000 0000 0000 -0495 0000 A 1845 1602 1724 017 0122 -0122 0621 0840 0122

B 1602 1176 1389 030 0213 -0213 -2613 1247 B 1602 1301 1452 021 0151 -0151 -0769 1041 0062

C 1699 1301 1500 028 0199 -0199 -1510 1165 C 1301 1845 1573 038 -0272 0272 -0148 1881 0073

D 1602 1778 1690 012 -0088 0088 0380 0516 D 1602 1602 1602 000 0000 0000 0000 0000 -0088

E 1699 1602 1651 007 0048 -0048 -0013 0284 E 1699 1301 1500 028 0199 -0199 -0522 1376 -0151

F 1778 1301 1540 034 0239 -0239 -1116 1397 F 1778 1699 1739 006 0040 -0040 0698 0274 0199

G 1845 1544 1695 021 0151 -0151 0425 0881 G 2079 1699 1889 027 0190 -0190 1467 1315 0194

H 2041 1602 1822 031 0220 -0220 1689 1286 H 2000 1954 1977 003 0023 -0023 1917 0158 0155

L 1544 1778 1661 017 -0117 0117 0092 0685 L 1301 1602 1452 021 -0151 0151 -0769 1041 -0210

M 1653 1653 1653 000 0000 0000 0013 0000 M 1477 1602 1540 009 -0062 0062 -0319 0432 -0114

m 165186 m 160206 mediane D 00678

Cn(2p) 1867 Cn(2p) 1867 Cp 16101

Cn(p) 16101 Cn(p) 16101 Qn(D) 0092926

n=2p n 20 n=2p n 20 Qdiff 01496169

f 11 f 11 t 114

l 55 l 55

n=p n 10 n=p n 10

f 6 f 6

l 15 l 15

Qn(2p) 0091465 Qn(2p) 007745

Qn(p) 0062469 Qn(p) 0121519

Qintra 017078 Qintra 014461

Qinter 010058 Qinter 019565

Sr(eacutecart-type de reacutepeacutetabiliteacute) 02415 Sr 02045

RSDr(coefficient de variation de la reacutepeacutetabiliteacute)1462 RSDr 1277

r(repeatability limit)) 0676 r 0573

SL(eacutecart-type interlaboratoire) -001905 SLlt0 SL 001737

SR(eacutecart-type de reproductibiliteacute) 02415 SR 02433

RSDR(coefficient de variation de la reproductibiliteacute)0146 RSDR 0152

R(limite de reproductibiliteacute)) 06762 R(limite de reproductibiliteacute))068123

h k h MoyenneLaboratoires

Niveau 1

si LaboratoiresDeviationMoyenne Dk DeviationsiAlt

Niveau 1

Ref

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3638 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Level 2

Nombre de laboratoires (p) 10

A 2681 2613 2647 005 0034 -0034 -0151 0891 A 2322 2531 2427 015 -0105 0105 -2119 1620 -0220

B 2544 2477 2511 005 0033 -0033 -2699 0872 B 2556 2653 2605 007 -0048 0048 -0581 0750 0094

C 2643 2633 2638 001 0005 -0005 -0311 0130 C 2681 2653 2667 002 0014 -0014 -0040 0217 0029

D 2690 2663 2676 002 0014 -0014 0400 0357 D 2778 2732 2755 003 0023 -0023 0721 0354 0079

E 2690 2716 2703 002 -0013 0013 0897 0336 E 2724 2748 2736 002 -0012 0012 0556 0185 0033

F 2568 2398 2483 012 0085 -0085 -3214 2217 F 2477 2114 2296 026 0182 -0182 -3255 2811 -0188

G 2643 2591 2617 004 0026 -0026 -0707 0682 G 2756 2568 2662 013 0094 -0094 -0085 1453 0045

H 2716 2643 2680 005 0036 -0036 0460 0945 H 2653 2732 2693 006 -0040 0040 0181 0613 0013

L 2591 2591 2591 000 0000 0000 -1196 0000 L 2230 2580 2405 025 -0175 0175 -2307 2704 -0186

M 2653 2690 2672 003 -0018 0018 0311 0481 M 2740 2613 2677 009 0064 -0064 0040 0987 0005

m 2655082 m 26719001 mediane D 00209

Cn(2p) 1867 Cn(2p) 1867 Cp 16101

Cn(p) 16101 Cn(p) 16101 Qn(D) 0039911

n=2p n 20 n=2p n 20 Qdiff 00642591

f 11 f 11 t 082

l 55 l 55

n=p n 10 n=p n 10

f 6 f 6

l 15 l 15

Qn(2p) 0020570 Qn(2p) 00345985

Qn(p) 00332437 Qn(p) 00718158

Qintra 003841 Qintra 006460

Qinter 005352 Qinter 011563

Sr(eacutecart-type de reacutepeacutetabiliteacute) 00543 Sr 00914

RSDr(coefficient de variation de la reacutepeacutetabiliteacute)205 RSDr 342

r(repeatability limit)) 0152 r 0256

SL(eacutecart-type interlaboratoire) 000139 SL 000920

SR(eacutecart-type de reproductibiliteacute) 00659 SR 01325

RSDR(coefficient de variation de la reproductibiliteacute)0025 RSDR 0050

R(limite de reproductibiliteacute)) 018446 R(limite de reproductibiliteacute))03708618

Laboratoires

Niveau 2

Moyenne si Laboratoires

Niveau 2

DRef Alt Moyenne si Deviation h Deviation h k k

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3738 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Level 2 bis

Nombre de laboratoires (p) 10

A 3000 2477 2739 037 0261 -0261 0832 1418 A 2602 2477 2540 009 0062 -0062 -0391 0465 -0199

B 2544 2813 2678 019 -0134 0134 0211 0729 B 2903 2778 2841 009 0062 -0062 1732 0465 0162

C 2477 2813 2645 024 -0168 0168 -0135 0911 C 2477 2699 2588 016 -0111 0111 -0049 0826 -0057

D 2398 2653 2526 018 -0128 0128 -1370 0692 D 2778 3000 2889 016 -0111 0111 2074 0826 0363

E 2875 2740 2808 010 0067 -0067 1547 0365 E 2602 2301 2452 021 0151 -0151 -1012 1120 -0356

F 2477 2544 2511 005 -0033 0033 -1525 0182 F 2778 2477 2628 021 0151 -0151 0230 1120 0117

G 2653 2740 2697 006 -0044 0044 0400 0236 G 2845 2845 2845 000 0000 0000 1764 0000 0148

H 2176 2813 2495 045 -0318 0318 -1692 1727 H 2477 2477 2477 000 0000 0000 -0832 0000 -0017

L 2813 2544 2678 019 0134 -0134 0211 0729 L 2903 2301 2602 043 0301 -0301 0049 2241 -0076

M 2602 2740 2671 010 -0069 0069 0135 0375 M 2903 2255 2579 046 0324 -0324 -0112 2411 -0092

m 2658114 m 259505 mediane D -00372

Cn(2p) 1867 Cn(2p) 1867 Cp 16101

Cn(p) 16101 Cn(p) 16101 Qn(D) 01656915

n=2p n 20 n=2p n 20 Qdiff 02667753

f 11 f 11 t 035

l 55 l 55

n=p n 10 n=p n 10

f 6 f 6

l 15 l 15

Qn(2p) 0098745 Qn(2p) 007195

Qn(p) 006007 Qn(p) 008805

Qintra 018437 Qintra 013435

Qinter 009672 Qinter 014176

Sr(eacutecart-type de reacutepeacutetabiliteacute) 02607 Sr 01900

RSDr(coefficient de variation de la reacutepeacutetabiliteacute)981 RSDr 732

r(repeatability limit)) 0730 r 0532

SL(eacutecart-type interlaboratoire) -002464 SLlt0 SL 000205

SR(eacutecart-type de reproductibiliteacute) 02607 SR 01953

RSDR(coefficient de variation de la reproductibiliteacute)0098 RSDR 0075

R(limite de reproductibiliteacute)) 072996 R(limite de reproductibiliteacute))054686

Laboratoires

Niveau 2 bis

Moyenne si Laboratoires

Niveau 2 bis

DRef Alt Moyenne si Deviation h Deviation h k k

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3838 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Level 3

Nombre de laboratoires (p) 10

A 3690 3663 3676 002 0014 -0014 -0311 0513 A 3690 3708 3699 001 -0009 0009 0354 0257 0022

B 3613 3672 3642 004 -0030 0030 -1851 1109 B 3708 3568 3638 010 0070 -0070 -0844 2061 -0005

C 3663 3732 3698 005 -0035 0035 0645 1302 C 3663 3699 3681 003 -0018 0018 0000 0536 -0017

D 3690 3690 3690 000 0000 0000 0311 0000 D 3643 3663 3653 001 -0010 0010 -0545 0286 -0037

E 3732 3653 3693 006 0040 -0040 0429 1480 E 3613 3732 3673 008 -0060 0060 -0162 1769 -0020

F 3643 3672 3658 002 -0014 0014 -1157 0536 F 3690 3771 3731 006 -0040 0040 0975 1193 0073

G 3699 3653 3676 003 0023 -0023 -0328 0856 G 3602 3643 3623 003 -0021 0021 -1141 0612 -0053

H 3623 3672 3648 003 -0024 0024 -1615 0913 H 3748 3681 3715 005 0033 -0033 0664 0990 0067

L 3724 3663 3694 004 0031 -0031 0461 1150 L 3748 3771 3760 002 -0011 0011 1544 0335 0066

M 3690 3690 3690 000 0000 0000 0311 0000 M 3806 3681 3744 009 0062 -0062 1234 1848 0054

m 368334 m 368086 mediane D 00089

Cn(2p) 1867 Cn(2p) 1867 Cp 16101

Cn(p) 16101 Cn(p) 16101 Qn(D) 00325359

n=2p n 20 n=2p n 20 Qdiff 00523852

f 11 f 11 t 043

l 55 l 55

n=p n 10 n=p n 10

f 6 f 6

l 15 l 15

Qn(2p) 0014323 Qn(2p) 001811

Qn(p) 0013719 Qn(p) 003164

Qintra 002674 Qintra 003381

Qinter 002209 Qinter 005094

Sr(eacutecart-type de reacutepeacutetabiliteacute) 00378 Sr 00478

RSDr(coefficient de variation de la reacutepeacutetabiliteacute)103 RSDr 130

r(repeatability limit)) 0106 r 0134

SL(eacutecart-type interlaboratoire) -000023 SLlt0 SL 000145

SR(eacutecart-type de reproductibiliteacute) 00378 SR 00611

RSDR(coefficient de variation de la reproductibiliteacute)0010 RSDR 0017

R(limite de reproductibiliteacute)) 0105894 R(limite de reproductibiliteacute))017119

Ref Alt DLaboratoires

Niveau 3

Moyenne si Deviation h k Laboratoires

Niveau 3

Moyenne si Deviation h k

Page 20: ÞÀ¿¸7 ú Y5zñLjÜ< /u +æü t¬Á ... - NF Validation · NF VALIDATION Validation of alternative analytical methods Application in food microbiology ... 500 – 1 000 1 000 –

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2038 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

212 Practicability

1 Packaging Ready to use Brilliance Listeria agar plates are available

(x 10 plates)

2 Reagent volume

3 Storage conditionsndash

Shelf life of products

not opened

The plates are stored at 6 - 12degC in darkness conditions The

expiration date is mentioned on the package and on the plates It

is around 3 months

4 Modalities of use

after first use

5 Required

equipments No specific equipment is required

6 Reagents Ready to use plates

7 Training 05 day

8 Real time handling

Reference method

9 min for one sample

55 min for 10 samples

Alternative method

7 min for one sample

35 minutes for 10 samples

9 Time to result

Negative results

Reference method Day 2

Alternative method Day 2

Negative results after confirmation or positive results

Reference method Day 4 ndash Day 7

Alternative method Day 4 ndash Day 7

Positive results

Reference method Day 4 ndash Day 7

Alternative method Day 2 ndash Day 3 (with OBIS Mono Test)

or Day 4 ndash Day 7 (by the classical tests described in the

standardised method)

10 Technician skill Identical to that required for the reference method

11 Steps common to

the reference method Mother suspension revivification step and decimal dilutions

12 Traceability

13 Maintenance

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2138 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

213 Inter-laboratory study

2131 Organisation

12 laboratories participated to this study Pasteurised milk was inoculated by

Listeria monocytogenes 12a isolated from raw milk The targeted

inoculation levels were

- 0 CFUml

- 50 CFU ml

- 500 CFU ml

- 5 000 CFU ml

Each laboratory received eight flasks of 10 ml sample ie two flasks per

inoculation level Furthermore one non-inoculated sample has been added

to the package for total viable count microflora (NF ISO 4833 method)

Detailed instructions were transmitted to the collaborators by the expert

laboratory

Collaborative study laboratories and the expert laboratory carried out the

analyses with the alternative and reference methods

2132 Verification of experimental parameters

Strain stability during transport

In order to evaluate the Listeria monocytogenes strain stability during

transport bacterial count of all inoculated flasks were checked at different

time ie inoculation time after 24 h of conservation at 2deg - 8degC Results are

reported in table 11

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2238 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Table 11 ndash Listeria monocytogenes count

by ISO 11290-2A1 method (log CFUml)

Day of the analyses Level contamination Reference method

Day 0

(29052006)

1 45

2 440

3 4 500

Day 1

(30052006)

1 60

2 430

3 4 900

No evolution of the strain was observed after storage for 24 h at 2degC ndash 8degC

Contamination level

The real contamination levels are provided in table 12

Table 12 ndash Real contamination level (CFUml)

Target inoculation

(CFU ml) Samples Ndeg

Real contamination levels

(CFUml)

0 1 - 8 0

50 5 - 6 45

500 3 - 7 440

5000 2 - 4 4 500

Sample temperature at receipt

Measured temperatures at receipt are listed in table 13

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2338 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Table 13 - Sample temperature on receipt

Laboratories Receipt

day

Hour

receipt

Measured temperature

at receipt (degC)

Measured temperature by the

temperature probe (degC)

A

B

C

D

E

F

G

H

I

J

K

L

M

30052006

10h45

10h15

11h15

10h00

09h30

8h30

11h50

10h00

08h30

14h45

08h30

09h45

08h45

82

55

45

63

60

62

81

38

48

-

14

67

76

03

03

-13

03

04

-14

05

04

01

05

-13

04

Conclusion

The temperatures during the transport were correct for all the labs except for

Lab J which didnrsquot provide the information

All the labs received their packages with several broken flasks

Three labs (B D and L) received some damaged samples but proceeded to

analyses

All the labs started the analyses at Day 1 except Lab J

2133 Results

Aerobic mesophilic flora

The count of aerobic mesophilic flora varied from 94 102 CFUml to

33 103 CFUml

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2438 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Expert lab results

The results obtained by the expert lab are the following

Table 14

Target inoculation level (CFUml)

Reference method ISO 11290-2

Alternative method Listeria Precis Enumeration

Rep 1 Rep 2 Rep 1 Rep 2

lt 10 lt 10 lt 10 lt 10 lt10

50 50 (Ne) 60 (Ne) 50 (Ne) 30 (Ne)

500 470 390 490 500

5 000 4 700 5 100 5 900 3 400

Ne estimated number

The obtained results are in agreement with those expected

Collaborator Labs results

A synthesis of the results obtained by the collaborator labs is provided

Table 15 (CFUml) and Table 16 (log CFUml)

Table 15 ndash Synthesis of results obtained by the alternative

and the reference methods (CFUml)

Labs Level 1 Level 2 Level 2 bis Level 3

Reference method

Alternative method

Reference method

Alternative method

Reference method

Alternative method

Reference method

Alternative method

A 40 40 70 40 480 410 210 340 1000 300 400 300 4900 4600 4900 5100

B 40 15 40 20 350 300 360 450 350 650 800 600 4100 4700 5100 3700

C 50 20 20 70 440 430 480 450 300 650 300 500 4600 5400 4600 5000

D 40 60 40 40 490 460 600 540 250 450 600 1000 4900 4900 4400 4600

E 50 40 50 20 490 520 530 560 750 550 400 200 5400 4500 4100 5400

F 60 20 60 50 370 250 300 130 300 350 600 300 4400 4700 4900 5900

G 70 35 120 50 440 390 570 370 450 550 700 700 5000 4500 4000 4400

H 110 40 100 90 520 440 450 540 150 650 300 300 4200 4700 5600 4800

L 35 60 20 40 390 390 170 380 650 350 800 200 5300 4600 5600 5900

M 45 45 30 40 450 490 550 410 400 550 800 180 4900 4900 6400 4800

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2538 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Table 16 ndash Synthesis of results obtained by the alternative

and the reference methods (log CFUml)

Labs Level 1 Level 2 Level 2 bis Level 3

Reference method

Alternative method

Reference method

Alternative method

Reference method

Alternative method

Reference method

Alternative method

A 1602 1602 1845 1602 2681 2613 2322 2531 3000 2477 2602 2477 3690 3663 3690 3708

B 1602 1176 1602 1301 2544 2477 2556 2653 2544 2813 2903 2778 3613 3672 3708 3568

C 1699 1301 1301 1845 2643 2633 2681 2653 2477 2813 2477 2699 3663 3732 3663 3699

D 1602 1778 1602 1602 2690 2663 2778 2732 2398 2653 2778 3000 3690 3690 3643 3663

E 1699 1602 1699 1301 2690 2716 2724 2748 2875 2740 2602 2301 3732 3653 3613 3732

F 1778 1301 1778 1699 2568 2398 2477 2114 2477 2544 2778 2477 3643 3672 3690 3771

G 1845 1544 2079 1699 2643 2591 2756 2568 2653 2740 2845 2845 3699 3653 3602 3643

H 2041 1602 2000 1954 2716 2643 2653 2732 2176 2813 2477 2477 3623 3672 3748 3681

L 1544 1778 1301 1602 2591 2591 2230 2580 2813 2544 2903 2301 3724 3663 3748 3771

M 1653 1653 1477 1602 2653 2690 2740 2613 2602 2740 2903 2255 3690 3690 3806 3681

Level 2 results obtained taking account the 3 plates inoculated with the mother-suspension

Level 2 bis results obtained taking account the plates inoculated with 01 ml of the mother-suspension

Scrutiny of the measurement results for consistency

In order to identify the measurement results or laboratories that could be

inconsistent two graphical consistency techniques were realised Mandelrsquos

h-ank-statistics Mandel indicators h and k at 5 significance highlighted

some possible inconsistent data (See tables and graphics in Appendix 3 and

Appendix 4)

Mandelrsquos values Number of values observed over the threshold

Reference method Alternative method

h gt 1 Lab F Level 2 Lab F Level 2

h gt 5 Lab F Level 2

Lab B Levels 1 and 2

Lab A Level 2

Lab D Level 3

Lab F Level 2

Lab L Level 2

kgt 1 Lab F Level 3

k gt 5 Lab F Level 2 Lab C Level 2

Lab F Level 3

Lab L Levels 1 and 3

Lab M Level 1

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2638 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

The emphasized results appear (i) coherent with the targeted contamination

levels (ii) concordant between both methods

All the results were thus kept for the statistical analysis

Comparison of the trueness and precision characteristics of

the reference method and alternative methods

The statistical values are summarised hereafter

Level p

Reference method Alternative method

Repeatability ratio

Reproducibility ratio Median

Repeatability standard deviation

Reproducibility standard deviation

Median Repeatability

standard deviation

Reproducibility standard deviation

1 10 16519 02415 02425 16021 02045 02433 0847 1007

2 10 26551 00543 00659 26719 00914 01325 1682 2011

2 bis 10 26581 02607 02607 25951 01900 01953 0729 0749

3 10 18671 00378 00378 18671 00478 00611 1264 1617

Bias of the alternative method

In order to estimate the bias of the alternative method with regard to the reference method for every

level Dij and t are calculated according to the following equations

RefAlt ijYijYDij

Diffp

Dijit

)2(

)(median

If the t value is superior to 2 the alternative method is significantly biased

with regard to the reference method

The values are given in table 17

Table 17 ndash t (d) values obtained by level

Level Bias t (d) Conclusion

1 0068 114 Non significant bias

2 0021 082 Non significant bias

2 bis - 0037 035 Non significant bias

3 - 0037 043 Non significant bias

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2738 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

The statistical test concludes to a non significant bias for all the inoculation

levels The bias varies from ndash 0037 to 0068 log CFUml

Comparison of the repeatability standard deviations

If the ratio Srj Alt Srj Ref of the repeatability standard deviations of the

alternative method and the reference method is larger than 2 the precision

under repeatability conditions of the alternative method is considered to be

lower than that of the reference method If this ratio is smaller than 05 the

precision under repeatability conditions of the alternative method is

considered to be greater than that of the reference method

The ratio values are given in table 18

Table 18

Level

Repeatability standard deviation

Reference method Alternative method Alternative method

reference method ratio

1 0242 0205 0847

2 0054 0091 1682

2 bis 0261 0190 0729

3 0038 0048 1264

Level Repeatability limit

Reference method Alternative method

1 0676 0573

2 0152 0256

2 bis 0730 0532

3 0106 0134

The ratios of the repeatability standard deviations are below 2 in all cases

The precision under repeatability conditions of the alternative method is

equivalent to that of the reference method

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2838 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Comparison of the reproducibility standard deviations

If the ratio Srj Alt Srj Ref of the reproducibility standard deviations of the alternative method and the

reference method is larger than 2 the precision under reproducibility conditions of the alternative

method is considered to be lower than that of the reference method If this ratio is smaller than 05 the

precision under reproducibility conditions of the alternative method is considered to be greater than

that of the reference method

The ratio values are given in table 19

Table 19

Level

Reproducibility standard deviation

Reference method Alternative method Alternative method

reference method ratio

1 0242 0243 1007

2 0066 0133 2011

2 bis 0261 0195 0749

3 0038 0061 1617

Level Reproducibility limit

Reference method Alternative method

1 0676 0681

2 0185 0371

2 bis 0730 0547

3 0106 0171

The ratios of the reproducibility standard deviations are below or close to 2

The precision under reproducibility conditions of the alternative method is

equivalent to that of the reference method

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2938 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

214 Conclusion

Method comparison study

The Listeria PrecisTM enumeration method shows satisfying linearity and

relative accuracy

The alternative method shows satisfying inclusivity and exclusivity results

Two Listeria monocytogenes strains gave negative results with the

alternative method These strains grew weakly on the reference method

media (OampA Palcam and Oxford)

The limits of repeatability of the alternative method are similar to those of the

reference method

The bias between both methods are low between - 0133 Log CFUg and

- 0008 Log CFUg

The Listeria PrecisTM enumeration method offers the saving for confirmatory

tests

Positive results are obtained with the alternative method in 2-3 days (with

OBIS Mono test) or in 4-7 days (by the classical tests described in the

standardized method) against 4-7 days for the reference method

Inter-laboratory study

The bias between the reference and the alternative methods shows satisfying

and correct values

- level 1 (100 - 1 000 CFUg) bias = + 007 log CFUg

- level 2 (1 000 - 10 000 CFUg) bias = 002 log CFUg

- level 2 bis (1 000 - 10 000 CFUg) bias = - 004 log CFUg

- level 3 (10 000 - 100 000 CFUg) bias = - 004 log CFUg

The repeatability and the reproducibility of the alternative method look similar

to those of the reference method

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3038 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

22 Extension study (realised by ASEPT 2007)

An extension study was performed in 2007 for a new confirmatory test OBIS

MONO

150 Listeria monocytogenes strains and 100 non-target strains were isolated

onto TSAYE and BrillianceTM Listeria Agar plates All the Listeria

monocytogenes and the non-target strains gave the expected results

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3138 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Appendix 1 ndash Flow diagram of the Listeria PrecisTM method

x g sample + 9 x ml buffered peptone water

Revivification step for 1 h plusmn 5 min at 20degC plusmn 1degC

Streaking 1 ml onto 3 Brilliance Listeria Agar plates (mother suspension)

or 01 ml onto 1 Brilliance Listeria Agar plates (mother suspension and decimal dilutions)

Incubation for 48 h plusmn 3 h at 37degC plusmn 1degC

Confirmatory tests on at least one typical colony

- by the tests described in the ISO method

- by applying the OBIS mono test on an isolated colony

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3238 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Appendix 2 ndash ISO 11290-2 (1998) amp ISO 11290-2A1 (2004) Horizontal

method for the detection and enumeration of Listeria monocytogenes -

Part 2 enumeration of Listeria monocytogenes in foods

10 g cheese + 90 ml buffered peptone water or Half Fraser Broth (without supplements) (110 dilution)

Stomach 1 min 30 seconds

Keep at 20degC plusmn 2degC for 1 h plusmn 5 min

Decimal dilutions in 9 ml peptone salt tubes

Dilution 110

Dilution 1100

Dilution 11000

1 ml on 3 Chromogenic Listeria medium (ISO) plates (x 2) (-1)

01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-2)

01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-3)

01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-4)

Incubation at 37degC plusmn 1degC

24 h - 48 h

Numerate typical colonies at 24 h

(bluegreen surrounded by an opaque halo)

Numerate at 48 h

Streak typical colonies on TSYEA

(5 coloniesplate 2 successive dilutions)

Confirmation tests

Catalase

Gram stain

For confirmation of L monocytogenes

Haemolysis

Carbohydrate (rhamnose and xylose) utilisation

CAMP Test

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3338 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Appendix 3 ndash Mandelrsquos graphics

Laboratoire h Ref

Niveau 1 h Ref

Niveau 2 h Ref

Niveau 3 h Alt

Niveau 1 h Alt

Niveau 2 h Alt

Niveau 3 h5 h1 h5 h1

A -050 -015 083 062 -212 -039 198 299 -198 -299

B -261 -270 021 -077 -058 173 198 299 -198 -299

C -151 -031 -014 -015 -004 -005 198 299 -198 -299

D 038 040 -137 000 072 207 198 299 -198 -299

E -001 090 155 -052 056 -101 198 299 -198 -299

F -112 -321 -153 070 -325 023 198 299 -198 -299

G 042 -071 040 147 -009 176 198 299 -198 -299

H 169 046 -169 192 018 -083 198 299 -198 -299

L 009 -120 021 -077 -231 005 198 299 -198 -299

M 001 031 014 -032 004 -011 198 299 -198 -299

-300

-200

-100

000

100

200

300

A B C D E F G H L M

Vale

urs

h d

e M

an

del(

rob

uste

s)

Laboratoires

Meacutethode de reacutefeacuterence analyse robuste

h Ref Niveau 1

h Ref Niveau 2

h Ref Niveau 3

h 5=198

h 1=299

-300

-200

-100

000

100

200

300

A B C D E F G H L M

Vale

urs

de h

de M

an

del(

rob

uste

s)

Laboratoires

Meacutethode alternative analyse robuste

h Alt Niveau 1

h Alt Niveau 2

h Alt Niveau 2

h 1=299

h 5=198

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3438 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Laboratoire k Ref

Niveau 1 k Ref

Niveau 2 k Ref

Niveau 3 k Alt

Niveau 1 k Alt

Niveau 2 k Alt

Niveau 3 k1 k5

A 000 089 142 084 162 046 259 181

B 125 087 073 104 075 046 259 181

C 117 013 091 188 022 083 259 181

D 052 036 069 000 035 083 259 181

E 028 034 037 138 019 112 259 181

F 140 222 018 027 281 112 259 181

G 088 068 024 131 145 000 259 181

H 129 094 173 016 061 000 259 181

L 069 000 073 104 270 224 259 181

M 000 048 038 043 099 241 259 181

000

050

100

150

200

250

300

350

400

A B C D E F G H L M

Vale

urs

k d

e M

an

del(

rob

uste

s)

Laboratoires

Meacutethode de reacutefeacuterence analyse robuste

k Ref Niveau 1k Ref Niveau 2k Ref Niveau 3

k 1=259

k 5=181

000

050

100

150

200

250

300

A B C D E F G H L M

Vale

urs

k d

e M

an

del

(ro

bu

ste

s)

Laboratoires

Meacutethode alternative analyse robuste

k Alt Niveau 3

k Alt Niveau 1

k Alt Niveau 2

k 1=259

k 5=181

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3538 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Appendix 4 ndash Statistical results

Level 1

Nombre de laboratoires (p) 10

A 1602 1602 1602 000 0000 0000 -0495 0000 A 1845 1602 1724 017 0122 -0122 0621 0840 0122

B 1602 1176 1389 030 0213 -0213 -2613 1247 B 1602 1301 1452 021 0151 -0151 -0769 1041 0062

C 1699 1301 1500 028 0199 -0199 -1510 1165 C 1301 1845 1573 038 -0272 0272 -0148 1881 0073

D 1602 1778 1690 012 -0088 0088 0380 0516 D 1602 1602 1602 000 0000 0000 0000 0000 -0088

E 1699 1602 1651 007 0048 -0048 -0013 0284 E 1699 1301 1500 028 0199 -0199 -0522 1376 -0151

F 1778 1301 1540 034 0239 -0239 -1116 1397 F 1778 1699 1739 006 0040 -0040 0698 0274 0199

G 1845 1544 1695 021 0151 -0151 0425 0881 G 2079 1699 1889 027 0190 -0190 1467 1315 0194

H 2041 1602 1822 031 0220 -0220 1689 1286 H 2000 1954 1977 003 0023 -0023 1917 0158 0155

L 1544 1778 1661 017 -0117 0117 0092 0685 L 1301 1602 1452 021 -0151 0151 -0769 1041 -0210

M 1653 1653 1653 000 0000 0000 0013 0000 M 1477 1602 1540 009 -0062 0062 -0319 0432 -0114

m 165186 m 160206 mediane D 00678

Cn(2p) 1867 Cn(2p) 1867 Cp 16101

Cn(p) 16101 Cn(p) 16101 Qn(D) 0092926

n=2p n 20 n=2p n 20 Qdiff 01496169

f 11 f 11 t 114

l 55 l 55

n=p n 10 n=p n 10

f 6 f 6

l 15 l 15

Qn(2p) 0091465 Qn(2p) 007745

Qn(p) 0062469 Qn(p) 0121519

Qintra 017078 Qintra 014461

Qinter 010058 Qinter 019565

Sr(eacutecart-type de reacutepeacutetabiliteacute) 02415 Sr 02045

RSDr(coefficient de variation de la reacutepeacutetabiliteacute)1462 RSDr 1277

r(repeatability limit)) 0676 r 0573

SL(eacutecart-type interlaboratoire) -001905 SLlt0 SL 001737

SR(eacutecart-type de reproductibiliteacute) 02415 SR 02433

RSDR(coefficient de variation de la reproductibiliteacute)0146 RSDR 0152

R(limite de reproductibiliteacute)) 06762 R(limite de reproductibiliteacute))068123

h k h MoyenneLaboratoires

Niveau 1

si LaboratoiresDeviationMoyenne Dk DeviationsiAlt

Niveau 1

Ref

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3638 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Level 2

Nombre de laboratoires (p) 10

A 2681 2613 2647 005 0034 -0034 -0151 0891 A 2322 2531 2427 015 -0105 0105 -2119 1620 -0220

B 2544 2477 2511 005 0033 -0033 -2699 0872 B 2556 2653 2605 007 -0048 0048 -0581 0750 0094

C 2643 2633 2638 001 0005 -0005 -0311 0130 C 2681 2653 2667 002 0014 -0014 -0040 0217 0029

D 2690 2663 2676 002 0014 -0014 0400 0357 D 2778 2732 2755 003 0023 -0023 0721 0354 0079

E 2690 2716 2703 002 -0013 0013 0897 0336 E 2724 2748 2736 002 -0012 0012 0556 0185 0033

F 2568 2398 2483 012 0085 -0085 -3214 2217 F 2477 2114 2296 026 0182 -0182 -3255 2811 -0188

G 2643 2591 2617 004 0026 -0026 -0707 0682 G 2756 2568 2662 013 0094 -0094 -0085 1453 0045

H 2716 2643 2680 005 0036 -0036 0460 0945 H 2653 2732 2693 006 -0040 0040 0181 0613 0013

L 2591 2591 2591 000 0000 0000 -1196 0000 L 2230 2580 2405 025 -0175 0175 -2307 2704 -0186

M 2653 2690 2672 003 -0018 0018 0311 0481 M 2740 2613 2677 009 0064 -0064 0040 0987 0005

m 2655082 m 26719001 mediane D 00209

Cn(2p) 1867 Cn(2p) 1867 Cp 16101

Cn(p) 16101 Cn(p) 16101 Qn(D) 0039911

n=2p n 20 n=2p n 20 Qdiff 00642591

f 11 f 11 t 082

l 55 l 55

n=p n 10 n=p n 10

f 6 f 6

l 15 l 15

Qn(2p) 0020570 Qn(2p) 00345985

Qn(p) 00332437 Qn(p) 00718158

Qintra 003841 Qintra 006460

Qinter 005352 Qinter 011563

Sr(eacutecart-type de reacutepeacutetabiliteacute) 00543 Sr 00914

RSDr(coefficient de variation de la reacutepeacutetabiliteacute)205 RSDr 342

r(repeatability limit)) 0152 r 0256

SL(eacutecart-type interlaboratoire) 000139 SL 000920

SR(eacutecart-type de reproductibiliteacute) 00659 SR 01325

RSDR(coefficient de variation de la reproductibiliteacute)0025 RSDR 0050

R(limite de reproductibiliteacute)) 018446 R(limite de reproductibiliteacute))03708618

Laboratoires

Niveau 2

Moyenne si Laboratoires

Niveau 2

DRef Alt Moyenne si Deviation h Deviation h k k

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3738 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Level 2 bis

Nombre de laboratoires (p) 10

A 3000 2477 2739 037 0261 -0261 0832 1418 A 2602 2477 2540 009 0062 -0062 -0391 0465 -0199

B 2544 2813 2678 019 -0134 0134 0211 0729 B 2903 2778 2841 009 0062 -0062 1732 0465 0162

C 2477 2813 2645 024 -0168 0168 -0135 0911 C 2477 2699 2588 016 -0111 0111 -0049 0826 -0057

D 2398 2653 2526 018 -0128 0128 -1370 0692 D 2778 3000 2889 016 -0111 0111 2074 0826 0363

E 2875 2740 2808 010 0067 -0067 1547 0365 E 2602 2301 2452 021 0151 -0151 -1012 1120 -0356

F 2477 2544 2511 005 -0033 0033 -1525 0182 F 2778 2477 2628 021 0151 -0151 0230 1120 0117

G 2653 2740 2697 006 -0044 0044 0400 0236 G 2845 2845 2845 000 0000 0000 1764 0000 0148

H 2176 2813 2495 045 -0318 0318 -1692 1727 H 2477 2477 2477 000 0000 0000 -0832 0000 -0017

L 2813 2544 2678 019 0134 -0134 0211 0729 L 2903 2301 2602 043 0301 -0301 0049 2241 -0076

M 2602 2740 2671 010 -0069 0069 0135 0375 M 2903 2255 2579 046 0324 -0324 -0112 2411 -0092

m 2658114 m 259505 mediane D -00372

Cn(2p) 1867 Cn(2p) 1867 Cp 16101

Cn(p) 16101 Cn(p) 16101 Qn(D) 01656915

n=2p n 20 n=2p n 20 Qdiff 02667753

f 11 f 11 t 035

l 55 l 55

n=p n 10 n=p n 10

f 6 f 6

l 15 l 15

Qn(2p) 0098745 Qn(2p) 007195

Qn(p) 006007 Qn(p) 008805

Qintra 018437 Qintra 013435

Qinter 009672 Qinter 014176

Sr(eacutecart-type de reacutepeacutetabiliteacute) 02607 Sr 01900

RSDr(coefficient de variation de la reacutepeacutetabiliteacute)981 RSDr 732

r(repeatability limit)) 0730 r 0532

SL(eacutecart-type interlaboratoire) -002464 SLlt0 SL 000205

SR(eacutecart-type de reproductibiliteacute) 02607 SR 01953

RSDR(coefficient de variation de la reproductibiliteacute)0098 RSDR 0075

R(limite de reproductibiliteacute)) 072996 R(limite de reproductibiliteacute))054686

Laboratoires

Niveau 2 bis

Moyenne si Laboratoires

Niveau 2 bis

DRef Alt Moyenne si Deviation h Deviation h k k

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3838 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Level 3

Nombre de laboratoires (p) 10

A 3690 3663 3676 002 0014 -0014 -0311 0513 A 3690 3708 3699 001 -0009 0009 0354 0257 0022

B 3613 3672 3642 004 -0030 0030 -1851 1109 B 3708 3568 3638 010 0070 -0070 -0844 2061 -0005

C 3663 3732 3698 005 -0035 0035 0645 1302 C 3663 3699 3681 003 -0018 0018 0000 0536 -0017

D 3690 3690 3690 000 0000 0000 0311 0000 D 3643 3663 3653 001 -0010 0010 -0545 0286 -0037

E 3732 3653 3693 006 0040 -0040 0429 1480 E 3613 3732 3673 008 -0060 0060 -0162 1769 -0020

F 3643 3672 3658 002 -0014 0014 -1157 0536 F 3690 3771 3731 006 -0040 0040 0975 1193 0073

G 3699 3653 3676 003 0023 -0023 -0328 0856 G 3602 3643 3623 003 -0021 0021 -1141 0612 -0053

H 3623 3672 3648 003 -0024 0024 -1615 0913 H 3748 3681 3715 005 0033 -0033 0664 0990 0067

L 3724 3663 3694 004 0031 -0031 0461 1150 L 3748 3771 3760 002 -0011 0011 1544 0335 0066

M 3690 3690 3690 000 0000 0000 0311 0000 M 3806 3681 3744 009 0062 -0062 1234 1848 0054

m 368334 m 368086 mediane D 00089

Cn(2p) 1867 Cn(2p) 1867 Cp 16101

Cn(p) 16101 Cn(p) 16101 Qn(D) 00325359

n=2p n 20 n=2p n 20 Qdiff 00523852

f 11 f 11 t 043

l 55 l 55

n=p n 10 n=p n 10

f 6 f 6

l 15 l 15

Qn(2p) 0014323 Qn(2p) 001811

Qn(p) 0013719 Qn(p) 003164

Qintra 002674 Qintra 003381

Qinter 002209 Qinter 005094

Sr(eacutecart-type de reacutepeacutetabiliteacute) 00378 Sr 00478

RSDr(coefficient de variation de la reacutepeacutetabiliteacute)103 RSDr 130

r(repeatability limit)) 0106 r 0134

SL(eacutecart-type interlaboratoire) -000023 SLlt0 SL 000145

SR(eacutecart-type de reproductibiliteacute) 00378 SR 00611

RSDR(coefficient de variation de la reproductibiliteacute)0010 RSDR 0017

R(limite de reproductibiliteacute)) 0105894 R(limite de reproductibiliteacute))017119

Ref Alt DLaboratoires

Niveau 3

Moyenne si Deviation h k Laboratoires

Niveau 3

Moyenne si Deviation h k

Page 21: ÞÀ¿¸7 ú Y5zñLjÜ< /u +æü t¬Á ... - NF Validation · NF VALIDATION Validation of alternative analytical methods Application in food microbiology ... 500 – 1 000 1 000 –

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2138 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

213 Inter-laboratory study

2131 Organisation

12 laboratories participated to this study Pasteurised milk was inoculated by

Listeria monocytogenes 12a isolated from raw milk The targeted

inoculation levels were

- 0 CFUml

- 50 CFU ml

- 500 CFU ml

- 5 000 CFU ml

Each laboratory received eight flasks of 10 ml sample ie two flasks per

inoculation level Furthermore one non-inoculated sample has been added

to the package for total viable count microflora (NF ISO 4833 method)

Detailed instructions were transmitted to the collaborators by the expert

laboratory

Collaborative study laboratories and the expert laboratory carried out the

analyses with the alternative and reference methods

2132 Verification of experimental parameters

Strain stability during transport

In order to evaluate the Listeria monocytogenes strain stability during

transport bacterial count of all inoculated flasks were checked at different

time ie inoculation time after 24 h of conservation at 2deg - 8degC Results are

reported in table 11

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2238 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Table 11 ndash Listeria monocytogenes count

by ISO 11290-2A1 method (log CFUml)

Day of the analyses Level contamination Reference method

Day 0

(29052006)

1 45

2 440

3 4 500

Day 1

(30052006)

1 60

2 430

3 4 900

No evolution of the strain was observed after storage for 24 h at 2degC ndash 8degC

Contamination level

The real contamination levels are provided in table 12

Table 12 ndash Real contamination level (CFUml)

Target inoculation

(CFU ml) Samples Ndeg

Real contamination levels

(CFUml)

0 1 - 8 0

50 5 - 6 45

500 3 - 7 440

5000 2 - 4 4 500

Sample temperature at receipt

Measured temperatures at receipt are listed in table 13

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2338 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Table 13 - Sample temperature on receipt

Laboratories Receipt

day

Hour

receipt

Measured temperature

at receipt (degC)

Measured temperature by the

temperature probe (degC)

A

B

C

D

E

F

G

H

I

J

K

L

M

30052006

10h45

10h15

11h15

10h00

09h30

8h30

11h50

10h00

08h30

14h45

08h30

09h45

08h45

82

55

45

63

60

62

81

38

48

-

14

67

76

03

03

-13

03

04

-14

05

04

01

05

-13

04

Conclusion

The temperatures during the transport were correct for all the labs except for

Lab J which didnrsquot provide the information

All the labs received their packages with several broken flasks

Three labs (B D and L) received some damaged samples but proceeded to

analyses

All the labs started the analyses at Day 1 except Lab J

2133 Results

Aerobic mesophilic flora

The count of aerobic mesophilic flora varied from 94 102 CFUml to

33 103 CFUml

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2438 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Expert lab results

The results obtained by the expert lab are the following

Table 14

Target inoculation level (CFUml)

Reference method ISO 11290-2

Alternative method Listeria Precis Enumeration

Rep 1 Rep 2 Rep 1 Rep 2

lt 10 lt 10 lt 10 lt 10 lt10

50 50 (Ne) 60 (Ne) 50 (Ne) 30 (Ne)

500 470 390 490 500

5 000 4 700 5 100 5 900 3 400

Ne estimated number

The obtained results are in agreement with those expected

Collaborator Labs results

A synthesis of the results obtained by the collaborator labs is provided

Table 15 (CFUml) and Table 16 (log CFUml)

Table 15 ndash Synthesis of results obtained by the alternative

and the reference methods (CFUml)

Labs Level 1 Level 2 Level 2 bis Level 3

Reference method

Alternative method

Reference method

Alternative method

Reference method

Alternative method

Reference method

Alternative method

A 40 40 70 40 480 410 210 340 1000 300 400 300 4900 4600 4900 5100

B 40 15 40 20 350 300 360 450 350 650 800 600 4100 4700 5100 3700

C 50 20 20 70 440 430 480 450 300 650 300 500 4600 5400 4600 5000

D 40 60 40 40 490 460 600 540 250 450 600 1000 4900 4900 4400 4600

E 50 40 50 20 490 520 530 560 750 550 400 200 5400 4500 4100 5400

F 60 20 60 50 370 250 300 130 300 350 600 300 4400 4700 4900 5900

G 70 35 120 50 440 390 570 370 450 550 700 700 5000 4500 4000 4400

H 110 40 100 90 520 440 450 540 150 650 300 300 4200 4700 5600 4800

L 35 60 20 40 390 390 170 380 650 350 800 200 5300 4600 5600 5900

M 45 45 30 40 450 490 550 410 400 550 800 180 4900 4900 6400 4800

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2538 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Table 16 ndash Synthesis of results obtained by the alternative

and the reference methods (log CFUml)

Labs Level 1 Level 2 Level 2 bis Level 3

Reference method

Alternative method

Reference method

Alternative method

Reference method

Alternative method

Reference method

Alternative method

A 1602 1602 1845 1602 2681 2613 2322 2531 3000 2477 2602 2477 3690 3663 3690 3708

B 1602 1176 1602 1301 2544 2477 2556 2653 2544 2813 2903 2778 3613 3672 3708 3568

C 1699 1301 1301 1845 2643 2633 2681 2653 2477 2813 2477 2699 3663 3732 3663 3699

D 1602 1778 1602 1602 2690 2663 2778 2732 2398 2653 2778 3000 3690 3690 3643 3663

E 1699 1602 1699 1301 2690 2716 2724 2748 2875 2740 2602 2301 3732 3653 3613 3732

F 1778 1301 1778 1699 2568 2398 2477 2114 2477 2544 2778 2477 3643 3672 3690 3771

G 1845 1544 2079 1699 2643 2591 2756 2568 2653 2740 2845 2845 3699 3653 3602 3643

H 2041 1602 2000 1954 2716 2643 2653 2732 2176 2813 2477 2477 3623 3672 3748 3681

L 1544 1778 1301 1602 2591 2591 2230 2580 2813 2544 2903 2301 3724 3663 3748 3771

M 1653 1653 1477 1602 2653 2690 2740 2613 2602 2740 2903 2255 3690 3690 3806 3681

Level 2 results obtained taking account the 3 plates inoculated with the mother-suspension

Level 2 bis results obtained taking account the plates inoculated with 01 ml of the mother-suspension

Scrutiny of the measurement results for consistency

In order to identify the measurement results or laboratories that could be

inconsistent two graphical consistency techniques were realised Mandelrsquos

h-ank-statistics Mandel indicators h and k at 5 significance highlighted

some possible inconsistent data (See tables and graphics in Appendix 3 and

Appendix 4)

Mandelrsquos values Number of values observed over the threshold

Reference method Alternative method

h gt 1 Lab F Level 2 Lab F Level 2

h gt 5 Lab F Level 2

Lab B Levels 1 and 2

Lab A Level 2

Lab D Level 3

Lab F Level 2

Lab L Level 2

kgt 1 Lab F Level 3

k gt 5 Lab F Level 2 Lab C Level 2

Lab F Level 3

Lab L Levels 1 and 3

Lab M Level 1

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2638 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

The emphasized results appear (i) coherent with the targeted contamination

levels (ii) concordant between both methods

All the results were thus kept for the statistical analysis

Comparison of the trueness and precision characteristics of

the reference method and alternative methods

The statistical values are summarised hereafter

Level p

Reference method Alternative method

Repeatability ratio

Reproducibility ratio Median

Repeatability standard deviation

Reproducibility standard deviation

Median Repeatability

standard deviation

Reproducibility standard deviation

1 10 16519 02415 02425 16021 02045 02433 0847 1007

2 10 26551 00543 00659 26719 00914 01325 1682 2011

2 bis 10 26581 02607 02607 25951 01900 01953 0729 0749

3 10 18671 00378 00378 18671 00478 00611 1264 1617

Bias of the alternative method

In order to estimate the bias of the alternative method with regard to the reference method for every

level Dij and t are calculated according to the following equations

RefAlt ijYijYDij

Diffp

Dijit

)2(

)(median

If the t value is superior to 2 the alternative method is significantly biased

with regard to the reference method

The values are given in table 17

Table 17 ndash t (d) values obtained by level

Level Bias t (d) Conclusion

1 0068 114 Non significant bias

2 0021 082 Non significant bias

2 bis - 0037 035 Non significant bias

3 - 0037 043 Non significant bias

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2738 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

The statistical test concludes to a non significant bias for all the inoculation

levels The bias varies from ndash 0037 to 0068 log CFUml

Comparison of the repeatability standard deviations

If the ratio Srj Alt Srj Ref of the repeatability standard deviations of the

alternative method and the reference method is larger than 2 the precision

under repeatability conditions of the alternative method is considered to be

lower than that of the reference method If this ratio is smaller than 05 the

precision under repeatability conditions of the alternative method is

considered to be greater than that of the reference method

The ratio values are given in table 18

Table 18

Level

Repeatability standard deviation

Reference method Alternative method Alternative method

reference method ratio

1 0242 0205 0847

2 0054 0091 1682

2 bis 0261 0190 0729

3 0038 0048 1264

Level Repeatability limit

Reference method Alternative method

1 0676 0573

2 0152 0256

2 bis 0730 0532

3 0106 0134

The ratios of the repeatability standard deviations are below 2 in all cases

The precision under repeatability conditions of the alternative method is

equivalent to that of the reference method

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2838 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Comparison of the reproducibility standard deviations

If the ratio Srj Alt Srj Ref of the reproducibility standard deviations of the alternative method and the

reference method is larger than 2 the precision under reproducibility conditions of the alternative

method is considered to be lower than that of the reference method If this ratio is smaller than 05 the

precision under reproducibility conditions of the alternative method is considered to be greater than

that of the reference method

The ratio values are given in table 19

Table 19

Level

Reproducibility standard deviation

Reference method Alternative method Alternative method

reference method ratio

1 0242 0243 1007

2 0066 0133 2011

2 bis 0261 0195 0749

3 0038 0061 1617

Level Reproducibility limit

Reference method Alternative method

1 0676 0681

2 0185 0371

2 bis 0730 0547

3 0106 0171

The ratios of the reproducibility standard deviations are below or close to 2

The precision under reproducibility conditions of the alternative method is

equivalent to that of the reference method

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2938 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

214 Conclusion

Method comparison study

The Listeria PrecisTM enumeration method shows satisfying linearity and

relative accuracy

The alternative method shows satisfying inclusivity and exclusivity results

Two Listeria monocytogenes strains gave negative results with the

alternative method These strains grew weakly on the reference method

media (OampA Palcam and Oxford)

The limits of repeatability of the alternative method are similar to those of the

reference method

The bias between both methods are low between - 0133 Log CFUg and

- 0008 Log CFUg

The Listeria PrecisTM enumeration method offers the saving for confirmatory

tests

Positive results are obtained with the alternative method in 2-3 days (with

OBIS Mono test) or in 4-7 days (by the classical tests described in the

standardized method) against 4-7 days for the reference method

Inter-laboratory study

The bias between the reference and the alternative methods shows satisfying

and correct values

- level 1 (100 - 1 000 CFUg) bias = + 007 log CFUg

- level 2 (1 000 - 10 000 CFUg) bias = 002 log CFUg

- level 2 bis (1 000 - 10 000 CFUg) bias = - 004 log CFUg

- level 3 (10 000 - 100 000 CFUg) bias = - 004 log CFUg

The repeatability and the reproducibility of the alternative method look similar

to those of the reference method

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3038 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

22 Extension study (realised by ASEPT 2007)

An extension study was performed in 2007 for a new confirmatory test OBIS

MONO

150 Listeria monocytogenes strains and 100 non-target strains were isolated

onto TSAYE and BrillianceTM Listeria Agar plates All the Listeria

monocytogenes and the non-target strains gave the expected results

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3138 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Appendix 1 ndash Flow diagram of the Listeria PrecisTM method

x g sample + 9 x ml buffered peptone water

Revivification step for 1 h plusmn 5 min at 20degC plusmn 1degC

Streaking 1 ml onto 3 Brilliance Listeria Agar plates (mother suspension)

or 01 ml onto 1 Brilliance Listeria Agar plates (mother suspension and decimal dilutions)

Incubation for 48 h plusmn 3 h at 37degC plusmn 1degC

Confirmatory tests on at least one typical colony

- by the tests described in the ISO method

- by applying the OBIS mono test on an isolated colony

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3238 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Appendix 2 ndash ISO 11290-2 (1998) amp ISO 11290-2A1 (2004) Horizontal

method for the detection and enumeration of Listeria monocytogenes -

Part 2 enumeration of Listeria monocytogenes in foods

10 g cheese + 90 ml buffered peptone water or Half Fraser Broth (without supplements) (110 dilution)

Stomach 1 min 30 seconds

Keep at 20degC plusmn 2degC for 1 h plusmn 5 min

Decimal dilutions in 9 ml peptone salt tubes

Dilution 110

Dilution 1100

Dilution 11000

1 ml on 3 Chromogenic Listeria medium (ISO) plates (x 2) (-1)

01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-2)

01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-3)

01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-4)

Incubation at 37degC plusmn 1degC

24 h - 48 h

Numerate typical colonies at 24 h

(bluegreen surrounded by an opaque halo)

Numerate at 48 h

Streak typical colonies on TSYEA

(5 coloniesplate 2 successive dilutions)

Confirmation tests

Catalase

Gram stain

For confirmation of L monocytogenes

Haemolysis

Carbohydrate (rhamnose and xylose) utilisation

CAMP Test

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3338 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Appendix 3 ndash Mandelrsquos graphics

Laboratoire h Ref

Niveau 1 h Ref

Niveau 2 h Ref

Niveau 3 h Alt

Niveau 1 h Alt

Niveau 2 h Alt

Niveau 3 h5 h1 h5 h1

A -050 -015 083 062 -212 -039 198 299 -198 -299

B -261 -270 021 -077 -058 173 198 299 -198 -299

C -151 -031 -014 -015 -004 -005 198 299 -198 -299

D 038 040 -137 000 072 207 198 299 -198 -299

E -001 090 155 -052 056 -101 198 299 -198 -299

F -112 -321 -153 070 -325 023 198 299 -198 -299

G 042 -071 040 147 -009 176 198 299 -198 -299

H 169 046 -169 192 018 -083 198 299 -198 -299

L 009 -120 021 -077 -231 005 198 299 -198 -299

M 001 031 014 -032 004 -011 198 299 -198 -299

-300

-200

-100

000

100

200

300

A B C D E F G H L M

Vale

urs

h d

e M

an

del(

rob

uste

s)

Laboratoires

Meacutethode de reacutefeacuterence analyse robuste

h Ref Niveau 1

h Ref Niveau 2

h Ref Niveau 3

h 5=198

h 1=299

-300

-200

-100

000

100

200

300

A B C D E F G H L M

Vale

urs

de h

de M

an

del(

rob

uste

s)

Laboratoires

Meacutethode alternative analyse robuste

h Alt Niveau 1

h Alt Niveau 2

h Alt Niveau 2

h 1=299

h 5=198

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3438 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Laboratoire k Ref

Niveau 1 k Ref

Niveau 2 k Ref

Niveau 3 k Alt

Niveau 1 k Alt

Niveau 2 k Alt

Niveau 3 k1 k5

A 000 089 142 084 162 046 259 181

B 125 087 073 104 075 046 259 181

C 117 013 091 188 022 083 259 181

D 052 036 069 000 035 083 259 181

E 028 034 037 138 019 112 259 181

F 140 222 018 027 281 112 259 181

G 088 068 024 131 145 000 259 181

H 129 094 173 016 061 000 259 181

L 069 000 073 104 270 224 259 181

M 000 048 038 043 099 241 259 181

000

050

100

150

200

250

300

350

400

A B C D E F G H L M

Vale

urs

k d

e M

an

del(

rob

uste

s)

Laboratoires

Meacutethode de reacutefeacuterence analyse robuste

k Ref Niveau 1k Ref Niveau 2k Ref Niveau 3

k 1=259

k 5=181

000

050

100

150

200

250

300

A B C D E F G H L M

Vale

urs

k d

e M

an

del

(ro

bu

ste

s)

Laboratoires

Meacutethode alternative analyse robuste

k Alt Niveau 3

k Alt Niveau 1

k Alt Niveau 2

k 1=259

k 5=181

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3538 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Appendix 4 ndash Statistical results

Level 1

Nombre de laboratoires (p) 10

A 1602 1602 1602 000 0000 0000 -0495 0000 A 1845 1602 1724 017 0122 -0122 0621 0840 0122

B 1602 1176 1389 030 0213 -0213 -2613 1247 B 1602 1301 1452 021 0151 -0151 -0769 1041 0062

C 1699 1301 1500 028 0199 -0199 -1510 1165 C 1301 1845 1573 038 -0272 0272 -0148 1881 0073

D 1602 1778 1690 012 -0088 0088 0380 0516 D 1602 1602 1602 000 0000 0000 0000 0000 -0088

E 1699 1602 1651 007 0048 -0048 -0013 0284 E 1699 1301 1500 028 0199 -0199 -0522 1376 -0151

F 1778 1301 1540 034 0239 -0239 -1116 1397 F 1778 1699 1739 006 0040 -0040 0698 0274 0199

G 1845 1544 1695 021 0151 -0151 0425 0881 G 2079 1699 1889 027 0190 -0190 1467 1315 0194

H 2041 1602 1822 031 0220 -0220 1689 1286 H 2000 1954 1977 003 0023 -0023 1917 0158 0155

L 1544 1778 1661 017 -0117 0117 0092 0685 L 1301 1602 1452 021 -0151 0151 -0769 1041 -0210

M 1653 1653 1653 000 0000 0000 0013 0000 M 1477 1602 1540 009 -0062 0062 -0319 0432 -0114

m 165186 m 160206 mediane D 00678

Cn(2p) 1867 Cn(2p) 1867 Cp 16101

Cn(p) 16101 Cn(p) 16101 Qn(D) 0092926

n=2p n 20 n=2p n 20 Qdiff 01496169

f 11 f 11 t 114

l 55 l 55

n=p n 10 n=p n 10

f 6 f 6

l 15 l 15

Qn(2p) 0091465 Qn(2p) 007745

Qn(p) 0062469 Qn(p) 0121519

Qintra 017078 Qintra 014461

Qinter 010058 Qinter 019565

Sr(eacutecart-type de reacutepeacutetabiliteacute) 02415 Sr 02045

RSDr(coefficient de variation de la reacutepeacutetabiliteacute)1462 RSDr 1277

r(repeatability limit)) 0676 r 0573

SL(eacutecart-type interlaboratoire) -001905 SLlt0 SL 001737

SR(eacutecart-type de reproductibiliteacute) 02415 SR 02433

RSDR(coefficient de variation de la reproductibiliteacute)0146 RSDR 0152

R(limite de reproductibiliteacute)) 06762 R(limite de reproductibiliteacute))068123

h k h MoyenneLaboratoires

Niveau 1

si LaboratoiresDeviationMoyenne Dk DeviationsiAlt

Niveau 1

Ref

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3638 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Level 2

Nombre de laboratoires (p) 10

A 2681 2613 2647 005 0034 -0034 -0151 0891 A 2322 2531 2427 015 -0105 0105 -2119 1620 -0220

B 2544 2477 2511 005 0033 -0033 -2699 0872 B 2556 2653 2605 007 -0048 0048 -0581 0750 0094

C 2643 2633 2638 001 0005 -0005 -0311 0130 C 2681 2653 2667 002 0014 -0014 -0040 0217 0029

D 2690 2663 2676 002 0014 -0014 0400 0357 D 2778 2732 2755 003 0023 -0023 0721 0354 0079

E 2690 2716 2703 002 -0013 0013 0897 0336 E 2724 2748 2736 002 -0012 0012 0556 0185 0033

F 2568 2398 2483 012 0085 -0085 -3214 2217 F 2477 2114 2296 026 0182 -0182 -3255 2811 -0188

G 2643 2591 2617 004 0026 -0026 -0707 0682 G 2756 2568 2662 013 0094 -0094 -0085 1453 0045

H 2716 2643 2680 005 0036 -0036 0460 0945 H 2653 2732 2693 006 -0040 0040 0181 0613 0013

L 2591 2591 2591 000 0000 0000 -1196 0000 L 2230 2580 2405 025 -0175 0175 -2307 2704 -0186

M 2653 2690 2672 003 -0018 0018 0311 0481 M 2740 2613 2677 009 0064 -0064 0040 0987 0005

m 2655082 m 26719001 mediane D 00209

Cn(2p) 1867 Cn(2p) 1867 Cp 16101

Cn(p) 16101 Cn(p) 16101 Qn(D) 0039911

n=2p n 20 n=2p n 20 Qdiff 00642591

f 11 f 11 t 082

l 55 l 55

n=p n 10 n=p n 10

f 6 f 6

l 15 l 15

Qn(2p) 0020570 Qn(2p) 00345985

Qn(p) 00332437 Qn(p) 00718158

Qintra 003841 Qintra 006460

Qinter 005352 Qinter 011563

Sr(eacutecart-type de reacutepeacutetabiliteacute) 00543 Sr 00914

RSDr(coefficient de variation de la reacutepeacutetabiliteacute)205 RSDr 342

r(repeatability limit)) 0152 r 0256

SL(eacutecart-type interlaboratoire) 000139 SL 000920

SR(eacutecart-type de reproductibiliteacute) 00659 SR 01325

RSDR(coefficient de variation de la reproductibiliteacute)0025 RSDR 0050

R(limite de reproductibiliteacute)) 018446 R(limite de reproductibiliteacute))03708618

Laboratoires

Niveau 2

Moyenne si Laboratoires

Niveau 2

DRef Alt Moyenne si Deviation h Deviation h k k

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3738 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Level 2 bis

Nombre de laboratoires (p) 10

A 3000 2477 2739 037 0261 -0261 0832 1418 A 2602 2477 2540 009 0062 -0062 -0391 0465 -0199

B 2544 2813 2678 019 -0134 0134 0211 0729 B 2903 2778 2841 009 0062 -0062 1732 0465 0162

C 2477 2813 2645 024 -0168 0168 -0135 0911 C 2477 2699 2588 016 -0111 0111 -0049 0826 -0057

D 2398 2653 2526 018 -0128 0128 -1370 0692 D 2778 3000 2889 016 -0111 0111 2074 0826 0363

E 2875 2740 2808 010 0067 -0067 1547 0365 E 2602 2301 2452 021 0151 -0151 -1012 1120 -0356

F 2477 2544 2511 005 -0033 0033 -1525 0182 F 2778 2477 2628 021 0151 -0151 0230 1120 0117

G 2653 2740 2697 006 -0044 0044 0400 0236 G 2845 2845 2845 000 0000 0000 1764 0000 0148

H 2176 2813 2495 045 -0318 0318 -1692 1727 H 2477 2477 2477 000 0000 0000 -0832 0000 -0017

L 2813 2544 2678 019 0134 -0134 0211 0729 L 2903 2301 2602 043 0301 -0301 0049 2241 -0076

M 2602 2740 2671 010 -0069 0069 0135 0375 M 2903 2255 2579 046 0324 -0324 -0112 2411 -0092

m 2658114 m 259505 mediane D -00372

Cn(2p) 1867 Cn(2p) 1867 Cp 16101

Cn(p) 16101 Cn(p) 16101 Qn(D) 01656915

n=2p n 20 n=2p n 20 Qdiff 02667753

f 11 f 11 t 035

l 55 l 55

n=p n 10 n=p n 10

f 6 f 6

l 15 l 15

Qn(2p) 0098745 Qn(2p) 007195

Qn(p) 006007 Qn(p) 008805

Qintra 018437 Qintra 013435

Qinter 009672 Qinter 014176

Sr(eacutecart-type de reacutepeacutetabiliteacute) 02607 Sr 01900

RSDr(coefficient de variation de la reacutepeacutetabiliteacute)981 RSDr 732

r(repeatability limit)) 0730 r 0532

SL(eacutecart-type interlaboratoire) -002464 SLlt0 SL 000205

SR(eacutecart-type de reproductibiliteacute) 02607 SR 01953

RSDR(coefficient de variation de la reproductibiliteacute)0098 RSDR 0075

R(limite de reproductibiliteacute)) 072996 R(limite de reproductibiliteacute))054686

Laboratoires

Niveau 2 bis

Moyenne si Laboratoires

Niveau 2 bis

DRef Alt Moyenne si Deviation h Deviation h k k

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3838 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Level 3

Nombre de laboratoires (p) 10

A 3690 3663 3676 002 0014 -0014 -0311 0513 A 3690 3708 3699 001 -0009 0009 0354 0257 0022

B 3613 3672 3642 004 -0030 0030 -1851 1109 B 3708 3568 3638 010 0070 -0070 -0844 2061 -0005

C 3663 3732 3698 005 -0035 0035 0645 1302 C 3663 3699 3681 003 -0018 0018 0000 0536 -0017

D 3690 3690 3690 000 0000 0000 0311 0000 D 3643 3663 3653 001 -0010 0010 -0545 0286 -0037

E 3732 3653 3693 006 0040 -0040 0429 1480 E 3613 3732 3673 008 -0060 0060 -0162 1769 -0020

F 3643 3672 3658 002 -0014 0014 -1157 0536 F 3690 3771 3731 006 -0040 0040 0975 1193 0073

G 3699 3653 3676 003 0023 -0023 -0328 0856 G 3602 3643 3623 003 -0021 0021 -1141 0612 -0053

H 3623 3672 3648 003 -0024 0024 -1615 0913 H 3748 3681 3715 005 0033 -0033 0664 0990 0067

L 3724 3663 3694 004 0031 -0031 0461 1150 L 3748 3771 3760 002 -0011 0011 1544 0335 0066

M 3690 3690 3690 000 0000 0000 0311 0000 M 3806 3681 3744 009 0062 -0062 1234 1848 0054

m 368334 m 368086 mediane D 00089

Cn(2p) 1867 Cn(2p) 1867 Cp 16101

Cn(p) 16101 Cn(p) 16101 Qn(D) 00325359

n=2p n 20 n=2p n 20 Qdiff 00523852

f 11 f 11 t 043

l 55 l 55

n=p n 10 n=p n 10

f 6 f 6

l 15 l 15

Qn(2p) 0014323 Qn(2p) 001811

Qn(p) 0013719 Qn(p) 003164

Qintra 002674 Qintra 003381

Qinter 002209 Qinter 005094

Sr(eacutecart-type de reacutepeacutetabiliteacute) 00378 Sr 00478

RSDr(coefficient de variation de la reacutepeacutetabiliteacute)103 RSDr 130

r(repeatability limit)) 0106 r 0134

SL(eacutecart-type interlaboratoire) -000023 SLlt0 SL 000145

SR(eacutecart-type de reproductibiliteacute) 00378 SR 00611

RSDR(coefficient de variation de la reproductibiliteacute)0010 RSDR 0017

R(limite de reproductibiliteacute)) 0105894 R(limite de reproductibiliteacute))017119

Ref Alt DLaboratoires

Niveau 3

Moyenne si Deviation h k Laboratoires

Niveau 3

Moyenne si Deviation h k

Page 22: ÞÀ¿¸7 ú Y5zñLjÜ< /u +æü t¬Á ... - NF Validation · NF VALIDATION Validation of alternative analytical methods Application in food microbiology ... 500 – 1 000 1 000 –

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2238 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Table 11 ndash Listeria monocytogenes count

by ISO 11290-2A1 method (log CFUml)

Day of the analyses Level contamination Reference method

Day 0

(29052006)

1 45

2 440

3 4 500

Day 1

(30052006)

1 60

2 430

3 4 900

No evolution of the strain was observed after storage for 24 h at 2degC ndash 8degC

Contamination level

The real contamination levels are provided in table 12

Table 12 ndash Real contamination level (CFUml)

Target inoculation

(CFU ml) Samples Ndeg

Real contamination levels

(CFUml)

0 1 - 8 0

50 5 - 6 45

500 3 - 7 440

5000 2 - 4 4 500

Sample temperature at receipt

Measured temperatures at receipt are listed in table 13

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2338 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Table 13 - Sample temperature on receipt

Laboratories Receipt

day

Hour

receipt

Measured temperature

at receipt (degC)

Measured temperature by the

temperature probe (degC)

A

B

C

D

E

F

G

H

I

J

K

L

M

30052006

10h45

10h15

11h15

10h00

09h30

8h30

11h50

10h00

08h30

14h45

08h30

09h45

08h45

82

55

45

63

60

62

81

38

48

-

14

67

76

03

03

-13

03

04

-14

05

04

01

05

-13

04

Conclusion

The temperatures during the transport were correct for all the labs except for

Lab J which didnrsquot provide the information

All the labs received their packages with several broken flasks

Three labs (B D and L) received some damaged samples but proceeded to

analyses

All the labs started the analyses at Day 1 except Lab J

2133 Results

Aerobic mesophilic flora

The count of aerobic mesophilic flora varied from 94 102 CFUml to

33 103 CFUml

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2438 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Expert lab results

The results obtained by the expert lab are the following

Table 14

Target inoculation level (CFUml)

Reference method ISO 11290-2

Alternative method Listeria Precis Enumeration

Rep 1 Rep 2 Rep 1 Rep 2

lt 10 lt 10 lt 10 lt 10 lt10

50 50 (Ne) 60 (Ne) 50 (Ne) 30 (Ne)

500 470 390 490 500

5 000 4 700 5 100 5 900 3 400

Ne estimated number

The obtained results are in agreement with those expected

Collaborator Labs results

A synthesis of the results obtained by the collaborator labs is provided

Table 15 (CFUml) and Table 16 (log CFUml)

Table 15 ndash Synthesis of results obtained by the alternative

and the reference methods (CFUml)

Labs Level 1 Level 2 Level 2 bis Level 3

Reference method

Alternative method

Reference method

Alternative method

Reference method

Alternative method

Reference method

Alternative method

A 40 40 70 40 480 410 210 340 1000 300 400 300 4900 4600 4900 5100

B 40 15 40 20 350 300 360 450 350 650 800 600 4100 4700 5100 3700

C 50 20 20 70 440 430 480 450 300 650 300 500 4600 5400 4600 5000

D 40 60 40 40 490 460 600 540 250 450 600 1000 4900 4900 4400 4600

E 50 40 50 20 490 520 530 560 750 550 400 200 5400 4500 4100 5400

F 60 20 60 50 370 250 300 130 300 350 600 300 4400 4700 4900 5900

G 70 35 120 50 440 390 570 370 450 550 700 700 5000 4500 4000 4400

H 110 40 100 90 520 440 450 540 150 650 300 300 4200 4700 5600 4800

L 35 60 20 40 390 390 170 380 650 350 800 200 5300 4600 5600 5900

M 45 45 30 40 450 490 550 410 400 550 800 180 4900 4900 6400 4800

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2538 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Table 16 ndash Synthesis of results obtained by the alternative

and the reference methods (log CFUml)

Labs Level 1 Level 2 Level 2 bis Level 3

Reference method

Alternative method

Reference method

Alternative method

Reference method

Alternative method

Reference method

Alternative method

A 1602 1602 1845 1602 2681 2613 2322 2531 3000 2477 2602 2477 3690 3663 3690 3708

B 1602 1176 1602 1301 2544 2477 2556 2653 2544 2813 2903 2778 3613 3672 3708 3568

C 1699 1301 1301 1845 2643 2633 2681 2653 2477 2813 2477 2699 3663 3732 3663 3699

D 1602 1778 1602 1602 2690 2663 2778 2732 2398 2653 2778 3000 3690 3690 3643 3663

E 1699 1602 1699 1301 2690 2716 2724 2748 2875 2740 2602 2301 3732 3653 3613 3732

F 1778 1301 1778 1699 2568 2398 2477 2114 2477 2544 2778 2477 3643 3672 3690 3771

G 1845 1544 2079 1699 2643 2591 2756 2568 2653 2740 2845 2845 3699 3653 3602 3643

H 2041 1602 2000 1954 2716 2643 2653 2732 2176 2813 2477 2477 3623 3672 3748 3681

L 1544 1778 1301 1602 2591 2591 2230 2580 2813 2544 2903 2301 3724 3663 3748 3771

M 1653 1653 1477 1602 2653 2690 2740 2613 2602 2740 2903 2255 3690 3690 3806 3681

Level 2 results obtained taking account the 3 plates inoculated with the mother-suspension

Level 2 bis results obtained taking account the plates inoculated with 01 ml of the mother-suspension

Scrutiny of the measurement results for consistency

In order to identify the measurement results or laboratories that could be

inconsistent two graphical consistency techniques were realised Mandelrsquos

h-ank-statistics Mandel indicators h and k at 5 significance highlighted

some possible inconsistent data (See tables and graphics in Appendix 3 and

Appendix 4)

Mandelrsquos values Number of values observed over the threshold

Reference method Alternative method

h gt 1 Lab F Level 2 Lab F Level 2

h gt 5 Lab F Level 2

Lab B Levels 1 and 2

Lab A Level 2

Lab D Level 3

Lab F Level 2

Lab L Level 2

kgt 1 Lab F Level 3

k gt 5 Lab F Level 2 Lab C Level 2

Lab F Level 3

Lab L Levels 1 and 3

Lab M Level 1

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2638 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

The emphasized results appear (i) coherent with the targeted contamination

levels (ii) concordant between both methods

All the results were thus kept for the statistical analysis

Comparison of the trueness and precision characteristics of

the reference method and alternative methods

The statistical values are summarised hereafter

Level p

Reference method Alternative method

Repeatability ratio

Reproducibility ratio Median

Repeatability standard deviation

Reproducibility standard deviation

Median Repeatability

standard deviation

Reproducibility standard deviation

1 10 16519 02415 02425 16021 02045 02433 0847 1007

2 10 26551 00543 00659 26719 00914 01325 1682 2011

2 bis 10 26581 02607 02607 25951 01900 01953 0729 0749

3 10 18671 00378 00378 18671 00478 00611 1264 1617

Bias of the alternative method

In order to estimate the bias of the alternative method with regard to the reference method for every

level Dij and t are calculated according to the following equations

RefAlt ijYijYDij

Diffp

Dijit

)2(

)(median

If the t value is superior to 2 the alternative method is significantly biased

with regard to the reference method

The values are given in table 17

Table 17 ndash t (d) values obtained by level

Level Bias t (d) Conclusion

1 0068 114 Non significant bias

2 0021 082 Non significant bias

2 bis - 0037 035 Non significant bias

3 - 0037 043 Non significant bias

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2738 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

The statistical test concludes to a non significant bias for all the inoculation

levels The bias varies from ndash 0037 to 0068 log CFUml

Comparison of the repeatability standard deviations

If the ratio Srj Alt Srj Ref of the repeatability standard deviations of the

alternative method and the reference method is larger than 2 the precision

under repeatability conditions of the alternative method is considered to be

lower than that of the reference method If this ratio is smaller than 05 the

precision under repeatability conditions of the alternative method is

considered to be greater than that of the reference method

The ratio values are given in table 18

Table 18

Level

Repeatability standard deviation

Reference method Alternative method Alternative method

reference method ratio

1 0242 0205 0847

2 0054 0091 1682

2 bis 0261 0190 0729

3 0038 0048 1264

Level Repeatability limit

Reference method Alternative method

1 0676 0573

2 0152 0256

2 bis 0730 0532

3 0106 0134

The ratios of the repeatability standard deviations are below 2 in all cases

The precision under repeatability conditions of the alternative method is

equivalent to that of the reference method

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2838 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Comparison of the reproducibility standard deviations

If the ratio Srj Alt Srj Ref of the reproducibility standard deviations of the alternative method and the

reference method is larger than 2 the precision under reproducibility conditions of the alternative

method is considered to be lower than that of the reference method If this ratio is smaller than 05 the

precision under reproducibility conditions of the alternative method is considered to be greater than

that of the reference method

The ratio values are given in table 19

Table 19

Level

Reproducibility standard deviation

Reference method Alternative method Alternative method

reference method ratio

1 0242 0243 1007

2 0066 0133 2011

2 bis 0261 0195 0749

3 0038 0061 1617

Level Reproducibility limit

Reference method Alternative method

1 0676 0681

2 0185 0371

2 bis 0730 0547

3 0106 0171

The ratios of the reproducibility standard deviations are below or close to 2

The precision under reproducibility conditions of the alternative method is

equivalent to that of the reference method

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2938 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

214 Conclusion

Method comparison study

The Listeria PrecisTM enumeration method shows satisfying linearity and

relative accuracy

The alternative method shows satisfying inclusivity and exclusivity results

Two Listeria monocytogenes strains gave negative results with the

alternative method These strains grew weakly on the reference method

media (OampA Palcam and Oxford)

The limits of repeatability of the alternative method are similar to those of the

reference method

The bias between both methods are low between - 0133 Log CFUg and

- 0008 Log CFUg

The Listeria PrecisTM enumeration method offers the saving for confirmatory

tests

Positive results are obtained with the alternative method in 2-3 days (with

OBIS Mono test) or in 4-7 days (by the classical tests described in the

standardized method) against 4-7 days for the reference method

Inter-laboratory study

The bias between the reference and the alternative methods shows satisfying

and correct values

- level 1 (100 - 1 000 CFUg) bias = + 007 log CFUg

- level 2 (1 000 - 10 000 CFUg) bias = 002 log CFUg

- level 2 bis (1 000 - 10 000 CFUg) bias = - 004 log CFUg

- level 3 (10 000 - 100 000 CFUg) bias = - 004 log CFUg

The repeatability and the reproducibility of the alternative method look similar

to those of the reference method

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3038 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

22 Extension study (realised by ASEPT 2007)

An extension study was performed in 2007 for a new confirmatory test OBIS

MONO

150 Listeria monocytogenes strains and 100 non-target strains were isolated

onto TSAYE and BrillianceTM Listeria Agar plates All the Listeria

monocytogenes and the non-target strains gave the expected results

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3138 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Appendix 1 ndash Flow diagram of the Listeria PrecisTM method

x g sample + 9 x ml buffered peptone water

Revivification step for 1 h plusmn 5 min at 20degC plusmn 1degC

Streaking 1 ml onto 3 Brilliance Listeria Agar plates (mother suspension)

or 01 ml onto 1 Brilliance Listeria Agar plates (mother suspension and decimal dilutions)

Incubation for 48 h plusmn 3 h at 37degC plusmn 1degC

Confirmatory tests on at least one typical colony

- by the tests described in the ISO method

- by applying the OBIS mono test on an isolated colony

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3238 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Appendix 2 ndash ISO 11290-2 (1998) amp ISO 11290-2A1 (2004) Horizontal

method for the detection and enumeration of Listeria monocytogenes -

Part 2 enumeration of Listeria monocytogenes in foods

10 g cheese + 90 ml buffered peptone water or Half Fraser Broth (without supplements) (110 dilution)

Stomach 1 min 30 seconds

Keep at 20degC plusmn 2degC for 1 h plusmn 5 min

Decimal dilutions in 9 ml peptone salt tubes

Dilution 110

Dilution 1100

Dilution 11000

1 ml on 3 Chromogenic Listeria medium (ISO) plates (x 2) (-1)

01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-2)

01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-3)

01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-4)

Incubation at 37degC plusmn 1degC

24 h - 48 h

Numerate typical colonies at 24 h

(bluegreen surrounded by an opaque halo)

Numerate at 48 h

Streak typical colonies on TSYEA

(5 coloniesplate 2 successive dilutions)

Confirmation tests

Catalase

Gram stain

For confirmation of L monocytogenes

Haemolysis

Carbohydrate (rhamnose and xylose) utilisation

CAMP Test

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3338 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Appendix 3 ndash Mandelrsquos graphics

Laboratoire h Ref

Niveau 1 h Ref

Niveau 2 h Ref

Niveau 3 h Alt

Niveau 1 h Alt

Niveau 2 h Alt

Niveau 3 h5 h1 h5 h1

A -050 -015 083 062 -212 -039 198 299 -198 -299

B -261 -270 021 -077 -058 173 198 299 -198 -299

C -151 -031 -014 -015 -004 -005 198 299 -198 -299

D 038 040 -137 000 072 207 198 299 -198 -299

E -001 090 155 -052 056 -101 198 299 -198 -299

F -112 -321 -153 070 -325 023 198 299 -198 -299

G 042 -071 040 147 -009 176 198 299 -198 -299

H 169 046 -169 192 018 -083 198 299 -198 -299

L 009 -120 021 -077 -231 005 198 299 -198 -299

M 001 031 014 -032 004 -011 198 299 -198 -299

-300

-200

-100

000

100

200

300

A B C D E F G H L M

Vale

urs

h d

e M

an

del(

rob

uste

s)

Laboratoires

Meacutethode de reacutefeacuterence analyse robuste

h Ref Niveau 1

h Ref Niveau 2

h Ref Niveau 3

h 5=198

h 1=299

-300

-200

-100

000

100

200

300

A B C D E F G H L M

Vale

urs

de h

de M

an

del(

rob

uste

s)

Laboratoires

Meacutethode alternative analyse robuste

h Alt Niveau 1

h Alt Niveau 2

h Alt Niveau 2

h 1=299

h 5=198

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3438 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Laboratoire k Ref

Niveau 1 k Ref

Niveau 2 k Ref

Niveau 3 k Alt

Niveau 1 k Alt

Niveau 2 k Alt

Niveau 3 k1 k5

A 000 089 142 084 162 046 259 181

B 125 087 073 104 075 046 259 181

C 117 013 091 188 022 083 259 181

D 052 036 069 000 035 083 259 181

E 028 034 037 138 019 112 259 181

F 140 222 018 027 281 112 259 181

G 088 068 024 131 145 000 259 181

H 129 094 173 016 061 000 259 181

L 069 000 073 104 270 224 259 181

M 000 048 038 043 099 241 259 181

000

050

100

150

200

250

300

350

400

A B C D E F G H L M

Vale

urs

k d

e M

an

del(

rob

uste

s)

Laboratoires

Meacutethode de reacutefeacuterence analyse robuste

k Ref Niveau 1k Ref Niveau 2k Ref Niveau 3

k 1=259

k 5=181

000

050

100

150

200

250

300

A B C D E F G H L M

Vale

urs

k d

e M

an

del

(ro

bu

ste

s)

Laboratoires

Meacutethode alternative analyse robuste

k Alt Niveau 3

k Alt Niveau 1

k Alt Niveau 2

k 1=259

k 5=181

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3538 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Appendix 4 ndash Statistical results

Level 1

Nombre de laboratoires (p) 10

A 1602 1602 1602 000 0000 0000 -0495 0000 A 1845 1602 1724 017 0122 -0122 0621 0840 0122

B 1602 1176 1389 030 0213 -0213 -2613 1247 B 1602 1301 1452 021 0151 -0151 -0769 1041 0062

C 1699 1301 1500 028 0199 -0199 -1510 1165 C 1301 1845 1573 038 -0272 0272 -0148 1881 0073

D 1602 1778 1690 012 -0088 0088 0380 0516 D 1602 1602 1602 000 0000 0000 0000 0000 -0088

E 1699 1602 1651 007 0048 -0048 -0013 0284 E 1699 1301 1500 028 0199 -0199 -0522 1376 -0151

F 1778 1301 1540 034 0239 -0239 -1116 1397 F 1778 1699 1739 006 0040 -0040 0698 0274 0199

G 1845 1544 1695 021 0151 -0151 0425 0881 G 2079 1699 1889 027 0190 -0190 1467 1315 0194

H 2041 1602 1822 031 0220 -0220 1689 1286 H 2000 1954 1977 003 0023 -0023 1917 0158 0155

L 1544 1778 1661 017 -0117 0117 0092 0685 L 1301 1602 1452 021 -0151 0151 -0769 1041 -0210

M 1653 1653 1653 000 0000 0000 0013 0000 M 1477 1602 1540 009 -0062 0062 -0319 0432 -0114

m 165186 m 160206 mediane D 00678

Cn(2p) 1867 Cn(2p) 1867 Cp 16101

Cn(p) 16101 Cn(p) 16101 Qn(D) 0092926

n=2p n 20 n=2p n 20 Qdiff 01496169

f 11 f 11 t 114

l 55 l 55

n=p n 10 n=p n 10

f 6 f 6

l 15 l 15

Qn(2p) 0091465 Qn(2p) 007745

Qn(p) 0062469 Qn(p) 0121519

Qintra 017078 Qintra 014461

Qinter 010058 Qinter 019565

Sr(eacutecart-type de reacutepeacutetabiliteacute) 02415 Sr 02045

RSDr(coefficient de variation de la reacutepeacutetabiliteacute)1462 RSDr 1277

r(repeatability limit)) 0676 r 0573

SL(eacutecart-type interlaboratoire) -001905 SLlt0 SL 001737

SR(eacutecart-type de reproductibiliteacute) 02415 SR 02433

RSDR(coefficient de variation de la reproductibiliteacute)0146 RSDR 0152

R(limite de reproductibiliteacute)) 06762 R(limite de reproductibiliteacute))068123

h k h MoyenneLaboratoires

Niveau 1

si LaboratoiresDeviationMoyenne Dk DeviationsiAlt

Niveau 1

Ref

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3638 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Level 2

Nombre de laboratoires (p) 10

A 2681 2613 2647 005 0034 -0034 -0151 0891 A 2322 2531 2427 015 -0105 0105 -2119 1620 -0220

B 2544 2477 2511 005 0033 -0033 -2699 0872 B 2556 2653 2605 007 -0048 0048 -0581 0750 0094

C 2643 2633 2638 001 0005 -0005 -0311 0130 C 2681 2653 2667 002 0014 -0014 -0040 0217 0029

D 2690 2663 2676 002 0014 -0014 0400 0357 D 2778 2732 2755 003 0023 -0023 0721 0354 0079

E 2690 2716 2703 002 -0013 0013 0897 0336 E 2724 2748 2736 002 -0012 0012 0556 0185 0033

F 2568 2398 2483 012 0085 -0085 -3214 2217 F 2477 2114 2296 026 0182 -0182 -3255 2811 -0188

G 2643 2591 2617 004 0026 -0026 -0707 0682 G 2756 2568 2662 013 0094 -0094 -0085 1453 0045

H 2716 2643 2680 005 0036 -0036 0460 0945 H 2653 2732 2693 006 -0040 0040 0181 0613 0013

L 2591 2591 2591 000 0000 0000 -1196 0000 L 2230 2580 2405 025 -0175 0175 -2307 2704 -0186

M 2653 2690 2672 003 -0018 0018 0311 0481 M 2740 2613 2677 009 0064 -0064 0040 0987 0005

m 2655082 m 26719001 mediane D 00209

Cn(2p) 1867 Cn(2p) 1867 Cp 16101

Cn(p) 16101 Cn(p) 16101 Qn(D) 0039911

n=2p n 20 n=2p n 20 Qdiff 00642591

f 11 f 11 t 082

l 55 l 55

n=p n 10 n=p n 10

f 6 f 6

l 15 l 15

Qn(2p) 0020570 Qn(2p) 00345985

Qn(p) 00332437 Qn(p) 00718158

Qintra 003841 Qintra 006460

Qinter 005352 Qinter 011563

Sr(eacutecart-type de reacutepeacutetabiliteacute) 00543 Sr 00914

RSDr(coefficient de variation de la reacutepeacutetabiliteacute)205 RSDr 342

r(repeatability limit)) 0152 r 0256

SL(eacutecart-type interlaboratoire) 000139 SL 000920

SR(eacutecart-type de reproductibiliteacute) 00659 SR 01325

RSDR(coefficient de variation de la reproductibiliteacute)0025 RSDR 0050

R(limite de reproductibiliteacute)) 018446 R(limite de reproductibiliteacute))03708618

Laboratoires

Niveau 2

Moyenne si Laboratoires

Niveau 2

DRef Alt Moyenne si Deviation h Deviation h k k

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3738 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Level 2 bis

Nombre de laboratoires (p) 10

A 3000 2477 2739 037 0261 -0261 0832 1418 A 2602 2477 2540 009 0062 -0062 -0391 0465 -0199

B 2544 2813 2678 019 -0134 0134 0211 0729 B 2903 2778 2841 009 0062 -0062 1732 0465 0162

C 2477 2813 2645 024 -0168 0168 -0135 0911 C 2477 2699 2588 016 -0111 0111 -0049 0826 -0057

D 2398 2653 2526 018 -0128 0128 -1370 0692 D 2778 3000 2889 016 -0111 0111 2074 0826 0363

E 2875 2740 2808 010 0067 -0067 1547 0365 E 2602 2301 2452 021 0151 -0151 -1012 1120 -0356

F 2477 2544 2511 005 -0033 0033 -1525 0182 F 2778 2477 2628 021 0151 -0151 0230 1120 0117

G 2653 2740 2697 006 -0044 0044 0400 0236 G 2845 2845 2845 000 0000 0000 1764 0000 0148

H 2176 2813 2495 045 -0318 0318 -1692 1727 H 2477 2477 2477 000 0000 0000 -0832 0000 -0017

L 2813 2544 2678 019 0134 -0134 0211 0729 L 2903 2301 2602 043 0301 -0301 0049 2241 -0076

M 2602 2740 2671 010 -0069 0069 0135 0375 M 2903 2255 2579 046 0324 -0324 -0112 2411 -0092

m 2658114 m 259505 mediane D -00372

Cn(2p) 1867 Cn(2p) 1867 Cp 16101

Cn(p) 16101 Cn(p) 16101 Qn(D) 01656915

n=2p n 20 n=2p n 20 Qdiff 02667753

f 11 f 11 t 035

l 55 l 55

n=p n 10 n=p n 10

f 6 f 6

l 15 l 15

Qn(2p) 0098745 Qn(2p) 007195

Qn(p) 006007 Qn(p) 008805

Qintra 018437 Qintra 013435

Qinter 009672 Qinter 014176

Sr(eacutecart-type de reacutepeacutetabiliteacute) 02607 Sr 01900

RSDr(coefficient de variation de la reacutepeacutetabiliteacute)981 RSDr 732

r(repeatability limit)) 0730 r 0532

SL(eacutecart-type interlaboratoire) -002464 SLlt0 SL 000205

SR(eacutecart-type de reproductibiliteacute) 02607 SR 01953

RSDR(coefficient de variation de la reproductibiliteacute)0098 RSDR 0075

R(limite de reproductibiliteacute)) 072996 R(limite de reproductibiliteacute))054686

Laboratoires

Niveau 2 bis

Moyenne si Laboratoires

Niveau 2 bis

DRef Alt Moyenne si Deviation h Deviation h k k

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3838 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Level 3

Nombre de laboratoires (p) 10

A 3690 3663 3676 002 0014 -0014 -0311 0513 A 3690 3708 3699 001 -0009 0009 0354 0257 0022

B 3613 3672 3642 004 -0030 0030 -1851 1109 B 3708 3568 3638 010 0070 -0070 -0844 2061 -0005

C 3663 3732 3698 005 -0035 0035 0645 1302 C 3663 3699 3681 003 -0018 0018 0000 0536 -0017

D 3690 3690 3690 000 0000 0000 0311 0000 D 3643 3663 3653 001 -0010 0010 -0545 0286 -0037

E 3732 3653 3693 006 0040 -0040 0429 1480 E 3613 3732 3673 008 -0060 0060 -0162 1769 -0020

F 3643 3672 3658 002 -0014 0014 -1157 0536 F 3690 3771 3731 006 -0040 0040 0975 1193 0073

G 3699 3653 3676 003 0023 -0023 -0328 0856 G 3602 3643 3623 003 -0021 0021 -1141 0612 -0053

H 3623 3672 3648 003 -0024 0024 -1615 0913 H 3748 3681 3715 005 0033 -0033 0664 0990 0067

L 3724 3663 3694 004 0031 -0031 0461 1150 L 3748 3771 3760 002 -0011 0011 1544 0335 0066

M 3690 3690 3690 000 0000 0000 0311 0000 M 3806 3681 3744 009 0062 -0062 1234 1848 0054

m 368334 m 368086 mediane D 00089

Cn(2p) 1867 Cn(2p) 1867 Cp 16101

Cn(p) 16101 Cn(p) 16101 Qn(D) 00325359

n=2p n 20 n=2p n 20 Qdiff 00523852

f 11 f 11 t 043

l 55 l 55

n=p n 10 n=p n 10

f 6 f 6

l 15 l 15

Qn(2p) 0014323 Qn(2p) 001811

Qn(p) 0013719 Qn(p) 003164

Qintra 002674 Qintra 003381

Qinter 002209 Qinter 005094

Sr(eacutecart-type de reacutepeacutetabiliteacute) 00378 Sr 00478

RSDr(coefficient de variation de la reacutepeacutetabiliteacute)103 RSDr 130

r(repeatability limit)) 0106 r 0134

SL(eacutecart-type interlaboratoire) -000023 SLlt0 SL 000145

SR(eacutecart-type de reproductibiliteacute) 00378 SR 00611

RSDR(coefficient de variation de la reproductibiliteacute)0010 RSDR 0017

R(limite de reproductibiliteacute)) 0105894 R(limite de reproductibiliteacute))017119

Ref Alt DLaboratoires

Niveau 3

Moyenne si Deviation h k Laboratoires

Niveau 3

Moyenne si Deviation h k

Page 23: ÞÀ¿¸7 ú Y5zñLjÜ< /u +æü t¬Á ... - NF Validation · NF VALIDATION Validation of alternative analytical methods Application in food microbiology ... 500 – 1 000 1 000 –

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2338 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Table 13 - Sample temperature on receipt

Laboratories Receipt

day

Hour

receipt

Measured temperature

at receipt (degC)

Measured temperature by the

temperature probe (degC)

A

B

C

D

E

F

G

H

I

J

K

L

M

30052006

10h45

10h15

11h15

10h00

09h30

8h30

11h50

10h00

08h30

14h45

08h30

09h45

08h45

82

55

45

63

60

62

81

38

48

-

14

67

76

03

03

-13

03

04

-14

05

04

01

05

-13

04

Conclusion

The temperatures during the transport were correct for all the labs except for

Lab J which didnrsquot provide the information

All the labs received their packages with several broken flasks

Three labs (B D and L) received some damaged samples but proceeded to

analyses

All the labs started the analyses at Day 1 except Lab J

2133 Results

Aerobic mesophilic flora

The count of aerobic mesophilic flora varied from 94 102 CFUml to

33 103 CFUml

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2438 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Expert lab results

The results obtained by the expert lab are the following

Table 14

Target inoculation level (CFUml)

Reference method ISO 11290-2

Alternative method Listeria Precis Enumeration

Rep 1 Rep 2 Rep 1 Rep 2

lt 10 lt 10 lt 10 lt 10 lt10

50 50 (Ne) 60 (Ne) 50 (Ne) 30 (Ne)

500 470 390 490 500

5 000 4 700 5 100 5 900 3 400

Ne estimated number

The obtained results are in agreement with those expected

Collaborator Labs results

A synthesis of the results obtained by the collaborator labs is provided

Table 15 (CFUml) and Table 16 (log CFUml)

Table 15 ndash Synthesis of results obtained by the alternative

and the reference methods (CFUml)

Labs Level 1 Level 2 Level 2 bis Level 3

Reference method

Alternative method

Reference method

Alternative method

Reference method

Alternative method

Reference method

Alternative method

A 40 40 70 40 480 410 210 340 1000 300 400 300 4900 4600 4900 5100

B 40 15 40 20 350 300 360 450 350 650 800 600 4100 4700 5100 3700

C 50 20 20 70 440 430 480 450 300 650 300 500 4600 5400 4600 5000

D 40 60 40 40 490 460 600 540 250 450 600 1000 4900 4900 4400 4600

E 50 40 50 20 490 520 530 560 750 550 400 200 5400 4500 4100 5400

F 60 20 60 50 370 250 300 130 300 350 600 300 4400 4700 4900 5900

G 70 35 120 50 440 390 570 370 450 550 700 700 5000 4500 4000 4400

H 110 40 100 90 520 440 450 540 150 650 300 300 4200 4700 5600 4800

L 35 60 20 40 390 390 170 380 650 350 800 200 5300 4600 5600 5900

M 45 45 30 40 450 490 550 410 400 550 800 180 4900 4900 6400 4800

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2538 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Table 16 ndash Synthesis of results obtained by the alternative

and the reference methods (log CFUml)

Labs Level 1 Level 2 Level 2 bis Level 3

Reference method

Alternative method

Reference method

Alternative method

Reference method

Alternative method

Reference method

Alternative method

A 1602 1602 1845 1602 2681 2613 2322 2531 3000 2477 2602 2477 3690 3663 3690 3708

B 1602 1176 1602 1301 2544 2477 2556 2653 2544 2813 2903 2778 3613 3672 3708 3568

C 1699 1301 1301 1845 2643 2633 2681 2653 2477 2813 2477 2699 3663 3732 3663 3699

D 1602 1778 1602 1602 2690 2663 2778 2732 2398 2653 2778 3000 3690 3690 3643 3663

E 1699 1602 1699 1301 2690 2716 2724 2748 2875 2740 2602 2301 3732 3653 3613 3732

F 1778 1301 1778 1699 2568 2398 2477 2114 2477 2544 2778 2477 3643 3672 3690 3771

G 1845 1544 2079 1699 2643 2591 2756 2568 2653 2740 2845 2845 3699 3653 3602 3643

H 2041 1602 2000 1954 2716 2643 2653 2732 2176 2813 2477 2477 3623 3672 3748 3681

L 1544 1778 1301 1602 2591 2591 2230 2580 2813 2544 2903 2301 3724 3663 3748 3771

M 1653 1653 1477 1602 2653 2690 2740 2613 2602 2740 2903 2255 3690 3690 3806 3681

Level 2 results obtained taking account the 3 plates inoculated with the mother-suspension

Level 2 bis results obtained taking account the plates inoculated with 01 ml of the mother-suspension

Scrutiny of the measurement results for consistency

In order to identify the measurement results or laboratories that could be

inconsistent two graphical consistency techniques were realised Mandelrsquos

h-ank-statistics Mandel indicators h and k at 5 significance highlighted

some possible inconsistent data (See tables and graphics in Appendix 3 and

Appendix 4)

Mandelrsquos values Number of values observed over the threshold

Reference method Alternative method

h gt 1 Lab F Level 2 Lab F Level 2

h gt 5 Lab F Level 2

Lab B Levels 1 and 2

Lab A Level 2

Lab D Level 3

Lab F Level 2

Lab L Level 2

kgt 1 Lab F Level 3

k gt 5 Lab F Level 2 Lab C Level 2

Lab F Level 3

Lab L Levels 1 and 3

Lab M Level 1

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2638 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

The emphasized results appear (i) coherent with the targeted contamination

levels (ii) concordant between both methods

All the results were thus kept for the statistical analysis

Comparison of the trueness and precision characteristics of

the reference method and alternative methods

The statistical values are summarised hereafter

Level p

Reference method Alternative method

Repeatability ratio

Reproducibility ratio Median

Repeatability standard deviation

Reproducibility standard deviation

Median Repeatability

standard deviation

Reproducibility standard deviation

1 10 16519 02415 02425 16021 02045 02433 0847 1007

2 10 26551 00543 00659 26719 00914 01325 1682 2011

2 bis 10 26581 02607 02607 25951 01900 01953 0729 0749

3 10 18671 00378 00378 18671 00478 00611 1264 1617

Bias of the alternative method

In order to estimate the bias of the alternative method with regard to the reference method for every

level Dij and t are calculated according to the following equations

RefAlt ijYijYDij

Diffp

Dijit

)2(

)(median

If the t value is superior to 2 the alternative method is significantly biased

with regard to the reference method

The values are given in table 17

Table 17 ndash t (d) values obtained by level

Level Bias t (d) Conclusion

1 0068 114 Non significant bias

2 0021 082 Non significant bias

2 bis - 0037 035 Non significant bias

3 - 0037 043 Non significant bias

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2738 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

The statistical test concludes to a non significant bias for all the inoculation

levels The bias varies from ndash 0037 to 0068 log CFUml

Comparison of the repeatability standard deviations

If the ratio Srj Alt Srj Ref of the repeatability standard deviations of the

alternative method and the reference method is larger than 2 the precision

under repeatability conditions of the alternative method is considered to be

lower than that of the reference method If this ratio is smaller than 05 the

precision under repeatability conditions of the alternative method is

considered to be greater than that of the reference method

The ratio values are given in table 18

Table 18

Level

Repeatability standard deviation

Reference method Alternative method Alternative method

reference method ratio

1 0242 0205 0847

2 0054 0091 1682

2 bis 0261 0190 0729

3 0038 0048 1264

Level Repeatability limit

Reference method Alternative method

1 0676 0573

2 0152 0256

2 bis 0730 0532

3 0106 0134

The ratios of the repeatability standard deviations are below 2 in all cases

The precision under repeatability conditions of the alternative method is

equivalent to that of the reference method

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2838 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Comparison of the reproducibility standard deviations

If the ratio Srj Alt Srj Ref of the reproducibility standard deviations of the alternative method and the

reference method is larger than 2 the precision under reproducibility conditions of the alternative

method is considered to be lower than that of the reference method If this ratio is smaller than 05 the

precision under reproducibility conditions of the alternative method is considered to be greater than

that of the reference method

The ratio values are given in table 19

Table 19

Level

Reproducibility standard deviation

Reference method Alternative method Alternative method

reference method ratio

1 0242 0243 1007

2 0066 0133 2011

2 bis 0261 0195 0749

3 0038 0061 1617

Level Reproducibility limit

Reference method Alternative method

1 0676 0681

2 0185 0371

2 bis 0730 0547

3 0106 0171

The ratios of the reproducibility standard deviations are below or close to 2

The precision under reproducibility conditions of the alternative method is

equivalent to that of the reference method

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2938 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

214 Conclusion

Method comparison study

The Listeria PrecisTM enumeration method shows satisfying linearity and

relative accuracy

The alternative method shows satisfying inclusivity and exclusivity results

Two Listeria monocytogenes strains gave negative results with the

alternative method These strains grew weakly on the reference method

media (OampA Palcam and Oxford)

The limits of repeatability of the alternative method are similar to those of the

reference method

The bias between both methods are low between - 0133 Log CFUg and

- 0008 Log CFUg

The Listeria PrecisTM enumeration method offers the saving for confirmatory

tests

Positive results are obtained with the alternative method in 2-3 days (with

OBIS Mono test) or in 4-7 days (by the classical tests described in the

standardized method) against 4-7 days for the reference method

Inter-laboratory study

The bias between the reference and the alternative methods shows satisfying

and correct values

- level 1 (100 - 1 000 CFUg) bias = + 007 log CFUg

- level 2 (1 000 - 10 000 CFUg) bias = 002 log CFUg

- level 2 bis (1 000 - 10 000 CFUg) bias = - 004 log CFUg

- level 3 (10 000 - 100 000 CFUg) bias = - 004 log CFUg

The repeatability and the reproducibility of the alternative method look similar

to those of the reference method

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3038 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

22 Extension study (realised by ASEPT 2007)

An extension study was performed in 2007 for a new confirmatory test OBIS

MONO

150 Listeria monocytogenes strains and 100 non-target strains were isolated

onto TSAYE and BrillianceTM Listeria Agar plates All the Listeria

monocytogenes and the non-target strains gave the expected results

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3138 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Appendix 1 ndash Flow diagram of the Listeria PrecisTM method

x g sample + 9 x ml buffered peptone water

Revivification step for 1 h plusmn 5 min at 20degC plusmn 1degC

Streaking 1 ml onto 3 Brilliance Listeria Agar plates (mother suspension)

or 01 ml onto 1 Brilliance Listeria Agar plates (mother suspension and decimal dilutions)

Incubation for 48 h plusmn 3 h at 37degC plusmn 1degC

Confirmatory tests on at least one typical colony

- by the tests described in the ISO method

- by applying the OBIS mono test on an isolated colony

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3238 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Appendix 2 ndash ISO 11290-2 (1998) amp ISO 11290-2A1 (2004) Horizontal

method for the detection and enumeration of Listeria monocytogenes -

Part 2 enumeration of Listeria monocytogenes in foods

10 g cheese + 90 ml buffered peptone water or Half Fraser Broth (without supplements) (110 dilution)

Stomach 1 min 30 seconds

Keep at 20degC plusmn 2degC for 1 h plusmn 5 min

Decimal dilutions in 9 ml peptone salt tubes

Dilution 110

Dilution 1100

Dilution 11000

1 ml on 3 Chromogenic Listeria medium (ISO) plates (x 2) (-1)

01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-2)

01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-3)

01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-4)

Incubation at 37degC plusmn 1degC

24 h - 48 h

Numerate typical colonies at 24 h

(bluegreen surrounded by an opaque halo)

Numerate at 48 h

Streak typical colonies on TSYEA

(5 coloniesplate 2 successive dilutions)

Confirmation tests

Catalase

Gram stain

For confirmation of L monocytogenes

Haemolysis

Carbohydrate (rhamnose and xylose) utilisation

CAMP Test

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3338 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Appendix 3 ndash Mandelrsquos graphics

Laboratoire h Ref

Niveau 1 h Ref

Niveau 2 h Ref

Niveau 3 h Alt

Niveau 1 h Alt

Niveau 2 h Alt

Niveau 3 h5 h1 h5 h1

A -050 -015 083 062 -212 -039 198 299 -198 -299

B -261 -270 021 -077 -058 173 198 299 -198 -299

C -151 -031 -014 -015 -004 -005 198 299 -198 -299

D 038 040 -137 000 072 207 198 299 -198 -299

E -001 090 155 -052 056 -101 198 299 -198 -299

F -112 -321 -153 070 -325 023 198 299 -198 -299

G 042 -071 040 147 -009 176 198 299 -198 -299

H 169 046 -169 192 018 -083 198 299 -198 -299

L 009 -120 021 -077 -231 005 198 299 -198 -299

M 001 031 014 -032 004 -011 198 299 -198 -299

-300

-200

-100

000

100

200

300

A B C D E F G H L M

Vale

urs

h d

e M

an

del(

rob

uste

s)

Laboratoires

Meacutethode de reacutefeacuterence analyse robuste

h Ref Niveau 1

h Ref Niveau 2

h Ref Niveau 3

h 5=198

h 1=299

-300

-200

-100

000

100

200

300

A B C D E F G H L M

Vale

urs

de h

de M

an

del(

rob

uste

s)

Laboratoires

Meacutethode alternative analyse robuste

h Alt Niveau 1

h Alt Niveau 2

h Alt Niveau 2

h 1=299

h 5=198

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3438 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Laboratoire k Ref

Niveau 1 k Ref

Niveau 2 k Ref

Niveau 3 k Alt

Niveau 1 k Alt

Niveau 2 k Alt

Niveau 3 k1 k5

A 000 089 142 084 162 046 259 181

B 125 087 073 104 075 046 259 181

C 117 013 091 188 022 083 259 181

D 052 036 069 000 035 083 259 181

E 028 034 037 138 019 112 259 181

F 140 222 018 027 281 112 259 181

G 088 068 024 131 145 000 259 181

H 129 094 173 016 061 000 259 181

L 069 000 073 104 270 224 259 181

M 000 048 038 043 099 241 259 181

000

050

100

150

200

250

300

350

400

A B C D E F G H L M

Vale

urs

k d

e M

an

del(

rob

uste

s)

Laboratoires

Meacutethode de reacutefeacuterence analyse robuste

k Ref Niveau 1k Ref Niveau 2k Ref Niveau 3

k 1=259

k 5=181

000

050

100

150

200

250

300

A B C D E F G H L M

Vale

urs

k d

e M

an

del

(ro

bu

ste

s)

Laboratoires

Meacutethode alternative analyse robuste

k Alt Niveau 3

k Alt Niveau 1

k Alt Niveau 2

k 1=259

k 5=181

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3538 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Appendix 4 ndash Statistical results

Level 1

Nombre de laboratoires (p) 10

A 1602 1602 1602 000 0000 0000 -0495 0000 A 1845 1602 1724 017 0122 -0122 0621 0840 0122

B 1602 1176 1389 030 0213 -0213 -2613 1247 B 1602 1301 1452 021 0151 -0151 -0769 1041 0062

C 1699 1301 1500 028 0199 -0199 -1510 1165 C 1301 1845 1573 038 -0272 0272 -0148 1881 0073

D 1602 1778 1690 012 -0088 0088 0380 0516 D 1602 1602 1602 000 0000 0000 0000 0000 -0088

E 1699 1602 1651 007 0048 -0048 -0013 0284 E 1699 1301 1500 028 0199 -0199 -0522 1376 -0151

F 1778 1301 1540 034 0239 -0239 -1116 1397 F 1778 1699 1739 006 0040 -0040 0698 0274 0199

G 1845 1544 1695 021 0151 -0151 0425 0881 G 2079 1699 1889 027 0190 -0190 1467 1315 0194

H 2041 1602 1822 031 0220 -0220 1689 1286 H 2000 1954 1977 003 0023 -0023 1917 0158 0155

L 1544 1778 1661 017 -0117 0117 0092 0685 L 1301 1602 1452 021 -0151 0151 -0769 1041 -0210

M 1653 1653 1653 000 0000 0000 0013 0000 M 1477 1602 1540 009 -0062 0062 -0319 0432 -0114

m 165186 m 160206 mediane D 00678

Cn(2p) 1867 Cn(2p) 1867 Cp 16101

Cn(p) 16101 Cn(p) 16101 Qn(D) 0092926

n=2p n 20 n=2p n 20 Qdiff 01496169

f 11 f 11 t 114

l 55 l 55

n=p n 10 n=p n 10

f 6 f 6

l 15 l 15

Qn(2p) 0091465 Qn(2p) 007745

Qn(p) 0062469 Qn(p) 0121519

Qintra 017078 Qintra 014461

Qinter 010058 Qinter 019565

Sr(eacutecart-type de reacutepeacutetabiliteacute) 02415 Sr 02045

RSDr(coefficient de variation de la reacutepeacutetabiliteacute)1462 RSDr 1277

r(repeatability limit)) 0676 r 0573

SL(eacutecart-type interlaboratoire) -001905 SLlt0 SL 001737

SR(eacutecart-type de reproductibiliteacute) 02415 SR 02433

RSDR(coefficient de variation de la reproductibiliteacute)0146 RSDR 0152

R(limite de reproductibiliteacute)) 06762 R(limite de reproductibiliteacute))068123

h k h MoyenneLaboratoires

Niveau 1

si LaboratoiresDeviationMoyenne Dk DeviationsiAlt

Niveau 1

Ref

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3638 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Level 2

Nombre de laboratoires (p) 10

A 2681 2613 2647 005 0034 -0034 -0151 0891 A 2322 2531 2427 015 -0105 0105 -2119 1620 -0220

B 2544 2477 2511 005 0033 -0033 -2699 0872 B 2556 2653 2605 007 -0048 0048 -0581 0750 0094

C 2643 2633 2638 001 0005 -0005 -0311 0130 C 2681 2653 2667 002 0014 -0014 -0040 0217 0029

D 2690 2663 2676 002 0014 -0014 0400 0357 D 2778 2732 2755 003 0023 -0023 0721 0354 0079

E 2690 2716 2703 002 -0013 0013 0897 0336 E 2724 2748 2736 002 -0012 0012 0556 0185 0033

F 2568 2398 2483 012 0085 -0085 -3214 2217 F 2477 2114 2296 026 0182 -0182 -3255 2811 -0188

G 2643 2591 2617 004 0026 -0026 -0707 0682 G 2756 2568 2662 013 0094 -0094 -0085 1453 0045

H 2716 2643 2680 005 0036 -0036 0460 0945 H 2653 2732 2693 006 -0040 0040 0181 0613 0013

L 2591 2591 2591 000 0000 0000 -1196 0000 L 2230 2580 2405 025 -0175 0175 -2307 2704 -0186

M 2653 2690 2672 003 -0018 0018 0311 0481 M 2740 2613 2677 009 0064 -0064 0040 0987 0005

m 2655082 m 26719001 mediane D 00209

Cn(2p) 1867 Cn(2p) 1867 Cp 16101

Cn(p) 16101 Cn(p) 16101 Qn(D) 0039911

n=2p n 20 n=2p n 20 Qdiff 00642591

f 11 f 11 t 082

l 55 l 55

n=p n 10 n=p n 10

f 6 f 6

l 15 l 15

Qn(2p) 0020570 Qn(2p) 00345985

Qn(p) 00332437 Qn(p) 00718158

Qintra 003841 Qintra 006460

Qinter 005352 Qinter 011563

Sr(eacutecart-type de reacutepeacutetabiliteacute) 00543 Sr 00914

RSDr(coefficient de variation de la reacutepeacutetabiliteacute)205 RSDr 342

r(repeatability limit)) 0152 r 0256

SL(eacutecart-type interlaboratoire) 000139 SL 000920

SR(eacutecart-type de reproductibiliteacute) 00659 SR 01325

RSDR(coefficient de variation de la reproductibiliteacute)0025 RSDR 0050

R(limite de reproductibiliteacute)) 018446 R(limite de reproductibiliteacute))03708618

Laboratoires

Niveau 2

Moyenne si Laboratoires

Niveau 2

DRef Alt Moyenne si Deviation h Deviation h k k

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3738 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Level 2 bis

Nombre de laboratoires (p) 10

A 3000 2477 2739 037 0261 -0261 0832 1418 A 2602 2477 2540 009 0062 -0062 -0391 0465 -0199

B 2544 2813 2678 019 -0134 0134 0211 0729 B 2903 2778 2841 009 0062 -0062 1732 0465 0162

C 2477 2813 2645 024 -0168 0168 -0135 0911 C 2477 2699 2588 016 -0111 0111 -0049 0826 -0057

D 2398 2653 2526 018 -0128 0128 -1370 0692 D 2778 3000 2889 016 -0111 0111 2074 0826 0363

E 2875 2740 2808 010 0067 -0067 1547 0365 E 2602 2301 2452 021 0151 -0151 -1012 1120 -0356

F 2477 2544 2511 005 -0033 0033 -1525 0182 F 2778 2477 2628 021 0151 -0151 0230 1120 0117

G 2653 2740 2697 006 -0044 0044 0400 0236 G 2845 2845 2845 000 0000 0000 1764 0000 0148

H 2176 2813 2495 045 -0318 0318 -1692 1727 H 2477 2477 2477 000 0000 0000 -0832 0000 -0017

L 2813 2544 2678 019 0134 -0134 0211 0729 L 2903 2301 2602 043 0301 -0301 0049 2241 -0076

M 2602 2740 2671 010 -0069 0069 0135 0375 M 2903 2255 2579 046 0324 -0324 -0112 2411 -0092

m 2658114 m 259505 mediane D -00372

Cn(2p) 1867 Cn(2p) 1867 Cp 16101

Cn(p) 16101 Cn(p) 16101 Qn(D) 01656915

n=2p n 20 n=2p n 20 Qdiff 02667753

f 11 f 11 t 035

l 55 l 55

n=p n 10 n=p n 10

f 6 f 6

l 15 l 15

Qn(2p) 0098745 Qn(2p) 007195

Qn(p) 006007 Qn(p) 008805

Qintra 018437 Qintra 013435

Qinter 009672 Qinter 014176

Sr(eacutecart-type de reacutepeacutetabiliteacute) 02607 Sr 01900

RSDr(coefficient de variation de la reacutepeacutetabiliteacute)981 RSDr 732

r(repeatability limit)) 0730 r 0532

SL(eacutecart-type interlaboratoire) -002464 SLlt0 SL 000205

SR(eacutecart-type de reproductibiliteacute) 02607 SR 01953

RSDR(coefficient de variation de la reproductibiliteacute)0098 RSDR 0075

R(limite de reproductibiliteacute)) 072996 R(limite de reproductibiliteacute))054686

Laboratoires

Niveau 2 bis

Moyenne si Laboratoires

Niveau 2 bis

DRef Alt Moyenne si Deviation h Deviation h k k

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3838 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Level 3

Nombre de laboratoires (p) 10

A 3690 3663 3676 002 0014 -0014 -0311 0513 A 3690 3708 3699 001 -0009 0009 0354 0257 0022

B 3613 3672 3642 004 -0030 0030 -1851 1109 B 3708 3568 3638 010 0070 -0070 -0844 2061 -0005

C 3663 3732 3698 005 -0035 0035 0645 1302 C 3663 3699 3681 003 -0018 0018 0000 0536 -0017

D 3690 3690 3690 000 0000 0000 0311 0000 D 3643 3663 3653 001 -0010 0010 -0545 0286 -0037

E 3732 3653 3693 006 0040 -0040 0429 1480 E 3613 3732 3673 008 -0060 0060 -0162 1769 -0020

F 3643 3672 3658 002 -0014 0014 -1157 0536 F 3690 3771 3731 006 -0040 0040 0975 1193 0073

G 3699 3653 3676 003 0023 -0023 -0328 0856 G 3602 3643 3623 003 -0021 0021 -1141 0612 -0053

H 3623 3672 3648 003 -0024 0024 -1615 0913 H 3748 3681 3715 005 0033 -0033 0664 0990 0067

L 3724 3663 3694 004 0031 -0031 0461 1150 L 3748 3771 3760 002 -0011 0011 1544 0335 0066

M 3690 3690 3690 000 0000 0000 0311 0000 M 3806 3681 3744 009 0062 -0062 1234 1848 0054

m 368334 m 368086 mediane D 00089

Cn(2p) 1867 Cn(2p) 1867 Cp 16101

Cn(p) 16101 Cn(p) 16101 Qn(D) 00325359

n=2p n 20 n=2p n 20 Qdiff 00523852

f 11 f 11 t 043

l 55 l 55

n=p n 10 n=p n 10

f 6 f 6

l 15 l 15

Qn(2p) 0014323 Qn(2p) 001811

Qn(p) 0013719 Qn(p) 003164

Qintra 002674 Qintra 003381

Qinter 002209 Qinter 005094

Sr(eacutecart-type de reacutepeacutetabiliteacute) 00378 Sr 00478

RSDr(coefficient de variation de la reacutepeacutetabiliteacute)103 RSDr 130

r(repeatability limit)) 0106 r 0134

SL(eacutecart-type interlaboratoire) -000023 SLlt0 SL 000145

SR(eacutecart-type de reproductibiliteacute) 00378 SR 00611

RSDR(coefficient de variation de la reproductibiliteacute)0010 RSDR 0017

R(limite de reproductibiliteacute)) 0105894 R(limite de reproductibiliteacute))017119

Ref Alt DLaboratoires

Niveau 3

Moyenne si Deviation h k Laboratoires

Niveau 3

Moyenne si Deviation h k

Page 24: ÞÀ¿¸7 ú Y5zñLjÜ< /u +æü t¬Á ... - NF Validation · NF VALIDATION Validation of alternative analytical methods Application in food microbiology ... 500 – 1 000 1 000 –

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2438 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Expert lab results

The results obtained by the expert lab are the following

Table 14

Target inoculation level (CFUml)

Reference method ISO 11290-2

Alternative method Listeria Precis Enumeration

Rep 1 Rep 2 Rep 1 Rep 2

lt 10 lt 10 lt 10 lt 10 lt10

50 50 (Ne) 60 (Ne) 50 (Ne) 30 (Ne)

500 470 390 490 500

5 000 4 700 5 100 5 900 3 400

Ne estimated number

The obtained results are in agreement with those expected

Collaborator Labs results

A synthesis of the results obtained by the collaborator labs is provided

Table 15 (CFUml) and Table 16 (log CFUml)

Table 15 ndash Synthesis of results obtained by the alternative

and the reference methods (CFUml)

Labs Level 1 Level 2 Level 2 bis Level 3

Reference method

Alternative method

Reference method

Alternative method

Reference method

Alternative method

Reference method

Alternative method

A 40 40 70 40 480 410 210 340 1000 300 400 300 4900 4600 4900 5100

B 40 15 40 20 350 300 360 450 350 650 800 600 4100 4700 5100 3700

C 50 20 20 70 440 430 480 450 300 650 300 500 4600 5400 4600 5000

D 40 60 40 40 490 460 600 540 250 450 600 1000 4900 4900 4400 4600

E 50 40 50 20 490 520 530 560 750 550 400 200 5400 4500 4100 5400

F 60 20 60 50 370 250 300 130 300 350 600 300 4400 4700 4900 5900

G 70 35 120 50 440 390 570 370 450 550 700 700 5000 4500 4000 4400

H 110 40 100 90 520 440 450 540 150 650 300 300 4200 4700 5600 4800

L 35 60 20 40 390 390 170 380 650 350 800 200 5300 4600 5600 5900

M 45 45 30 40 450 490 550 410 400 550 800 180 4900 4900 6400 4800

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2538 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Table 16 ndash Synthesis of results obtained by the alternative

and the reference methods (log CFUml)

Labs Level 1 Level 2 Level 2 bis Level 3

Reference method

Alternative method

Reference method

Alternative method

Reference method

Alternative method

Reference method

Alternative method

A 1602 1602 1845 1602 2681 2613 2322 2531 3000 2477 2602 2477 3690 3663 3690 3708

B 1602 1176 1602 1301 2544 2477 2556 2653 2544 2813 2903 2778 3613 3672 3708 3568

C 1699 1301 1301 1845 2643 2633 2681 2653 2477 2813 2477 2699 3663 3732 3663 3699

D 1602 1778 1602 1602 2690 2663 2778 2732 2398 2653 2778 3000 3690 3690 3643 3663

E 1699 1602 1699 1301 2690 2716 2724 2748 2875 2740 2602 2301 3732 3653 3613 3732

F 1778 1301 1778 1699 2568 2398 2477 2114 2477 2544 2778 2477 3643 3672 3690 3771

G 1845 1544 2079 1699 2643 2591 2756 2568 2653 2740 2845 2845 3699 3653 3602 3643

H 2041 1602 2000 1954 2716 2643 2653 2732 2176 2813 2477 2477 3623 3672 3748 3681

L 1544 1778 1301 1602 2591 2591 2230 2580 2813 2544 2903 2301 3724 3663 3748 3771

M 1653 1653 1477 1602 2653 2690 2740 2613 2602 2740 2903 2255 3690 3690 3806 3681

Level 2 results obtained taking account the 3 plates inoculated with the mother-suspension

Level 2 bis results obtained taking account the plates inoculated with 01 ml of the mother-suspension

Scrutiny of the measurement results for consistency

In order to identify the measurement results or laboratories that could be

inconsistent two graphical consistency techniques were realised Mandelrsquos

h-ank-statistics Mandel indicators h and k at 5 significance highlighted

some possible inconsistent data (See tables and graphics in Appendix 3 and

Appendix 4)

Mandelrsquos values Number of values observed over the threshold

Reference method Alternative method

h gt 1 Lab F Level 2 Lab F Level 2

h gt 5 Lab F Level 2

Lab B Levels 1 and 2

Lab A Level 2

Lab D Level 3

Lab F Level 2

Lab L Level 2

kgt 1 Lab F Level 3

k gt 5 Lab F Level 2 Lab C Level 2

Lab F Level 3

Lab L Levels 1 and 3

Lab M Level 1

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2638 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

The emphasized results appear (i) coherent with the targeted contamination

levels (ii) concordant between both methods

All the results were thus kept for the statistical analysis

Comparison of the trueness and precision characteristics of

the reference method and alternative methods

The statistical values are summarised hereafter

Level p

Reference method Alternative method

Repeatability ratio

Reproducibility ratio Median

Repeatability standard deviation

Reproducibility standard deviation

Median Repeatability

standard deviation

Reproducibility standard deviation

1 10 16519 02415 02425 16021 02045 02433 0847 1007

2 10 26551 00543 00659 26719 00914 01325 1682 2011

2 bis 10 26581 02607 02607 25951 01900 01953 0729 0749

3 10 18671 00378 00378 18671 00478 00611 1264 1617

Bias of the alternative method

In order to estimate the bias of the alternative method with regard to the reference method for every

level Dij and t are calculated according to the following equations

RefAlt ijYijYDij

Diffp

Dijit

)2(

)(median

If the t value is superior to 2 the alternative method is significantly biased

with regard to the reference method

The values are given in table 17

Table 17 ndash t (d) values obtained by level

Level Bias t (d) Conclusion

1 0068 114 Non significant bias

2 0021 082 Non significant bias

2 bis - 0037 035 Non significant bias

3 - 0037 043 Non significant bias

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2738 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

The statistical test concludes to a non significant bias for all the inoculation

levels The bias varies from ndash 0037 to 0068 log CFUml

Comparison of the repeatability standard deviations

If the ratio Srj Alt Srj Ref of the repeatability standard deviations of the

alternative method and the reference method is larger than 2 the precision

under repeatability conditions of the alternative method is considered to be

lower than that of the reference method If this ratio is smaller than 05 the

precision under repeatability conditions of the alternative method is

considered to be greater than that of the reference method

The ratio values are given in table 18

Table 18

Level

Repeatability standard deviation

Reference method Alternative method Alternative method

reference method ratio

1 0242 0205 0847

2 0054 0091 1682

2 bis 0261 0190 0729

3 0038 0048 1264

Level Repeatability limit

Reference method Alternative method

1 0676 0573

2 0152 0256

2 bis 0730 0532

3 0106 0134

The ratios of the repeatability standard deviations are below 2 in all cases

The precision under repeatability conditions of the alternative method is

equivalent to that of the reference method

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2838 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Comparison of the reproducibility standard deviations

If the ratio Srj Alt Srj Ref of the reproducibility standard deviations of the alternative method and the

reference method is larger than 2 the precision under reproducibility conditions of the alternative

method is considered to be lower than that of the reference method If this ratio is smaller than 05 the

precision under reproducibility conditions of the alternative method is considered to be greater than

that of the reference method

The ratio values are given in table 19

Table 19

Level

Reproducibility standard deviation

Reference method Alternative method Alternative method

reference method ratio

1 0242 0243 1007

2 0066 0133 2011

2 bis 0261 0195 0749

3 0038 0061 1617

Level Reproducibility limit

Reference method Alternative method

1 0676 0681

2 0185 0371

2 bis 0730 0547

3 0106 0171

The ratios of the reproducibility standard deviations are below or close to 2

The precision under reproducibility conditions of the alternative method is

equivalent to that of the reference method

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2938 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

214 Conclusion

Method comparison study

The Listeria PrecisTM enumeration method shows satisfying linearity and

relative accuracy

The alternative method shows satisfying inclusivity and exclusivity results

Two Listeria monocytogenes strains gave negative results with the

alternative method These strains grew weakly on the reference method

media (OampA Palcam and Oxford)

The limits of repeatability of the alternative method are similar to those of the

reference method

The bias between both methods are low between - 0133 Log CFUg and

- 0008 Log CFUg

The Listeria PrecisTM enumeration method offers the saving for confirmatory

tests

Positive results are obtained with the alternative method in 2-3 days (with

OBIS Mono test) or in 4-7 days (by the classical tests described in the

standardized method) against 4-7 days for the reference method

Inter-laboratory study

The bias between the reference and the alternative methods shows satisfying

and correct values

- level 1 (100 - 1 000 CFUg) bias = + 007 log CFUg

- level 2 (1 000 - 10 000 CFUg) bias = 002 log CFUg

- level 2 bis (1 000 - 10 000 CFUg) bias = - 004 log CFUg

- level 3 (10 000 - 100 000 CFUg) bias = - 004 log CFUg

The repeatability and the reproducibility of the alternative method look similar

to those of the reference method

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3038 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

22 Extension study (realised by ASEPT 2007)

An extension study was performed in 2007 for a new confirmatory test OBIS

MONO

150 Listeria monocytogenes strains and 100 non-target strains were isolated

onto TSAYE and BrillianceTM Listeria Agar plates All the Listeria

monocytogenes and the non-target strains gave the expected results

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3138 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Appendix 1 ndash Flow diagram of the Listeria PrecisTM method

x g sample + 9 x ml buffered peptone water

Revivification step for 1 h plusmn 5 min at 20degC plusmn 1degC

Streaking 1 ml onto 3 Brilliance Listeria Agar plates (mother suspension)

or 01 ml onto 1 Brilliance Listeria Agar plates (mother suspension and decimal dilutions)

Incubation for 48 h plusmn 3 h at 37degC plusmn 1degC

Confirmatory tests on at least one typical colony

- by the tests described in the ISO method

- by applying the OBIS mono test on an isolated colony

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3238 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Appendix 2 ndash ISO 11290-2 (1998) amp ISO 11290-2A1 (2004) Horizontal

method for the detection and enumeration of Listeria monocytogenes -

Part 2 enumeration of Listeria monocytogenes in foods

10 g cheese + 90 ml buffered peptone water or Half Fraser Broth (without supplements) (110 dilution)

Stomach 1 min 30 seconds

Keep at 20degC plusmn 2degC for 1 h plusmn 5 min

Decimal dilutions in 9 ml peptone salt tubes

Dilution 110

Dilution 1100

Dilution 11000

1 ml on 3 Chromogenic Listeria medium (ISO) plates (x 2) (-1)

01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-2)

01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-3)

01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-4)

Incubation at 37degC plusmn 1degC

24 h - 48 h

Numerate typical colonies at 24 h

(bluegreen surrounded by an opaque halo)

Numerate at 48 h

Streak typical colonies on TSYEA

(5 coloniesplate 2 successive dilutions)

Confirmation tests

Catalase

Gram stain

For confirmation of L monocytogenes

Haemolysis

Carbohydrate (rhamnose and xylose) utilisation

CAMP Test

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3338 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Appendix 3 ndash Mandelrsquos graphics

Laboratoire h Ref

Niveau 1 h Ref

Niveau 2 h Ref

Niveau 3 h Alt

Niveau 1 h Alt

Niveau 2 h Alt

Niveau 3 h5 h1 h5 h1

A -050 -015 083 062 -212 -039 198 299 -198 -299

B -261 -270 021 -077 -058 173 198 299 -198 -299

C -151 -031 -014 -015 -004 -005 198 299 -198 -299

D 038 040 -137 000 072 207 198 299 -198 -299

E -001 090 155 -052 056 -101 198 299 -198 -299

F -112 -321 -153 070 -325 023 198 299 -198 -299

G 042 -071 040 147 -009 176 198 299 -198 -299

H 169 046 -169 192 018 -083 198 299 -198 -299

L 009 -120 021 -077 -231 005 198 299 -198 -299

M 001 031 014 -032 004 -011 198 299 -198 -299

-300

-200

-100

000

100

200

300

A B C D E F G H L M

Vale

urs

h d

e M

an

del(

rob

uste

s)

Laboratoires

Meacutethode de reacutefeacuterence analyse robuste

h Ref Niveau 1

h Ref Niveau 2

h Ref Niveau 3

h 5=198

h 1=299

-300

-200

-100

000

100

200

300

A B C D E F G H L M

Vale

urs

de h

de M

an

del(

rob

uste

s)

Laboratoires

Meacutethode alternative analyse robuste

h Alt Niveau 1

h Alt Niveau 2

h Alt Niveau 2

h 1=299

h 5=198

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3438 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Laboratoire k Ref

Niveau 1 k Ref

Niveau 2 k Ref

Niveau 3 k Alt

Niveau 1 k Alt

Niveau 2 k Alt

Niveau 3 k1 k5

A 000 089 142 084 162 046 259 181

B 125 087 073 104 075 046 259 181

C 117 013 091 188 022 083 259 181

D 052 036 069 000 035 083 259 181

E 028 034 037 138 019 112 259 181

F 140 222 018 027 281 112 259 181

G 088 068 024 131 145 000 259 181

H 129 094 173 016 061 000 259 181

L 069 000 073 104 270 224 259 181

M 000 048 038 043 099 241 259 181

000

050

100

150

200

250

300

350

400

A B C D E F G H L M

Vale

urs

k d

e M

an

del(

rob

uste

s)

Laboratoires

Meacutethode de reacutefeacuterence analyse robuste

k Ref Niveau 1k Ref Niveau 2k Ref Niveau 3

k 1=259

k 5=181

000

050

100

150

200

250

300

A B C D E F G H L M

Vale

urs

k d

e M

an

del

(ro

bu

ste

s)

Laboratoires

Meacutethode alternative analyse robuste

k Alt Niveau 3

k Alt Niveau 1

k Alt Niveau 2

k 1=259

k 5=181

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3538 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Appendix 4 ndash Statistical results

Level 1

Nombre de laboratoires (p) 10

A 1602 1602 1602 000 0000 0000 -0495 0000 A 1845 1602 1724 017 0122 -0122 0621 0840 0122

B 1602 1176 1389 030 0213 -0213 -2613 1247 B 1602 1301 1452 021 0151 -0151 -0769 1041 0062

C 1699 1301 1500 028 0199 -0199 -1510 1165 C 1301 1845 1573 038 -0272 0272 -0148 1881 0073

D 1602 1778 1690 012 -0088 0088 0380 0516 D 1602 1602 1602 000 0000 0000 0000 0000 -0088

E 1699 1602 1651 007 0048 -0048 -0013 0284 E 1699 1301 1500 028 0199 -0199 -0522 1376 -0151

F 1778 1301 1540 034 0239 -0239 -1116 1397 F 1778 1699 1739 006 0040 -0040 0698 0274 0199

G 1845 1544 1695 021 0151 -0151 0425 0881 G 2079 1699 1889 027 0190 -0190 1467 1315 0194

H 2041 1602 1822 031 0220 -0220 1689 1286 H 2000 1954 1977 003 0023 -0023 1917 0158 0155

L 1544 1778 1661 017 -0117 0117 0092 0685 L 1301 1602 1452 021 -0151 0151 -0769 1041 -0210

M 1653 1653 1653 000 0000 0000 0013 0000 M 1477 1602 1540 009 -0062 0062 -0319 0432 -0114

m 165186 m 160206 mediane D 00678

Cn(2p) 1867 Cn(2p) 1867 Cp 16101

Cn(p) 16101 Cn(p) 16101 Qn(D) 0092926

n=2p n 20 n=2p n 20 Qdiff 01496169

f 11 f 11 t 114

l 55 l 55

n=p n 10 n=p n 10

f 6 f 6

l 15 l 15

Qn(2p) 0091465 Qn(2p) 007745

Qn(p) 0062469 Qn(p) 0121519

Qintra 017078 Qintra 014461

Qinter 010058 Qinter 019565

Sr(eacutecart-type de reacutepeacutetabiliteacute) 02415 Sr 02045

RSDr(coefficient de variation de la reacutepeacutetabiliteacute)1462 RSDr 1277

r(repeatability limit)) 0676 r 0573

SL(eacutecart-type interlaboratoire) -001905 SLlt0 SL 001737

SR(eacutecart-type de reproductibiliteacute) 02415 SR 02433

RSDR(coefficient de variation de la reproductibiliteacute)0146 RSDR 0152

R(limite de reproductibiliteacute)) 06762 R(limite de reproductibiliteacute))068123

h k h MoyenneLaboratoires

Niveau 1

si LaboratoiresDeviationMoyenne Dk DeviationsiAlt

Niveau 1

Ref

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3638 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Level 2

Nombre de laboratoires (p) 10

A 2681 2613 2647 005 0034 -0034 -0151 0891 A 2322 2531 2427 015 -0105 0105 -2119 1620 -0220

B 2544 2477 2511 005 0033 -0033 -2699 0872 B 2556 2653 2605 007 -0048 0048 -0581 0750 0094

C 2643 2633 2638 001 0005 -0005 -0311 0130 C 2681 2653 2667 002 0014 -0014 -0040 0217 0029

D 2690 2663 2676 002 0014 -0014 0400 0357 D 2778 2732 2755 003 0023 -0023 0721 0354 0079

E 2690 2716 2703 002 -0013 0013 0897 0336 E 2724 2748 2736 002 -0012 0012 0556 0185 0033

F 2568 2398 2483 012 0085 -0085 -3214 2217 F 2477 2114 2296 026 0182 -0182 -3255 2811 -0188

G 2643 2591 2617 004 0026 -0026 -0707 0682 G 2756 2568 2662 013 0094 -0094 -0085 1453 0045

H 2716 2643 2680 005 0036 -0036 0460 0945 H 2653 2732 2693 006 -0040 0040 0181 0613 0013

L 2591 2591 2591 000 0000 0000 -1196 0000 L 2230 2580 2405 025 -0175 0175 -2307 2704 -0186

M 2653 2690 2672 003 -0018 0018 0311 0481 M 2740 2613 2677 009 0064 -0064 0040 0987 0005

m 2655082 m 26719001 mediane D 00209

Cn(2p) 1867 Cn(2p) 1867 Cp 16101

Cn(p) 16101 Cn(p) 16101 Qn(D) 0039911

n=2p n 20 n=2p n 20 Qdiff 00642591

f 11 f 11 t 082

l 55 l 55

n=p n 10 n=p n 10

f 6 f 6

l 15 l 15

Qn(2p) 0020570 Qn(2p) 00345985

Qn(p) 00332437 Qn(p) 00718158

Qintra 003841 Qintra 006460

Qinter 005352 Qinter 011563

Sr(eacutecart-type de reacutepeacutetabiliteacute) 00543 Sr 00914

RSDr(coefficient de variation de la reacutepeacutetabiliteacute)205 RSDr 342

r(repeatability limit)) 0152 r 0256

SL(eacutecart-type interlaboratoire) 000139 SL 000920

SR(eacutecart-type de reproductibiliteacute) 00659 SR 01325

RSDR(coefficient de variation de la reproductibiliteacute)0025 RSDR 0050

R(limite de reproductibiliteacute)) 018446 R(limite de reproductibiliteacute))03708618

Laboratoires

Niveau 2

Moyenne si Laboratoires

Niveau 2

DRef Alt Moyenne si Deviation h Deviation h k k

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3738 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Level 2 bis

Nombre de laboratoires (p) 10

A 3000 2477 2739 037 0261 -0261 0832 1418 A 2602 2477 2540 009 0062 -0062 -0391 0465 -0199

B 2544 2813 2678 019 -0134 0134 0211 0729 B 2903 2778 2841 009 0062 -0062 1732 0465 0162

C 2477 2813 2645 024 -0168 0168 -0135 0911 C 2477 2699 2588 016 -0111 0111 -0049 0826 -0057

D 2398 2653 2526 018 -0128 0128 -1370 0692 D 2778 3000 2889 016 -0111 0111 2074 0826 0363

E 2875 2740 2808 010 0067 -0067 1547 0365 E 2602 2301 2452 021 0151 -0151 -1012 1120 -0356

F 2477 2544 2511 005 -0033 0033 -1525 0182 F 2778 2477 2628 021 0151 -0151 0230 1120 0117

G 2653 2740 2697 006 -0044 0044 0400 0236 G 2845 2845 2845 000 0000 0000 1764 0000 0148

H 2176 2813 2495 045 -0318 0318 -1692 1727 H 2477 2477 2477 000 0000 0000 -0832 0000 -0017

L 2813 2544 2678 019 0134 -0134 0211 0729 L 2903 2301 2602 043 0301 -0301 0049 2241 -0076

M 2602 2740 2671 010 -0069 0069 0135 0375 M 2903 2255 2579 046 0324 -0324 -0112 2411 -0092

m 2658114 m 259505 mediane D -00372

Cn(2p) 1867 Cn(2p) 1867 Cp 16101

Cn(p) 16101 Cn(p) 16101 Qn(D) 01656915

n=2p n 20 n=2p n 20 Qdiff 02667753

f 11 f 11 t 035

l 55 l 55

n=p n 10 n=p n 10

f 6 f 6

l 15 l 15

Qn(2p) 0098745 Qn(2p) 007195

Qn(p) 006007 Qn(p) 008805

Qintra 018437 Qintra 013435

Qinter 009672 Qinter 014176

Sr(eacutecart-type de reacutepeacutetabiliteacute) 02607 Sr 01900

RSDr(coefficient de variation de la reacutepeacutetabiliteacute)981 RSDr 732

r(repeatability limit)) 0730 r 0532

SL(eacutecart-type interlaboratoire) -002464 SLlt0 SL 000205

SR(eacutecart-type de reproductibiliteacute) 02607 SR 01953

RSDR(coefficient de variation de la reproductibiliteacute)0098 RSDR 0075

R(limite de reproductibiliteacute)) 072996 R(limite de reproductibiliteacute))054686

Laboratoires

Niveau 2 bis

Moyenne si Laboratoires

Niveau 2 bis

DRef Alt Moyenne si Deviation h Deviation h k k

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3838 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Level 3

Nombre de laboratoires (p) 10

A 3690 3663 3676 002 0014 -0014 -0311 0513 A 3690 3708 3699 001 -0009 0009 0354 0257 0022

B 3613 3672 3642 004 -0030 0030 -1851 1109 B 3708 3568 3638 010 0070 -0070 -0844 2061 -0005

C 3663 3732 3698 005 -0035 0035 0645 1302 C 3663 3699 3681 003 -0018 0018 0000 0536 -0017

D 3690 3690 3690 000 0000 0000 0311 0000 D 3643 3663 3653 001 -0010 0010 -0545 0286 -0037

E 3732 3653 3693 006 0040 -0040 0429 1480 E 3613 3732 3673 008 -0060 0060 -0162 1769 -0020

F 3643 3672 3658 002 -0014 0014 -1157 0536 F 3690 3771 3731 006 -0040 0040 0975 1193 0073

G 3699 3653 3676 003 0023 -0023 -0328 0856 G 3602 3643 3623 003 -0021 0021 -1141 0612 -0053

H 3623 3672 3648 003 -0024 0024 -1615 0913 H 3748 3681 3715 005 0033 -0033 0664 0990 0067

L 3724 3663 3694 004 0031 -0031 0461 1150 L 3748 3771 3760 002 -0011 0011 1544 0335 0066

M 3690 3690 3690 000 0000 0000 0311 0000 M 3806 3681 3744 009 0062 -0062 1234 1848 0054

m 368334 m 368086 mediane D 00089

Cn(2p) 1867 Cn(2p) 1867 Cp 16101

Cn(p) 16101 Cn(p) 16101 Qn(D) 00325359

n=2p n 20 n=2p n 20 Qdiff 00523852

f 11 f 11 t 043

l 55 l 55

n=p n 10 n=p n 10

f 6 f 6

l 15 l 15

Qn(2p) 0014323 Qn(2p) 001811

Qn(p) 0013719 Qn(p) 003164

Qintra 002674 Qintra 003381

Qinter 002209 Qinter 005094

Sr(eacutecart-type de reacutepeacutetabiliteacute) 00378 Sr 00478

RSDr(coefficient de variation de la reacutepeacutetabiliteacute)103 RSDr 130

r(repeatability limit)) 0106 r 0134

SL(eacutecart-type interlaboratoire) -000023 SLlt0 SL 000145

SR(eacutecart-type de reproductibiliteacute) 00378 SR 00611

RSDR(coefficient de variation de la reproductibiliteacute)0010 RSDR 0017

R(limite de reproductibiliteacute)) 0105894 R(limite de reproductibiliteacute))017119

Ref Alt DLaboratoires

Niveau 3

Moyenne si Deviation h k Laboratoires

Niveau 3

Moyenne si Deviation h k

Page 25: ÞÀ¿¸7 ú Y5zñLjÜ< /u +æü t¬Á ... - NF Validation · NF VALIDATION Validation of alternative analytical methods Application in food microbiology ... 500 – 1 000 1 000 –

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2538 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Table 16 ndash Synthesis of results obtained by the alternative

and the reference methods (log CFUml)

Labs Level 1 Level 2 Level 2 bis Level 3

Reference method

Alternative method

Reference method

Alternative method

Reference method

Alternative method

Reference method

Alternative method

A 1602 1602 1845 1602 2681 2613 2322 2531 3000 2477 2602 2477 3690 3663 3690 3708

B 1602 1176 1602 1301 2544 2477 2556 2653 2544 2813 2903 2778 3613 3672 3708 3568

C 1699 1301 1301 1845 2643 2633 2681 2653 2477 2813 2477 2699 3663 3732 3663 3699

D 1602 1778 1602 1602 2690 2663 2778 2732 2398 2653 2778 3000 3690 3690 3643 3663

E 1699 1602 1699 1301 2690 2716 2724 2748 2875 2740 2602 2301 3732 3653 3613 3732

F 1778 1301 1778 1699 2568 2398 2477 2114 2477 2544 2778 2477 3643 3672 3690 3771

G 1845 1544 2079 1699 2643 2591 2756 2568 2653 2740 2845 2845 3699 3653 3602 3643

H 2041 1602 2000 1954 2716 2643 2653 2732 2176 2813 2477 2477 3623 3672 3748 3681

L 1544 1778 1301 1602 2591 2591 2230 2580 2813 2544 2903 2301 3724 3663 3748 3771

M 1653 1653 1477 1602 2653 2690 2740 2613 2602 2740 2903 2255 3690 3690 3806 3681

Level 2 results obtained taking account the 3 plates inoculated with the mother-suspension

Level 2 bis results obtained taking account the plates inoculated with 01 ml of the mother-suspension

Scrutiny of the measurement results for consistency

In order to identify the measurement results or laboratories that could be

inconsistent two graphical consistency techniques were realised Mandelrsquos

h-ank-statistics Mandel indicators h and k at 5 significance highlighted

some possible inconsistent data (See tables and graphics in Appendix 3 and

Appendix 4)

Mandelrsquos values Number of values observed over the threshold

Reference method Alternative method

h gt 1 Lab F Level 2 Lab F Level 2

h gt 5 Lab F Level 2

Lab B Levels 1 and 2

Lab A Level 2

Lab D Level 3

Lab F Level 2

Lab L Level 2

kgt 1 Lab F Level 3

k gt 5 Lab F Level 2 Lab C Level 2

Lab F Level 3

Lab L Levels 1 and 3

Lab M Level 1

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2638 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

The emphasized results appear (i) coherent with the targeted contamination

levels (ii) concordant between both methods

All the results were thus kept for the statistical analysis

Comparison of the trueness and precision characteristics of

the reference method and alternative methods

The statistical values are summarised hereafter

Level p

Reference method Alternative method

Repeatability ratio

Reproducibility ratio Median

Repeatability standard deviation

Reproducibility standard deviation

Median Repeatability

standard deviation

Reproducibility standard deviation

1 10 16519 02415 02425 16021 02045 02433 0847 1007

2 10 26551 00543 00659 26719 00914 01325 1682 2011

2 bis 10 26581 02607 02607 25951 01900 01953 0729 0749

3 10 18671 00378 00378 18671 00478 00611 1264 1617

Bias of the alternative method

In order to estimate the bias of the alternative method with regard to the reference method for every

level Dij and t are calculated according to the following equations

RefAlt ijYijYDij

Diffp

Dijit

)2(

)(median

If the t value is superior to 2 the alternative method is significantly biased

with regard to the reference method

The values are given in table 17

Table 17 ndash t (d) values obtained by level

Level Bias t (d) Conclusion

1 0068 114 Non significant bias

2 0021 082 Non significant bias

2 bis - 0037 035 Non significant bias

3 - 0037 043 Non significant bias

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2738 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

The statistical test concludes to a non significant bias for all the inoculation

levels The bias varies from ndash 0037 to 0068 log CFUml

Comparison of the repeatability standard deviations

If the ratio Srj Alt Srj Ref of the repeatability standard deviations of the

alternative method and the reference method is larger than 2 the precision

under repeatability conditions of the alternative method is considered to be

lower than that of the reference method If this ratio is smaller than 05 the

precision under repeatability conditions of the alternative method is

considered to be greater than that of the reference method

The ratio values are given in table 18

Table 18

Level

Repeatability standard deviation

Reference method Alternative method Alternative method

reference method ratio

1 0242 0205 0847

2 0054 0091 1682

2 bis 0261 0190 0729

3 0038 0048 1264

Level Repeatability limit

Reference method Alternative method

1 0676 0573

2 0152 0256

2 bis 0730 0532

3 0106 0134

The ratios of the repeatability standard deviations are below 2 in all cases

The precision under repeatability conditions of the alternative method is

equivalent to that of the reference method

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2838 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Comparison of the reproducibility standard deviations

If the ratio Srj Alt Srj Ref of the reproducibility standard deviations of the alternative method and the

reference method is larger than 2 the precision under reproducibility conditions of the alternative

method is considered to be lower than that of the reference method If this ratio is smaller than 05 the

precision under reproducibility conditions of the alternative method is considered to be greater than

that of the reference method

The ratio values are given in table 19

Table 19

Level

Reproducibility standard deviation

Reference method Alternative method Alternative method

reference method ratio

1 0242 0243 1007

2 0066 0133 2011

2 bis 0261 0195 0749

3 0038 0061 1617

Level Reproducibility limit

Reference method Alternative method

1 0676 0681

2 0185 0371

2 bis 0730 0547

3 0106 0171

The ratios of the reproducibility standard deviations are below or close to 2

The precision under reproducibility conditions of the alternative method is

equivalent to that of the reference method

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2938 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

214 Conclusion

Method comparison study

The Listeria PrecisTM enumeration method shows satisfying linearity and

relative accuracy

The alternative method shows satisfying inclusivity and exclusivity results

Two Listeria monocytogenes strains gave negative results with the

alternative method These strains grew weakly on the reference method

media (OampA Palcam and Oxford)

The limits of repeatability of the alternative method are similar to those of the

reference method

The bias between both methods are low between - 0133 Log CFUg and

- 0008 Log CFUg

The Listeria PrecisTM enumeration method offers the saving for confirmatory

tests

Positive results are obtained with the alternative method in 2-3 days (with

OBIS Mono test) or in 4-7 days (by the classical tests described in the

standardized method) against 4-7 days for the reference method

Inter-laboratory study

The bias between the reference and the alternative methods shows satisfying

and correct values

- level 1 (100 - 1 000 CFUg) bias = + 007 log CFUg

- level 2 (1 000 - 10 000 CFUg) bias = 002 log CFUg

- level 2 bis (1 000 - 10 000 CFUg) bias = - 004 log CFUg

- level 3 (10 000 - 100 000 CFUg) bias = - 004 log CFUg

The repeatability and the reproducibility of the alternative method look similar

to those of the reference method

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3038 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

22 Extension study (realised by ASEPT 2007)

An extension study was performed in 2007 for a new confirmatory test OBIS

MONO

150 Listeria monocytogenes strains and 100 non-target strains were isolated

onto TSAYE and BrillianceTM Listeria Agar plates All the Listeria

monocytogenes and the non-target strains gave the expected results

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3138 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Appendix 1 ndash Flow diagram of the Listeria PrecisTM method

x g sample + 9 x ml buffered peptone water

Revivification step for 1 h plusmn 5 min at 20degC plusmn 1degC

Streaking 1 ml onto 3 Brilliance Listeria Agar plates (mother suspension)

or 01 ml onto 1 Brilliance Listeria Agar plates (mother suspension and decimal dilutions)

Incubation for 48 h plusmn 3 h at 37degC plusmn 1degC

Confirmatory tests on at least one typical colony

- by the tests described in the ISO method

- by applying the OBIS mono test on an isolated colony

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3238 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Appendix 2 ndash ISO 11290-2 (1998) amp ISO 11290-2A1 (2004) Horizontal

method for the detection and enumeration of Listeria monocytogenes -

Part 2 enumeration of Listeria monocytogenes in foods

10 g cheese + 90 ml buffered peptone water or Half Fraser Broth (without supplements) (110 dilution)

Stomach 1 min 30 seconds

Keep at 20degC plusmn 2degC for 1 h plusmn 5 min

Decimal dilutions in 9 ml peptone salt tubes

Dilution 110

Dilution 1100

Dilution 11000

1 ml on 3 Chromogenic Listeria medium (ISO) plates (x 2) (-1)

01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-2)

01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-3)

01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-4)

Incubation at 37degC plusmn 1degC

24 h - 48 h

Numerate typical colonies at 24 h

(bluegreen surrounded by an opaque halo)

Numerate at 48 h

Streak typical colonies on TSYEA

(5 coloniesplate 2 successive dilutions)

Confirmation tests

Catalase

Gram stain

For confirmation of L monocytogenes

Haemolysis

Carbohydrate (rhamnose and xylose) utilisation

CAMP Test

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3338 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Appendix 3 ndash Mandelrsquos graphics

Laboratoire h Ref

Niveau 1 h Ref

Niveau 2 h Ref

Niveau 3 h Alt

Niveau 1 h Alt

Niveau 2 h Alt

Niveau 3 h5 h1 h5 h1

A -050 -015 083 062 -212 -039 198 299 -198 -299

B -261 -270 021 -077 -058 173 198 299 -198 -299

C -151 -031 -014 -015 -004 -005 198 299 -198 -299

D 038 040 -137 000 072 207 198 299 -198 -299

E -001 090 155 -052 056 -101 198 299 -198 -299

F -112 -321 -153 070 -325 023 198 299 -198 -299

G 042 -071 040 147 -009 176 198 299 -198 -299

H 169 046 -169 192 018 -083 198 299 -198 -299

L 009 -120 021 -077 -231 005 198 299 -198 -299

M 001 031 014 -032 004 -011 198 299 -198 -299

-300

-200

-100

000

100

200

300

A B C D E F G H L M

Vale

urs

h d

e M

an

del(

rob

uste

s)

Laboratoires

Meacutethode de reacutefeacuterence analyse robuste

h Ref Niveau 1

h Ref Niveau 2

h Ref Niveau 3

h 5=198

h 1=299

-300

-200

-100

000

100

200

300

A B C D E F G H L M

Vale

urs

de h

de M

an

del(

rob

uste

s)

Laboratoires

Meacutethode alternative analyse robuste

h Alt Niveau 1

h Alt Niveau 2

h Alt Niveau 2

h 1=299

h 5=198

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3438 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Laboratoire k Ref

Niveau 1 k Ref

Niveau 2 k Ref

Niveau 3 k Alt

Niveau 1 k Alt

Niveau 2 k Alt

Niveau 3 k1 k5

A 000 089 142 084 162 046 259 181

B 125 087 073 104 075 046 259 181

C 117 013 091 188 022 083 259 181

D 052 036 069 000 035 083 259 181

E 028 034 037 138 019 112 259 181

F 140 222 018 027 281 112 259 181

G 088 068 024 131 145 000 259 181

H 129 094 173 016 061 000 259 181

L 069 000 073 104 270 224 259 181

M 000 048 038 043 099 241 259 181

000

050

100

150

200

250

300

350

400

A B C D E F G H L M

Vale

urs

k d

e M

an

del(

rob

uste

s)

Laboratoires

Meacutethode de reacutefeacuterence analyse robuste

k Ref Niveau 1k Ref Niveau 2k Ref Niveau 3

k 1=259

k 5=181

000

050

100

150

200

250

300

A B C D E F G H L M

Vale

urs

k d

e M

an

del

(ro

bu

ste

s)

Laboratoires

Meacutethode alternative analyse robuste

k Alt Niveau 3

k Alt Niveau 1

k Alt Niveau 2

k 1=259

k 5=181

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3538 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Appendix 4 ndash Statistical results

Level 1

Nombre de laboratoires (p) 10

A 1602 1602 1602 000 0000 0000 -0495 0000 A 1845 1602 1724 017 0122 -0122 0621 0840 0122

B 1602 1176 1389 030 0213 -0213 -2613 1247 B 1602 1301 1452 021 0151 -0151 -0769 1041 0062

C 1699 1301 1500 028 0199 -0199 -1510 1165 C 1301 1845 1573 038 -0272 0272 -0148 1881 0073

D 1602 1778 1690 012 -0088 0088 0380 0516 D 1602 1602 1602 000 0000 0000 0000 0000 -0088

E 1699 1602 1651 007 0048 -0048 -0013 0284 E 1699 1301 1500 028 0199 -0199 -0522 1376 -0151

F 1778 1301 1540 034 0239 -0239 -1116 1397 F 1778 1699 1739 006 0040 -0040 0698 0274 0199

G 1845 1544 1695 021 0151 -0151 0425 0881 G 2079 1699 1889 027 0190 -0190 1467 1315 0194

H 2041 1602 1822 031 0220 -0220 1689 1286 H 2000 1954 1977 003 0023 -0023 1917 0158 0155

L 1544 1778 1661 017 -0117 0117 0092 0685 L 1301 1602 1452 021 -0151 0151 -0769 1041 -0210

M 1653 1653 1653 000 0000 0000 0013 0000 M 1477 1602 1540 009 -0062 0062 -0319 0432 -0114

m 165186 m 160206 mediane D 00678

Cn(2p) 1867 Cn(2p) 1867 Cp 16101

Cn(p) 16101 Cn(p) 16101 Qn(D) 0092926

n=2p n 20 n=2p n 20 Qdiff 01496169

f 11 f 11 t 114

l 55 l 55

n=p n 10 n=p n 10

f 6 f 6

l 15 l 15

Qn(2p) 0091465 Qn(2p) 007745

Qn(p) 0062469 Qn(p) 0121519

Qintra 017078 Qintra 014461

Qinter 010058 Qinter 019565

Sr(eacutecart-type de reacutepeacutetabiliteacute) 02415 Sr 02045

RSDr(coefficient de variation de la reacutepeacutetabiliteacute)1462 RSDr 1277

r(repeatability limit)) 0676 r 0573

SL(eacutecart-type interlaboratoire) -001905 SLlt0 SL 001737

SR(eacutecart-type de reproductibiliteacute) 02415 SR 02433

RSDR(coefficient de variation de la reproductibiliteacute)0146 RSDR 0152

R(limite de reproductibiliteacute)) 06762 R(limite de reproductibiliteacute))068123

h k h MoyenneLaboratoires

Niveau 1

si LaboratoiresDeviationMoyenne Dk DeviationsiAlt

Niveau 1

Ref

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3638 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Level 2

Nombre de laboratoires (p) 10

A 2681 2613 2647 005 0034 -0034 -0151 0891 A 2322 2531 2427 015 -0105 0105 -2119 1620 -0220

B 2544 2477 2511 005 0033 -0033 -2699 0872 B 2556 2653 2605 007 -0048 0048 -0581 0750 0094

C 2643 2633 2638 001 0005 -0005 -0311 0130 C 2681 2653 2667 002 0014 -0014 -0040 0217 0029

D 2690 2663 2676 002 0014 -0014 0400 0357 D 2778 2732 2755 003 0023 -0023 0721 0354 0079

E 2690 2716 2703 002 -0013 0013 0897 0336 E 2724 2748 2736 002 -0012 0012 0556 0185 0033

F 2568 2398 2483 012 0085 -0085 -3214 2217 F 2477 2114 2296 026 0182 -0182 -3255 2811 -0188

G 2643 2591 2617 004 0026 -0026 -0707 0682 G 2756 2568 2662 013 0094 -0094 -0085 1453 0045

H 2716 2643 2680 005 0036 -0036 0460 0945 H 2653 2732 2693 006 -0040 0040 0181 0613 0013

L 2591 2591 2591 000 0000 0000 -1196 0000 L 2230 2580 2405 025 -0175 0175 -2307 2704 -0186

M 2653 2690 2672 003 -0018 0018 0311 0481 M 2740 2613 2677 009 0064 -0064 0040 0987 0005

m 2655082 m 26719001 mediane D 00209

Cn(2p) 1867 Cn(2p) 1867 Cp 16101

Cn(p) 16101 Cn(p) 16101 Qn(D) 0039911

n=2p n 20 n=2p n 20 Qdiff 00642591

f 11 f 11 t 082

l 55 l 55

n=p n 10 n=p n 10

f 6 f 6

l 15 l 15

Qn(2p) 0020570 Qn(2p) 00345985

Qn(p) 00332437 Qn(p) 00718158

Qintra 003841 Qintra 006460

Qinter 005352 Qinter 011563

Sr(eacutecart-type de reacutepeacutetabiliteacute) 00543 Sr 00914

RSDr(coefficient de variation de la reacutepeacutetabiliteacute)205 RSDr 342

r(repeatability limit)) 0152 r 0256

SL(eacutecart-type interlaboratoire) 000139 SL 000920

SR(eacutecart-type de reproductibiliteacute) 00659 SR 01325

RSDR(coefficient de variation de la reproductibiliteacute)0025 RSDR 0050

R(limite de reproductibiliteacute)) 018446 R(limite de reproductibiliteacute))03708618

Laboratoires

Niveau 2

Moyenne si Laboratoires

Niveau 2

DRef Alt Moyenne si Deviation h Deviation h k k

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3738 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Level 2 bis

Nombre de laboratoires (p) 10

A 3000 2477 2739 037 0261 -0261 0832 1418 A 2602 2477 2540 009 0062 -0062 -0391 0465 -0199

B 2544 2813 2678 019 -0134 0134 0211 0729 B 2903 2778 2841 009 0062 -0062 1732 0465 0162

C 2477 2813 2645 024 -0168 0168 -0135 0911 C 2477 2699 2588 016 -0111 0111 -0049 0826 -0057

D 2398 2653 2526 018 -0128 0128 -1370 0692 D 2778 3000 2889 016 -0111 0111 2074 0826 0363

E 2875 2740 2808 010 0067 -0067 1547 0365 E 2602 2301 2452 021 0151 -0151 -1012 1120 -0356

F 2477 2544 2511 005 -0033 0033 -1525 0182 F 2778 2477 2628 021 0151 -0151 0230 1120 0117

G 2653 2740 2697 006 -0044 0044 0400 0236 G 2845 2845 2845 000 0000 0000 1764 0000 0148

H 2176 2813 2495 045 -0318 0318 -1692 1727 H 2477 2477 2477 000 0000 0000 -0832 0000 -0017

L 2813 2544 2678 019 0134 -0134 0211 0729 L 2903 2301 2602 043 0301 -0301 0049 2241 -0076

M 2602 2740 2671 010 -0069 0069 0135 0375 M 2903 2255 2579 046 0324 -0324 -0112 2411 -0092

m 2658114 m 259505 mediane D -00372

Cn(2p) 1867 Cn(2p) 1867 Cp 16101

Cn(p) 16101 Cn(p) 16101 Qn(D) 01656915

n=2p n 20 n=2p n 20 Qdiff 02667753

f 11 f 11 t 035

l 55 l 55

n=p n 10 n=p n 10

f 6 f 6

l 15 l 15

Qn(2p) 0098745 Qn(2p) 007195

Qn(p) 006007 Qn(p) 008805

Qintra 018437 Qintra 013435

Qinter 009672 Qinter 014176

Sr(eacutecart-type de reacutepeacutetabiliteacute) 02607 Sr 01900

RSDr(coefficient de variation de la reacutepeacutetabiliteacute)981 RSDr 732

r(repeatability limit)) 0730 r 0532

SL(eacutecart-type interlaboratoire) -002464 SLlt0 SL 000205

SR(eacutecart-type de reproductibiliteacute) 02607 SR 01953

RSDR(coefficient de variation de la reproductibiliteacute)0098 RSDR 0075

R(limite de reproductibiliteacute)) 072996 R(limite de reproductibiliteacute))054686

Laboratoires

Niveau 2 bis

Moyenne si Laboratoires

Niveau 2 bis

DRef Alt Moyenne si Deviation h Deviation h k k

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3838 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Level 3

Nombre de laboratoires (p) 10

A 3690 3663 3676 002 0014 -0014 -0311 0513 A 3690 3708 3699 001 -0009 0009 0354 0257 0022

B 3613 3672 3642 004 -0030 0030 -1851 1109 B 3708 3568 3638 010 0070 -0070 -0844 2061 -0005

C 3663 3732 3698 005 -0035 0035 0645 1302 C 3663 3699 3681 003 -0018 0018 0000 0536 -0017

D 3690 3690 3690 000 0000 0000 0311 0000 D 3643 3663 3653 001 -0010 0010 -0545 0286 -0037

E 3732 3653 3693 006 0040 -0040 0429 1480 E 3613 3732 3673 008 -0060 0060 -0162 1769 -0020

F 3643 3672 3658 002 -0014 0014 -1157 0536 F 3690 3771 3731 006 -0040 0040 0975 1193 0073

G 3699 3653 3676 003 0023 -0023 -0328 0856 G 3602 3643 3623 003 -0021 0021 -1141 0612 -0053

H 3623 3672 3648 003 -0024 0024 -1615 0913 H 3748 3681 3715 005 0033 -0033 0664 0990 0067

L 3724 3663 3694 004 0031 -0031 0461 1150 L 3748 3771 3760 002 -0011 0011 1544 0335 0066

M 3690 3690 3690 000 0000 0000 0311 0000 M 3806 3681 3744 009 0062 -0062 1234 1848 0054

m 368334 m 368086 mediane D 00089

Cn(2p) 1867 Cn(2p) 1867 Cp 16101

Cn(p) 16101 Cn(p) 16101 Qn(D) 00325359

n=2p n 20 n=2p n 20 Qdiff 00523852

f 11 f 11 t 043

l 55 l 55

n=p n 10 n=p n 10

f 6 f 6

l 15 l 15

Qn(2p) 0014323 Qn(2p) 001811

Qn(p) 0013719 Qn(p) 003164

Qintra 002674 Qintra 003381

Qinter 002209 Qinter 005094

Sr(eacutecart-type de reacutepeacutetabiliteacute) 00378 Sr 00478

RSDr(coefficient de variation de la reacutepeacutetabiliteacute)103 RSDr 130

r(repeatability limit)) 0106 r 0134

SL(eacutecart-type interlaboratoire) -000023 SLlt0 SL 000145

SR(eacutecart-type de reproductibiliteacute) 00378 SR 00611

RSDR(coefficient de variation de la reproductibiliteacute)0010 RSDR 0017

R(limite de reproductibiliteacute)) 0105894 R(limite de reproductibiliteacute))017119

Ref Alt DLaboratoires

Niveau 3

Moyenne si Deviation h k Laboratoires

Niveau 3

Moyenne si Deviation h k

Page 26: ÞÀ¿¸7 ú Y5zñLjÜ< /u +æü t¬Á ... - NF Validation · NF VALIDATION Validation of alternative analytical methods Application in food microbiology ... 500 – 1 000 1 000 –

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2638 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

The emphasized results appear (i) coherent with the targeted contamination

levels (ii) concordant between both methods

All the results were thus kept for the statistical analysis

Comparison of the trueness and precision characteristics of

the reference method and alternative methods

The statistical values are summarised hereafter

Level p

Reference method Alternative method

Repeatability ratio

Reproducibility ratio Median

Repeatability standard deviation

Reproducibility standard deviation

Median Repeatability

standard deviation

Reproducibility standard deviation

1 10 16519 02415 02425 16021 02045 02433 0847 1007

2 10 26551 00543 00659 26719 00914 01325 1682 2011

2 bis 10 26581 02607 02607 25951 01900 01953 0729 0749

3 10 18671 00378 00378 18671 00478 00611 1264 1617

Bias of the alternative method

In order to estimate the bias of the alternative method with regard to the reference method for every

level Dij and t are calculated according to the following equations

RefAlt ijYijYDij

Diffp

Dijit

)2(

)(median

If the t value is superior to 2 the alternative method is significantly biased

with regard to the reference method

The values are given in table 17

Table 17 ndash t (d) values obtained by level

Level Bias t (d) Conclusion

1 0068 114 Non significant bias

2 0021 082 Non significant bias

2 bis - 0037 035 Non significant bias

3 - 0037 043 Non significant bias

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2738 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

The statistical test concludes to a non significant bias for all the inoculation

levels The bias varies from ndash 0037 to 0068 log CFUml

Comparison of the repeatability standard deviations

If the ratio Srj Alt Srj Ref of the repeatability standard deviations of the

alternative method and the reference method is larger than 2 the precision

under repeatability conditions of the alternative method is considered to be

lower than that of the reference method If this ratio is smaller than 05 the

precision under repeatability conditions of the alternative method is

considered to be greater than that of the reference method

The ratio values are given in table 18

Table 18

Level

Repeatability standard deviation

Reference method Alternative method Alternative method

reference method ratio

1 0242 0205 0847

2 0054 0091 1682

2 bis 0261 0190 0729

3 0038 0048 1264

Level Repeatability limit

Reference method Alternative method

1 0676 0573

2 0152 0256

2 bis 0730 0532

3 0106 0134

The ratios of the repeatability standard deviations are below 2 in all cases

The precision under repeatability conditions of the alternative method is

equivalent to that of the reference method

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2838 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Comparison of the reproducibility standard deviations

If the ratio Srj Alt Srj Ref of the reproducibility standard deviations of the alternative method and the

reference method is larger than 2 the precision under reproducibility conditions of the alternative

method is considered to be lower than that of the reference method If this ratio is smaller than 05 the

precision under reproducibility conditions of the alternative method is considered to be greater than

that of the reference method

The ratio values are given in table 19

Table 19

Level

Reproducibility standard deviation

Reference method Alternative method Alternative method

reference method ratio

1 0242 0243 1007

2 0066 0133 2011

2 bis 0261 0195 0749

3 0038 0061 1617

Level Reproducibility limit

Reference method Alternative method

1 0676 0681

2 0185 0371

2 bis 0730 0547

3 0106 0171

The ratios of the reproducibility standard deviations are below or close to 2

The precision under reproducibility conditions of the alternative method is

equivalent to that of the reference method

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2938 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

214 Conclusion

Method comparison study

The Listeria PrecisTM enumeration method shows satisfying linearity and

relative accuracy

The alternative method shows satisfying inclusivity and exclusivity results

Two Listeria monocytogenes strains gave negative results with the

alternative method These strains grew weakly on the reference method

media (OampA Palcam and Oxford)

The limits of repeatability of the alternative method are similar to those of the

reference method

The bias between both methods are low between - 0133 Log CFUg and

- 0008 Log CFUg

The Listeria PrecisTM enumeration method offers the saving for confirmatory

tests

Positive results are obtained with the alternative method in 2-3 days (with

OBIS Mono test) or in 4-7 days (by the classical tests described in the

standardized method) against 4-7 days for the reference method

Inter-laboratory study

The bias between the reference and the alternative methods shows satisfying

and correct values

- level 1 (100 - 1 000 CFUg) bias = + 007 log CFUg

- level 2 (1 000 - 10 000 CFUg) bias = 002 log CFUg

- level 2 bis (1 000 - 10 000 CFUg) bias = - 004 log CFUg

- level 3 (10 000 - 100 000 CFUg) bias = - 004 log CFUg

The repeatability and the reproducibility of the alternative method look similar

to those of the reference method

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3038 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

22 Extension study (realised by ASEPT 2007)

An extension study was performed in 2007 for a new confirmatory test OBIS

MONO

150 Listeria monocytogenes strains and 100 non-target strains were isolated

onto TSAYE and BrillianceTM Listeria Agar plates All the Listeria

monocytogenes and the non-target strains gave the expected results

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3138 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Appendix 1 ndash Flow diagram of the Listeria PrecisTM method

x g sample + 9 x ml buffered peptone water

Revivification step for 1 h plusmn 5 min at 20degC plusmn 1degC

Streaking 1 ml onto 3 Brilliance Listeria Agar plates (mother suspension)

or 01 ml onto 1 Brilliance Listeria Agar plates (mother suspension and decimal dilutions)

Incubation for 48 h plusmn 3 h at 37degC plusmn 1degC

Confirmatory tests on at least one typical colony

- by the tests described in the ISO method

- by applying the OBIS mono test on an isolated colony

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3238 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Appendix 2 ndash ISO 11290-2 (1998) amp ISO 11290-2A1 (2004) Horizontal

method for the detection and enumeration of Listeria monocytogenes -

Part 2 enumeration of Listeria monocytogenes in foods

10 g cheese + 90 ml buffered peptone water or Half Fraser Broth (without supplements) (110 dilution)

Stomach 1 min 30 seconds

Keep at 20degC plusmn 2degC for 1 h plusmn 5 min

Decimal dilutions in 9 ml peptone salt tubes

Dilution 110

Dilution 1100

Dilution 11000

1 ml on 3 Chromogenic Listeria medium (ISO) plates (x 2) (-1)

01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-2)

01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-3)

01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-4)

Incubation at 37degC plusmn 1degC

24 h - 48 h

Numerate typical colonies at 24 h

(bluegreen surrounded by an opaque halo)

Numerate at 48 h

Streak typical colonies on TSYEA

(5 coloniesplate 2 successive dilutions)

Confirmation tests

Catalase

Gram stain

For confirmation of L monocytogenes

Haemolysis

Carbohydrate (rhamnose and xylose) utilisation

CAMP Test

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3338 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Appendix 3 ndash Mandelrsquos graphics

Laboratoire h Ref

Niveau 1 h Ref

Niveau 2 h Ref

Niveau 3 h Alt

Niveau 1 h Alt

Niveau 2 h Alt

Niveau 3 h5 h1 h5 h1

A -050 -015 083 062 -212 -039 198 299 -198 -299

B -261 -270 021 -077 -058 173 198 299 -198 -299

C -151 -031 -014 -015 -004 -005 198 299 -198 -299

D 038 040 -137 000 072 207 198 299 -198 -299

E -001 090 155 -052 056 -101 198 299 -198 -299

F -112 -321 -153 070 -325 023 198 299 -198 -299

G 042 -071 040 147 -009 176 198 299 -198 -299

H 169 046 -169 192 018 -083 198 299 -198 -299

L 009 -120 021 -077 -231 005 198 299 -198 -299

M 001 031 014 -032 004 -011 198 299 -198 -299

-300

-200

-100

000

100

200

300

A B C D E F G H L M

Vale

urs

h d

e M

an

del(

rob

uste

s)

Laboratoires

Meacutethode de reacutefeacuterence analyse robuste

h Ref Niveau 1

h Ref Niveau 2

h Ref Niveau 3

h 5=198

h 1=299

-300

-200

-100

000

100

200

300

A B C D E F G H L M

Vale

urs

de h

de M

an

del(

rob

uste

s)

Laboratoires

Meacutethode alternative analyse robuste

h Alt Niveau 1

h Alt Niveau 2

h Alt Niveau 2

h 1=299

h 5=198

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3438 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Laboratoire k Ref

Niveau 1 k Ref

Niveau 2 k Ref

Niveau 3 k Alt

Niveau 1 k Alt

Niveau 2 k Alt

Niveau 3 k1 k5

A 000 089 142 084 162 046 259 181

B 125 087 073 104 075 046 259 181

C 117 013 091 188 022 083 259 181

D 052 036 069 000 035 083 259 181

E 028 034 037 138 019 112 259 181

F 140 222 018 027 281 112 259 181

G 088 068 024 131 145 000 259 181

H 129 094 173 016 061 000 259 181

L 069 000 073 104 270 224 259 181

M 000 048 038 043 099 241 259 181

000

050

100

150

200

250

300

350

400

A B C D E F G H L M

Vale

urs

k d

e M

an

del(

rob

uste

s)

Laboratoires

Meacutethode de reacutefeacuterence analyse robuste

k Ref Niveau 1k Ref Niveau 2k Ref Niveau 3

k 1=259

k 5=181

000

050

100

150

200

250

300

A B C D E F G H L M

Vale

urs

k d

e M

an

del

(ro

bu

ste

s)

Laboratoires

Meacutethode alternative analyse robuste

k Alt Niveau 3

k Alt Niveau 1

k Alt Niveau 2

k 1=259

k 5=181

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3538 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Appendix 4 ndash Statistical results

Level 1

Nombre de laboratoires (p) 10

A 1602 1602 1602 000 0000 0000 -0495 0000 A 1845 1602 1724 017 0122 -0122 0621 0840 0122

B 1602 1176 1389 030 0213 -0213 -2613 1247 B 1602 1301 1452 021 0151 -0151 -0769 1041 0062

C 1699 1301 1500 028 0199 -0199 -1510 1165 C 1301 1845 1573 038 -0272 0272 -0148 1881 0073

D 1602 1778 1690 012 -0088 0088 0380 0516 D 1602 1602 1602 000 0000 0000 0000 0000 -0088

E 1699 1602 1651 007 0048 -0048 -0013 0284 E 1699 1301 1500 028 0199 -0199 -0522 1376 -0151

F 1778 1301 1540 034 0239 -0239 -1116 1397 F 1778 1699 1739 006 0040 -0040 0698 0274 0199

G 1845 1544 1695 021 0151 -0151 0425 0881 G 2079 1699 1889 027 0190 -0190 1467 1315 0194

H 2041 1602 1822 031 0220 -0220 1689 1286 H 2000 1954 1977 003 0023 -0023 1917 0158 0155

L 1544 1778 1661 017 -0117 0117 0092 0685 L 1301 1602 1452 021 -0151 0151 -0769 1041 -0210

M 1653 1653 1653 000 0000 0000 0013 0000 M 1477 1602 1540 009 -0062 0062 -0319 0432 -0114

m 165186 m 160206 mediane D 00678

Cn(2p) 1867 Cn(2p) 1867 Cp 16101

Cn(p) 16101 Cn(p) 16101 Qn(D) 0092926

n=2p n 20 n=2p n 20 Qdiff 01496169

f 11 f 11 t 114

l 55 l 55

n=p n 10 n=p n 10

f 6 f 6

l 15 l 15

Qn(2p) 0091465 Qn(2p) 007745

Qn(p) 0062469 Qn(p) 0121519

Qintra 017078 Qintra 014461

Qinter 010058 Qinter 019565

Sr(eacutecart-type de reacutepeacutetabiliteacute) 02415 Sr 02045

RSDr(coefficient de variation de la reacutepeacutetabiliteacute)1462 RSDr 1277

r(repeatability limit)) 0676 r 0573

SL(eacutecart-type interlaboratoire) -001905 SLlt0 SL 001737

SR(eacutecart-type de reproductibiliteacute) 02415 SR 02433

RSDR(coefficient de variation de la reproductibiliteacute)0146 RSDR 0152

R(limite de reproductibiliteacute)) 06762 R(limite de reproductibiliteacute))068123

h k h MoyenneLaboratoires

Niveau 1

si LaboratoiresDeviationMoyenne Dk DeviationsiAlt

Niveau 1

Ref

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3638 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Level 2

Nombre de laboratoires (p) 10

A 2681 2613 2647 005 0034 -0034 -0151 0891 A 2322 2531 2427 015 -0105 0105 -2119 1620 -0220

B 2544 2477 2511 005 0033 -0033 -2699 0872 B 2556 2653 2605 007 -0048 0048 -0581 0750 0094

C 2643 2633 2638 001 0005 -0005 -0311 0130 C 2681 2653 2667 002 0014 -0014 -0040 0217 0029

D 2690 2663 2676 002 0014 -0014 0400 0357 D 2778 2732 2755 003 0023 -0023 0721 0354 0079

E 2690 2716 2703 002 -0013 0013 0897 0336 E 2724 2748 2736 002 -0012 0012 0556 0185 0033

F 2568 2398 2483 012 0085 -0085 -3214 2217 F 2477 2114 2296 026 0182 -0182 -3255 2811 -0188

G 2643 2591 2617 004 0026 -0026 -0707 0682 G 2756 2568 2662 013 0094 -0094 -0085 1453 0045

H 2716 2643 2680 005 0036 -0036 0460 0945 H 2653 2732 2693 006 -0040 0040 0181 0613 0013

L 2591 2591 2591 000 0000 0000 -1196 0000 L 2230 2580 2405 025 -0175 0175 -2307 2704 -0186

M 2653 2690 2672 003 -0018 0018 0311 0481 M 2740 2613 2677 009 0064 -0064 0040 0987 0005

m 2655082 m 26719001 mediane D 00209

Cn(2p) 1867 Cn(2p) 1867 Cp 16101

Cn(p) 16101 Cn(p) 16101 Qn(D) 0039911

n=2p n 20 n=2p n 20 Qdiff 00642591

f 11 f 11 t 082

l 55 l 55

n=p n 10 n=p n 10

f 6 f 6

l 15 l 15

Qn(2p) 0020570 Qn(2p) 00345985

Qn(p) 00332437 Qn(p) 00718158

Qintra 003841 Qintra 006460

Qinter 005352 Qinter 011563

Sr(eacutecart-type de reacutepeacutetabiliteacute) 00543 Sr 00914

RSDr(coefficient de variation de la reacutepeacutetabiliteacute)205 RSDr 342

r(repeatability limit)) 0152 r 0256

SL(eacutecart-type interlaboratoire) 000139 SL 000920

SR(eacutecart-type de reproductibiliteacute) 00659 SR 01325

RSDR(coefficient de variation de la reproductibiliteacute)0025 RSDR 0050

R(limite de reproductibiliteacute)) 018446 R(limite de reproductibiliteacute))03708618

Laboratoires

Niveau 2

Moyenne si Laboratoires

Niveau 2

DRef Alt Moyenne si Deviation h Deviation h k k

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3738 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Level 2 bis

Nombre de laboratoires (p) 10

A 3000 2477 2739 037 0261 -0261 0832 1418 A 2602 2477 2540 009 0062 -0062 -0391 0465 -0199

B 2544 2813 2678 019 -0134 0134 0211 0729 B 2903 2778 2841 009 0062 -0062 1732 0465 0162

C 2477 2813 2645 024 -0168 0168 -0135 0911 C 2477 2699 2588 016 -0111 0111 -0049 0826 -0057

D 2398 2653 2526 018 -0128 0128 -1370 0692 D 2778 3000 2889 016 -0111 0111 2074 0826 0363

E 2875 2740 2808 010 0067 -0067 1547 0365 E 2602 2301 2452 021 0151 -0151 -1012 1120 -0356

F 2477 2544 2511 005 -0033 0033 -1525 0182 F 2778 2477 2628 021 0151 -0151 0230 1120 0117

G 2653 2740 2697 006 -0044 0044 0400 0236 G 2845 2845 2845 000 0000 0000 1764 0000 0148

H 2176 2813 2495 045 -0318 0318 -1692 1727 H 2477 2477 2477 000 0000 0000 -0832 0000 -0017

L 2813 2544 2678 019 0134 -0134 0211 0729 L 2903 2301 2602 043 0301 -0301 0049 2241 -0076

M 2602 2740 2671 010 -0069 0069 0135 0375 M 2903 2255 2579 046 0324 -0324 -0112 2411 -0092

m 2658114 m 259505 mediane D -00372

Cn(2p) 1867 Cn(2p) 1867 Cp 16101

Cn(p) 16101 Cn(p) 16101 Qn(D) 01656915

n=2p n 20 n=2p n 20 Qdiff 02667753

f 11 f 11 t 035

l 55 l 55

n=p n 10 n=p n 10

f 6 f 6

l 15 l 15

Qn(2p) 0098745 Qn(2p) 007195

Qn(p) 006007 Qn(p) 008805

Qintra 018437 Qintra 013435

Qinter 009672 Qinter 014176

Sr(eacutecart-type de reacutepeacutetabiliteacute) 02607 Sr 01900

RSDr(coefficient de variation de la reacutepeacutetabiliteacute)981 RSDr 732

r(repeatability limit)) 0730 r 0532

SL(eacutecart-type interlaboratoire) -002464 SLlt0 SL 000205

SR(eacutecart-type de reproductibiliteacute) 02607 SR 01953

RSDR(coefficient de variation de la reproductibiliteacute)0098 RSDR 0075

R(limite de reproductibiliteacute)) 072996 R(limite de reproductibiliteacute))054686

Laboratoires

Niveau 2 bis

Moyenne si Laboratoires

Niveau 2 bis

DRef Alt Moyenne si Deviation h Deviation h k k

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3838 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Level 3

Nombre de laboratoires (p) 10

A 3690 3663 3676 002 0014 -0014 -0311 0513 A 3690 3708 3699 001 -0009 0009 0354 0257 0022

B 3613 3672 3642 004 -0030 0030 -1851 1109 B 3708 3568 3638 010 0070 -0070 -0844 2061 -0005

C 3663 3732 3698 005 -0035 0035 0645 1302 C 3663 3699 3681 003 -0018 0018 0000 0536 -0017

D 3690 3690 3690 000 0000 0000 0311 0000 D 3643 3663 3653 001 -0010 0010 -0545 0286 -0037

E 3732 3653 3693 006 0040 -0040 0429 1480 E 3613 3732 3673 008 -0060 0060 -0162 1769 -0020

F 3643 3672 3658 002 -0014 0014 -1157 0536 F 3690 3771 3731 006 -0040 0040 0975 1193 0073

G 3699 3653 3676 003 0023 -0023 -0328 0856 G 3602 3643 3623 003 -0021 0021 -1141 0612 -0053

H 3623 3672 3648 003 -0024 0024 -1615 0913 H 3748 3681 3715 005 0033 -0033 0664 0990 0067

L 3724 3663 3694 004 0031 -0031 0461 1150 L 3748 3771 3760 002 -0011 0011 1544 0335 0066

M 3690 3690 3690 000 0000 0000 0311 0000 M 3806 3681 3744 009 0062 -0062 1234 1848 0054

m 368334 m 368086 mediane D 00089

Cn(2p) 1867 Cn(2p) 1867 Cp 16101

Cn(p) 16101 Cn(p) 16101 Qn(D) 00325359

n=2p n 20 n=2p n 20 Qdiff 00523852

f 11 f 11 t 043

l 55 l 55

n=p n 10 n=p n 10

f 6 f 6

l 15 l 15

Qn(2p) 0014323 Qn(2p) 001811

Qn(p) 0013719 Qn(p) 003164

Qintra 002674 Qintra 003381

Qinter 002209 Qinter 005094

Sr(eacutecart-type de reacutepeacutetabiliteacute) 00378 Sr 00478

RSDr(coefficient de variation de la reacutepeacutetabiliteacute)103 RSDr 130

r(repeatability limit)) 0106 r 0134

SL(eacutecart-type interlaboratoire) -000023 SLlt0 SL 000145

SR(eacutecart-type de reproductibiliteacute) 00378 SR 00611

RSDR(coefficient de variation de la reproductibiliteacute)0010 RSDR 0017

R(limite de reproductibiliteacute)) 0105894 R(limite de reproductibiliteacute))017119

Ref Alt DLaboratoires

Niveau 3

Moyenne si Deviation h k Laboratoires

Niveau 3

Moyenne si Deviation h k

Page 27: ÞÀ¿¸7 ú Y5zñLjÜ< /u +æü t¬Á ... - NF Validation · NF VALIDATION Validation of alternative analytical methods Application in food microbiology ... 500 – 1 000 1 000 –

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2738 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

The statistical test concludes to a non significant bias for all the inoculation

levels The bias varies from ndash 0037 to 0068 log CFUml

Comparison of the repeatability standard deviations

If the ratio Srj Alt Srj Ref of the repeatability standard deviations of the

alternative method and the reference method is larger than 2 the precision

under repeatability conditions of the alternative method is considered to be

lower than that of the reference method If this ratio is smaller than 05 the

precision under repeatability conditions of the alternative method is

considered to be greater than that of the reference method

The ratio values are given in table 18

Table 18

Level

Repeatability standard deviation

Reference method Alternative method Alternative method

reference method ratio

1 0242 0205 0847

2 0054 0091 1682

2 bis 0261 0190 0729

3 0038 0048 1264

Level Repeatability limit

Reference method Alternative method

1 0676 0573

2 0152 0256

2 bis 0730 0532

3 0106 0134

The ratios of the repeatability standard deviations are below 2 in all cases

The precision under repeatability conditions of the alternative method is

equivalent to that of the reference method

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2838 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Comparison of the reproducibility standard deviations

If the ratio Srj Alt Srj Ref of the reproducibility standard deviations of the alternative method and the

reference method is larger than 2 the precision under reproducibility conditions of the alternative

method is considered to be lower than that of the reference method If this ratio is smaller than 05 the

precision under reproducibility conditions of the alternative method is considered to be greater than

that of the reference method

The ratio values are given in table 19

Table 19

Level

Reproducibility standard deviation

Reference method Alternative method Alternative method

reference method ratio

1 0242 0243 1007

2 0066 0133 2011

2 bis 0261 0195 0749

3 0038 0061 1617

Level Reproducibility limit

Reference method Alternative method

1 0676 0681

2 0185 0371

2 bis 0730 0547

3 0106 0171

The ratios of the reproducibility standard deviations are below or close to 2

The precision under reproducibility conditions of the alternative method is

equivalent to that of the reference method

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2938 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

214 Conclusion

Method comparison study

The Listeria PrecisTM enumeration method shows satisfying linearity and

relative accuracy

The alternative method shows satisfying inclusivity and exclusivity results

Two Listeria monocytogenes strains gave negative results with the

alternative method These strains grew weakly on the reference method

media (OampA Palcam and Oxford)

The limits of repeatability of the alternative method are similar to those of the

reference method

The bias between both methods are low between - 0133 Log CFUg and

- 0008 Log CFUg

The Listeria PrecisTM enumeration method offers the saving for confirmatory

tests

Positive results are obtained with the alternative method in 2-3 days (with

OBIS Mono test) or in 4-7 days (by the classical tests described in the

standardized method) against 4-7 days for the reference method

Inter-laboratory study

The bias between the reference and the alternative methods shows satisfying

and correct values

- level 1 (100 - 1 000 CFUg) bias = + 007 log CFUg

- level 2 (1 000 - 10 000 CFUg) bias = 002 log CFUg

- level 2 bis (1 000 - 10 000 CFUg) bias = - 004 log CFUg

- level 3 (10 000 - 100 000 CFUg) bias = - 004 log CFUg

The repeatability and the reproducibility of the alternative method look similar

to those of the reference method

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3038 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

22 Extension study (realised by ASEPT 2007)

An extension study was performed in 2007 for a new confirmatory test OBIS

MONO

150 Listeria monocytogenes strains and 100 non-target strains were isolated

onto TSAYE and BrillianceTM Listeria Agar plates All the Listeria

monocytogenes and the non-target strains gave the expected results

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3138 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Appendix 1 ndash Flow diagram of the Listeria PrecisTM method

x g sample + 9 x ml buffered peptone water

Revivification step for 1 h plusmn 5 min at 20degC plusmn 1degC

Streaking 1 ml onto 3 Brilliance Listeria Agar plates (mother suspension)

or 01 ml onto 1 Brilliance Listeria Agar plates (mother suspension and decimal dilutions)

Incubation for 48 h plusmn 3 h at 37degC plusmn 1degC

Confirmatory tests on at least one typical colony

- by the tests described in the ISO method

- by applying the OBIS mono test on an isolated colony

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3238 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Appendix 2 ndash ISO 11290-2 (1998) amp ISO 11290-2A1 (2004) Horizontal

method for the detection and enumeration of Listeria monocytogenes -

Part 2 enumeration of Listeria monocytogenes in foods

10 g cheese + 90 ml buffered peptone water or Half Fraser Broth (without supplements) (110 dilution)

Stomach 1 min 30 seconds

Keep at 20degC plusmn 2degC for 1 h plusmn 5 min

Decimal dilutions in 9 ml peptone salt tubes

Dilution 110

Dilution 1100

Dilution 11000

1 ml on 3 Chromogenic Listeria medium (ISO) plates (x 2) (-1)

01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-2)

01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-3)

01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-4)

Incubation at 37degC plusmn 1degC

24 h - 48 h

Numerate typical colonies at 24 h

(bluegreen surrounded by an opaque halo)

Numerate at 48 h

Streak typical colonies on TSYEA

(5 coloniesplate 2 successive dilutions)

Confirmation tests

Catalase

Gram stain

For confirmation of L monocytogenes

Haemolysis

Carbohydrate (rhamnose and xylose) utilisation

CAMP Test

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3338 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Appendix 3 ndash Mandelrsquos graphics

Laboratoire h Ref

Niveau 1 h Ref

Niveau 2 h Ref

Niveau 3 h Alt

Niveau 1 h Alt

Niveau 2 h Alt

Niveau 3 h5 h1 h5 h1

A -050 -015 083 062 -212 -039 198 299 -198 -299

B -261 -270 021 -077 -058 173 198 299 -198 -299

C -151 -031 -014 -015 -004 -005 198 299 -198 -299

D 038 040 -137 000 072 207 198 299 -198 -299

E -001 090 155 -052 056 -101 198 299 -198 -299

F -112 -321 -153 070 -325 023 198 299 -198 -299

G 042 -071 040 147 -009 176 198 299 -198 -299

H 169 046 -169 192 018 -083 198 299 -198 -299

L 009 -120 021 -077 -231 005 198 299 -198 -299

M 001 031 014 -032 004 -011 198 299 -198 -299

-300

-200

-100

000

100

200

300

A B C D E F G H L M

Vale

urs

h d

e M

an

del(

rob

uste

s)

Laboratoires

Meacutethode de reacutefeacuterence analyse robuste

h Ref Niveau 1

h Ref Niveau 2

h Ref Niveau 3

h 5=198

h 1=299

-300

-200

-100

000

100

200

300

A B C D E F G H L M

Vale

urs

de h

de M

an

del(

rob

uste

s)

Laboratoires

Meacutethode alternative analyse robuste

h Alt Niveau 1

h Alt Niveau 2

h Alt Niveau 2

h 1=299

h 5=198

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3438 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Laboratoire k Ref

Niveau 1 k Ref

Niveau 2 k Ref

Niveau 3 k Alt

Niveau 1 k Alt

Niveau 2 k Alt

Niveau 3 k1 k5

A 000 089 142 084 162 046 259 181

B 125 087 073 104 075 046 259 181

C 117 013 091 188 022 083 259 181

D 052 036 069 000 035 083 259 181

E 028 034 037 138 019 112 259 181

F 140 222 018 027 281 112 259 181

G 088 068 024 131 145 000 259 181

H 129 094 173 016 061 000 259 181

L 069 000 073 104 270 224 259 181

M 000 048 038 043 099 241 259 181

000

050

100

150

200

250

300

350

400

A B C D E F G H L M

Vale

urs

k d

e M

an

del(

rob

uste

s)

Laboratoires

Meacutethode de reacutefeacuterence analyse robuste

k Ref Niveau 1k Ref Niveau 2k Ref Niveau 3

k 1=259

k 5=181

000

050

100

150

200

250

300

A B C D E F G H L M

Vale

urs

k d

e M

an

del

(ro

bu

ste

s)

Laboratoires

Meacutethode alternative analyse robuste

k Alt Niveau 3

k Alt Niveau 1

k Alt Niveau 2

k 1=259

k 5=181

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3538 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Appendix 4 ndash Statistical results

Level 1

Nombre de laboratoires (p) 10

A 1602 1602 1602 000 0000 0000 -0495 0000 A 1845 1602 1724 017 0122 -0122 0621 0840 0122

B 1602 1176 1389 030 0213 -0213 -2613 1247 B 1602 1301 1452 021 0151 -0151 -0769 1041 0062

C 1699 1301 1500 028 0199 -0199 -1510 1165 C 1301 1845 1573 038 -0272 0272 -0148 1881 0073

D 1602 1778 1690 012 -0088 0088 0380 0516 D 1602 1602 1602 000 0000 0000 0000 0000 -0088

E 1699 1602 1651 007 0048 -0048 -0013 0284 E 1699 1301 1500 028 0199 -0199 -0522 1376 -0151

F 1778 1301 1540 034 0239 -0239 -1116 1397 F 1778 1699 1739 006 0040 -0040 0698 0274 0199

G 1845 1544 1695 021 0151 -0151 0425 0881 G 2079 1699 1889 027 0190 -0190 1467 1315 0194

H 2041 1602 1822 031 0220 -0220 1689 1286 H 2000 1954 1977 003 0023 -0023 1917 0158 0155

L 1544 1778 1661 017 -0117 0117 0092 0685 L 1301 1602 1452 021 -0151 0151 -0769 1041 -0210

M 1653 1653 1653 000 0000 0000 0013 0000 M 1477 1602 1540 009 -0062 0062 -0319 0432 -0114

m 165186 m 160206 mediane D 00678

Cn(2p) 1867 Cn(2p) 1867 Cp 16101

Cn(p) 16101 Cn(p) 16101 Qn(D) 0092926

n=2p n 20 n=2p n 20 Qdiff 01496169

f 11 f 11 t 114

l 55 l 55

n=p n 10 n=p n 10

f 6 f 6

l 15 l 15

Qn(2p) 0091465 Qn(2p) 007745

Qn(p) 0062469 Qn(p) 0121519

Qintra 017078 Qintra 014461

Qinter 010058 Qinter 019565

Sr(eacutecart-type de reacutepeacutetabiliteacute) 02415 Sr 02045

RSDr(coefficient de variation de la reacutepeacutetabiliteacute)1462 RSDr 1277

r(repeatability limit)) 0676 r 0573

SL(eacutecart-type interlaboratoire) -001905 SLlt0 SL 001737

SR(eacutecart-type de reproductibiliteacute) 02415 SR 02433

RSDR(coefficient de variation de la reproductibiliteacute)0146 RSDR 0152

R(limite de reproductibiliteacute)) 06762 R(limite de reproductibiliteacute))068123

h k h MoyenneLaboratoires

Niveau 1

si LaboratoiresDeviationMoyenne Dk DeviationsiAlt

Niveau 1

Ref

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3638 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Level 2

Nombre de laboratoires (p) 10

A 2681 2613 2647 005 0034 -0034 -0151 0891 A 2322 2531 2427 015 -0105 0105 -2119 1620 -0220

B 2544 2477 2511 005 0033 -0033 -2699 0872 B 2556 2653 2605 007 -0048 0048 -0581 0750 0094

C 2643 2633 2638 001 0005 -0005 -0311 0130 C 2681 2653 2667 002 0014 -0014 -0040 0217 0029

D 2690 2663 2676 002 0014 -0014 0400 0357 D 2778 2732 2755 003 0023 -0023 0721 0354 0079

E 2690 2716 2703 002 -0013 0013 0897 0336 E 2724 2748 2736 002 -0012 0012 0556 0185 0033

F 2568 2398 2483 012 0085 -0085 -3214 2217 F 2477 2114 2296 026 0182 -0182 -3255 2811 -0188

G 2643 2591 2617 004 0026 -0026 -0707 0682 G 2756 2568 2662 013 0094 -0094 -0085 1453 0045

H 2716 2643 2680 005 0036 -0036 0460 0945 H 2653 2732 2693 006 -0040 0040 0181 0613 0013

L 2591 2591 2591 000 0000 0000 -1196 0000 L 2230 2580 2405 025 -0175 0175 -2307 2704 -0186

M 2653 2690 2672 003 -0018 0018 0311 0481 M 2740 2613 2677 009 0064 -0064 0040 0987 0005

m 2655082 m 26719001 mediane D 00209

Cn(2p) 1867 Cn(2p) 1867 Cp 16101

Cn(p) 16101 Cn(p) 16101 Qn(D) 0039911

n=2p n 20 n=2p n 20 Qdiff 00642591

f 11 f 11 t 082

l 55 l 55

n=p n 10 n=p n 10

f 6 f 6

l 15 l 15

Qn(2p) 0020570 Qn(2p) 00345985

Qn(p) 00332437 Qn(p) 00718158

Qintra 003841 Qintra 006460

Qinter 005352 Qinter 011563

Sr(eacutecart-type de reacutepeacutetabiliteacute) 00543 Sr 00914

RSDr(coefficient de variation de la reacutepeacutetabiliteacute)205 RSDr 342

r(repeatability limit)) 0152 r 0256

SL(eacutecart-type interlaboratoire) 000139 SL 000920

SR(eacutecart-type de reproductibiliteacute) 00659 SR 01325

RSDR(coefficient de variation de la reproductibiliteacute)0025 RSDR 0050

R(limite de reproductibiliteacute)) 018446 R(limite de reproductibiliteacute))03708618

Laboratoires

Niveau 2

Moyenne si Laboratoires

Niveau 2

DRef Alt Moyenne si Deviation h Deviation h k k

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3738 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Level 2 bis

Nombre de laboratoires (p) 10

A 3000 2477 2739 037 0261 -0261 0832 1418 A 2602 2477 2540 009 0062 -0062 -0391 0465 -0199

B 2544 2813 2678 019 -0134 0134 0211 0729 B 2903 2778 2841 009 0062 -0062 1732 0465 0162

C 2477 2813 2645 024 -0168 0168 -0135 0911 C 2477 2699 2588 016 -0111 0111 -0049 0826 -0057

D 2398 2653 2526 018 -0128 0128 -1370 0692 D 2778 3000 2889 016 -0111 0111 2074 0826 0363

E 2875 2740 2808 010 0067 -0067 1547 0365 E 2602 2301 2452 021 0151 -0151 -1012 1120 -0356

F 2477 2544 2511 005 -0033 0033 -1525 0182 F 2778 2477 2628 021 0151 -0151 0230 1120 0117

G 2653 2740 2697 006 -0044 0044 0400 0236 G 2845 2845 2845 000 0000 0000 1764 0000 0148

H 2176 2813 2495 045 -0318 0318 -1692 1727 H 2477 2477 2477 000 0000 0000 -0832 0000 -0017

L 2813 2544 2678 019 0134 -0134 0211 0729 L 2903 2301 2602 043 0301 -0301 0049 2241 -0076

M 2602 2740 2671 010 -0069 0069 0135 0375 M 2903 2255 2579 046 0324 -0324 -0112 2411 -0092

m 2658114 m 259505 mediane D -00372

Cn(2p) 1867 Cn(2p) 1867 Cp 16101

Cn(p) 16101 Cn(p) 16101 Qn(D) 01656915

n=2p n 20 n=2p n 20 Qdiff 02667753

f 11 f 11 t 035

l 55 l 55

n=p n 10 n=p n 10

f 6 f 6

l 15 l 15

Qn(2p) 0098745 Qn(2p) 007195

Qn(p) 006007 Qn(p) 008805

Qintra 018437 Qintra 013435

Qinter 009672 Qinter 014176

Sr(eacutecart-type de reacutepeacutetabiliteacute) 02607 Sr 01900

RSDr(coefficient de variation de la reacutepeacutetabiliteacute)981 RSDr 732

r(repeatability limit)) 0730 r 0532

SL(eacutecart-type interlaboratoire) -002464 SLlt0 SL 000205

SR(eacutecart-type de reproductibiliteacute) 02607 SR 01953

RSDR(coefficient de variation de la reproductibiliteacute)0098 RSDR 0075

R(limite de reproductibiliteacute)) 072996 R(limite de reproductibiliteacute))054686

Laboratoires

Niveau 2 bis

Moyenne si Laboratoires

Niveau 2 bis

DRef Alt Moyenne si Deviation h Deviation h k k

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3838 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Level 3

Nombre de laboratoires (p) 10

A 3690 3663 3676 002 0014 -0014 -0311 0513 A 3690 3708 3699 001 -0009 0009 0354 0257 0022

B 3613 3672 3642 004 -0030 0030 -1851 1109 B 3708 3568 3638 010 0070 -0070 -0844 2061 -0005

C 3663 3732 3698 005 -0035 0035 0645 1302 C 3663 3699 3681 003 -0018 0018 0000 0536 -0017

D 3690 3690 3690 000 0000 0000 0311 0000 D 3643 3663 3653 001 -0010 0010 -0545 0286 -0037

E 3732 3653 3693 006 0040 -0040 0429 1480 E 3613 3732 3673 008 -0060 0060 -0162 1769 -0020

F 3643 3672 3658 002 -0014 0014 -1157 0536 F 3690 3771 3731 006 -0040 0040 0975 1193 0073

G 3699 3653 3676 003 0023 -0023 -0328 0856 G 3602 3643 3623 003 -0021 0021 -1141 0612 -0053

H 3623 3672 3648 003 -0024 0024 -1615 0913 H 3748 3681 3715 005 0033 -0033 0664 0990 0067

L 3724 3663 3694 004 0031 -0031 0461 1150 L 3748 3771 3760 002 -0011 0011 1544 0335 0066

M 3690 3690 3690 000 0000 0000 0311 0000 M 3806 3681 3744 009 0062 -0062 1234 1848 0054

m 368334 m 368086 mediane D 00089

Cn(2p) 1867 Cn(2p) 1867 Cp 16101

Cn(p) 16101 Cn(p) 16101 Qn(D) 00325359

n=2p n 20 n=2p n 20 Qdiff 00523852

f 11 f 11 t 043

l 55 l 55

n=p n 10 n=p n 10

f 6 f 6

l 15 l 15

Qn(2p) 0014323 Qn(2p) 001811

Qn(p) 0013719 Qn(p) 003164

Qintra 002674 Qintra 003381

Qinter 002209 Qinter 005094

Sr(eacutecart-type de reacutepeacutetabiliteacute) 00378 Sr 00478

RSDr(coefficient de variation de la reacutepeacutetabiliteacute)103 RSDr 130

r(repeatability limit)) 0106 r 0134

SL(eacutecart-type interlaboratoire) -000023 SLlt0 SL 000145

SR(eacutecart-type de reproductibiliteacute) 00378 SR 00611

RSDR(coefficient de variation de la reproductibiliteacute)0010 RSDR 0017

R(limite de reproductibiliteacute)) 0105894 R(limite de reproductibiliteacute))017119

Ref Alt DLaboratoires

Niveau 3

Moyenne si Deviation h k Laboratoires

Niveau 3

Moyenne si Deviation h k

Page 28: ÞÀ¿¸7 ú Y5zñLjÜ< /u +æü t¬Á ... - NF Validation · NF VALIDATION Validation of alternative analytical methods Application in food microbiology ... 500 – 1 000 1 000 –

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2838 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Comparison of the reproducibility standard deviations

If the ratio Srj Alt Srj Ref of the reproducibility standard deviations of the alternative method and the

reference method is larger than 2 the precision under reproducibility conditions of the alternative

method is considered to be lower than that of the reference method If this ratio is smaller than 05 the

precision under reproducibility conditions of the alternative method is considered to be greater than

that of the reference method

The ratio values are given in table 19

Table 19

Level

Reproducibility standard deviation

Reference method Alternative method Alternative method

reference method ratio

1 0242 0243 1007

2 0066 0133 2011

2 bis 0261 0195 0749

3 0038 0061 1617

Level Reproducibility limit

Reference method Alternative method

1 0676 0681

2 0185 0371

2 bis 0730 0547

3 0106 0171

The ratios of the reproducibility standard deviations are below or close to 2

The precision under reproducibility conditions of the alternative method is

equivalent to that of the reference method

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2938 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

214 Conclusion

Method comparison study

The Listeria PrecisTM enumeration method shows satisfying linearity and

relative accuracy

The alternative method shows satisfying inclusivity and exclusivity results

Two Listeria monocytogenes strains gave negative results with the

alternative method These strains grew weakly on the reference method

media (OampA Palcam and Oxford)

The limits of repeatability of the alternative method are similar to those of the

reference method

The bias between both methods are low between - 0133 Log CFUg and

- 0008 Log CFUg

The Listeria PrecisTM enumeration method offers the saving for confirmatory

tests

Positive results are obtained with the alternative method in 2-3 days (with

OBIS Mono test) or in 4-7 days (by the classical tests described in the

standardized method) against 4-7 days for the reference method

Inter-laboratory study

The bias between the reference and the alternative methods shows satisfying

and correct values

- level 1 (100 - 1 000 CFUg) bias = + 007 log CFUg

- level 2 (1 000 - 10 000 CFUg) bias = 002 log CFUg

- level 2 bis (1 000 - 10 000 CFUg) bias = - 004 log CFUg

- level 3 (10 000 - 100 000 CFUg) bias = - 004 log CFUg

The repeatability and the reproducibility of the alternative method look similar

to those of the reference method

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3038 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

22 Extension study (realised by ASEPT 2007)

An extension study was performed in 2007 for a new confirmatory test OBIS

MONO

150 Listeria monocytogenes strains and 100 non-target strains were isolated

onto TSAYE and BrillianceTM Listeria Agar plates All the Listeria

monocytogenes and the non-target strains gave the expected results

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3138 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Appendix 1 ndash Flow diagram of the Listeria PrecisTM method

x g sample + 9 x ml buffered peptone water

Revivification step for 1 h plusmn 5 min at 20degC plusmn 1degC

Streaking 1 ml onto 3 Brilliance Listeria Agar plates (mother suspension)

or 01 ml onto 1 Brilliance Listeria Agar plates (mother suspension and decimal dilutions)

Incubation for 48 h plusmn 3 h at 37degC plusmn 1degC

Confirmatory tests on at least one typical colony

- by the tests described in the ISO method

- by applying the OBIS mono test on an isolated colony

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3238 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Appendix 2 ndash ISO 11290-2 (1998) amp ISO 11290-2A1 (2004) Horizontal

method for the detection and enumeration of Listeria monocytogenes -

Part 2 enumeration of Listeria monocytogenes in foods

10 g cheese + 90 ml buffered peptone water or Half Fraser Broth (without supplements) (110 dilution)

Stomach 1 min 30 seconds

Keep at 20degC plusmn 2degC for 1 h plusmn 5 min

Decimal dilutions in 9 ml peptone salt tubes

Dilution 110

Dilution 1100

Dilution 11000

1 ml on 3 Chromogenic Listeria medium (ISO) plates (x 2) (-1)

01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-2)

01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-3)

01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-4)

Incubation at 37degC plusmn 1degC

24 h - 48 h

Numerate typical colonies at 24 h

(bluegreen surrounded by an opaque halo)

Numerate at 48 h

Streak typical colonies on TSYEA

(5 coloniesplate 2 successive dilutions)

Confirmation tests

Catalase

Gram stain

For confirmation of L monocytogenes

Haemolysis

Carbohydrate (rhamnose and xylose) utilisation

CAMP Test

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3338 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Appendix 3 ndash Mandelrsquos graphics

Laboratoire h Ref

Niveau 1 h Ref

Niveau 2 h Ref

Niveau 3 h Alt

Niveau 1 h Alt

Niveau 2 h Alt

Niveau 3 h5 h1 h5 h1

A -050 -015 083 062 -212 -039 198 299 -198 -299

B -261 -270 021 -077 -058 173 198 299 -198 -299

C -151 -031 -014 -015 -004 -005 198 299 -198 -299

D 038 040 -137 000 072 207 198 299 -198 -299

E -001 090 155 -052 056 -101 198 299 -198 -299

F -112 -321 -153 070 -325 023 198 299 -198 -299

G 042 -071 040 147 -009 176 198 299 -198 -299

H 169 046 -169 192 018 -083 198 299 -198 -299

L 009 -120 021 -077 -231 005 198 299 -198 -299

M 001 031 014 -032 004 -011 198 299 -198 -299

-300

-200

-100

000

100

200

300

A B C D E F G H L M

Vale

urs

h d

e M

an

del(

rob

uste

s)

Laboratoires

Meacutethode de reacutefeacuterence analyse robuste

h Ref Niveau 1

h Ref Niveau 2

h Ref Niveau 3

h 5=198

h 1=299

-300

-200

-100

000

100

200

300

A B C D E F G H L M

Vale

urs

de h

de M

an

del(

rob

uste

s)

Laboratoires

Meacutethode alternative analyse robuste

h Alt Niveau 1

h Alt Niveau 2

h Alt Niveau 2

h 1=299

h 5=198

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3438 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Laboratoire k Ref

Niveau 1 k Ref

Niveau 2 k Ref

Niveau 3 k Alt

Niveau 1 k Alt

Niveau 2 k Alt

Niveau 3 k1 k5

A 000 089 142 084 162 046 259 181

B 125 087 073 104 075 046 259 181

C 117 013 091 188 022 083 259 181

D 052 036 069 000 035 083 259 181

E 028 034 037 138 019 112 259 181

F 140 222 018 027 281 112 259 181

G 088 068 024 131 145 000 259 181

H 129 094 173 016 061 000 259 181

L 069 000 073 104 270 224 259 181

M 000 048 038 043 099 241 259 181

000

050

100

150

200

250

300

350

400

A B C D E F G H L M

Vale

urs

k d

e M

an

del(

rob

uste

s)

Laboratoires

Meacutethode de reacutefeacuterence analyse robuste

k Ref Niveau 1k Ref Niveau 2k Ref Niveau 3

k 1=259

k 5=181

000

050

100

150

200

250

300

A B C D E F G H L M

Vale

urs

k d

e M

an

del

(ro

bu

ste

s)

Laboratoires

Meacutethode alternative analyse robuste

k Alt Niveau 3

k Alt Niveau 1

k Alt Niveau 2

k 1=259

k 5=181

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3538 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Appendix 4 ndash Statistical results

Level 1

Nombre de laboratoires (p) 10

A 1602 1602 1602 000 0000 0000 -0495 0000 A 1845 1602 1724 017 0122 -0122 0621 0840 0122

B 1602 1176 1389 030 0213 -0213 -2613 1247 B 1602 1301 1452 021 0151 -0151 -0769 1041 0062

C 1699 1301 1500 028 0199 -0199 -1510 1165 C 1301 1845 1573 038 -0272 0272 -0148 1881 0073

D 1602 1778 1690 012 -0088 0088 0380 0516 D 1602 1602 1602 000 0000 0000 0000 0000 -0088

E 1699 1602 1651 007 0048 -0048 -0013 0284 E 1699 1301 1500 028 0199 -0199 -0522 1376 -0151

F 1778 1301 1540 034 0239 -0239 -1116 1397 F 1778 1699 1739 006 0040 -0040 0698 0274 0199

G 1845 1544 1695 021 0151 -0151 0425 0881 G 2079 1699 1889 027 0190 -0190 1467 1315 0194

H 2041 1602 1822 031 0220 -0220 1689 1286 H 2000 1954 1977 003 0023 -0023 1917 0158 0155

L 1544 1778 1661 017 -0117 0117 0092 0685 L 1301 1602 1452 021 -0151 0151 -0769 1041 -0210

M 1653 1653 1653 000 0000 0000 0013 0000 M 1477 1602 1540 009 -0062 0062 -0319 0432 -0114

m 165186 m 160206 mediane D 00678

Cn(2p) 1867 Cn(2p) 1867 Cp 16101

Cn(p) 16101 Cn(p) 16101 Qn(D) 0092926

n=2p n 20 n=2p n 20 Qdiff 01496169

f 11 f 11 t 114

l 55 l 55

n=p n 10 n=p n 10

f 6 f 6

l 15 l 15

Qn(2p) 0091465 Qn(2p) 007745

Qn(p) 0062469 Qn(p) 0121519

Qintra 017078 Qintra 014461

Qinter 010058 Qinter 019565

Sr(eacutecart-type de reacutepeacutetabiliteacute) 02415 Sr 02045

RSDr(coefficient de variation de la reacutepeacutetabiliteacute)1462 RSDr 1277

r(repeatability limit)) 0676 r 0573

SL(eacutecart-type interlaboratoire) -001905 SLlt0 SL 001737

SR(eacutecart-type de reproductibiliteacute) 02415 SR 02433

RSDR(coefficient de variation de la reproductibiliteacute)0146 RSDR 0152

R(limite de reproductibiliteacute)) 06762 R(limite de reproductibiliteacute))068123

h k h MoyenneLaboratoires

Niveau 1

si LaboratoiresDeviationMoyenne Dk DeviationsiAlt

Niveau 1

Ref

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3638 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Level 2

Nombre de laboratoires (p) 10

A 2681 2613 2647 005 0034 -0034 -0151 0891 A 2322 2531 2427 015 -0105 0105 -2119 1620 -0220

B 2544 2477 2511 005 0033 -0033 -2699 0872 B 2556 2653 2605 007 -0048 0048 -0581 0750 0094

C 2643 2633 2638 001 0005 -0005 -0311 0130 C 2681 2653 2667 002 0014 -0014 -0040 0217 0029

D 2690 2663 2676 002 0014 -0014 0400 0357 D 2778 2732 2755 003 0023 -0023 0721 0354 0079

E 2690 2716 2703 002 -0013 0013 0897 0336 E 2724 2748 2736 002 -0012 0012 0556 0185 0033

F 2568 2398 2483 012 0085 -0085 -3214 2217 F 2477 2114 2296 026 0182 -0182 -3255 2811 -0188

G 2643 2591 2617 004 0026 -0026 -0707 0682 G 2756 2568 2662 013 0094 -0094 -0085 1453 0045

H 2716 2643 2680 005 0036 -0036 0460 0945 H 2653 2732 2693 006 -0040 0040 0181 0613 0013

L 2591 2591 2591 000 0000 0000 -1196 0000 L 2230 2580 2405 025 -0175 0175 -2307 2704 -0186

M 2653 2690 2672 003 -0018 0018 0311 0481 M 2740 2613 2677 009 0064 -0064 0040 0987 0005

m 2655082 m 26719001 mediane D 00209

Cn(2p) 1867 Cn(2p) 1867 Cp 16101

Cn(p) 16101 Cn(p) 16101 Qn(D) 0039911

n=2p n 20 n=2p n 20 Qdiff 00642591

f 11 f 11 t 082

l 55 l 55

n=p n 10 n=p n 10

f 6 f 6

l 15 l 15

Qn(2p) 0020570 Qn(2p) 00345985

Qn(p) 00332437 Qn(p) 00718158

Qintra 003841 Qintra 006460

Qinter 005352 Qinter 011563

Sr(eacutecart-type de reacutepeacutetabiliteacute) 00543 Sr 00914

RSDr(coefficient de variation de la reacutepeacutetabiliteacute)205 RSDr 342

r(repeatability limit)) 0152 r 0256

SL(eacutecart-type interlaboratoire) 000139 SL 000920

SR(eacutecart-type de reproductibiliteacute) 00659 SR 01325

RSDR(coefficient de variation de la reproductibiliteacute)0025 RSDR 0050

R(limite de reproductibiliteacute)) 018446 R(limite de reproductibiliteacute))03708618

Laboratoires

Niveau 2

Moyenne si Laboratoires

Niveau 2

DRef Alt Moyenne si Deviation h Deviation h k k

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3738 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Level 2 bis

Nombre de laboratoires (p) 10

A 3000 2477 2739 037 0261 -0261 0832 1418 A 2602 2477 2540 009 0062 -0062 -0391 0465 -0199

B 2544 2813 2678 019 -0134 0134 0211 0729 B 2903 2778 2841 009 0062 -0062 1732 0465 0162

C 2477 2813 2645 024 -0168 0168 -0135 0911 C 2477 2699 2588 016 -0111 0111 -0049 0826 -0057

D 2398 2653 2526 018 -0128 0128 -1370 0692 D 2778 3000 2889 016 -0111 0111 2074 0826 0363

E 2875 2740 2808 010 0067 -0067 1547 0365 E 2602 2301 2452 021 0151 -0151 -1012 1120 -0356

F 2477 2544 2511 005 -0033 0033 -1525 0182 F 2778 2477 2628 021 0151 -0151 0230 1120 0117

G 2653 2740 2697 006 -0044 0044 0400 0236 G 2845 2845 2845 000 0000 0000 1764 0000 0148

H 2176 2813 2495 045 -0318 0318 -1692 1727 H 2477 2477 2477 000 0000 0000 -0832 0000 -0017

L 2813 2544 2678 019 0134 -0134 0211 0729 L 2903 2301 2602 043 0301 -0301 0049 2241 -0076

M 2602 2740 2671 010 -0069 0069 0135 0375 M 2903 2255 2579 046 0324 -0324 -0112 2411 -0092

m 2658114 m 259505 mediane D -00372

Cn(2p) 1867 Cn(2p) 1867 Cp 16101

Cn(p) 16101 Cn(p) 16101 Qn(D) 01656915

n=2p n 20 n=2p n 20 Qdiff 02667753

f 11 f 11 t 035

l 55 l 55

n=p n 10 n=p n 10

f 6 f 6

l 15 l 15

Qn(2p) 0098745 Qn(2p) 007195

Qn(p) 006007 Qn(p) 008805

Qintra 018437 Qintra 013435

Qinter 009672 Qinter 014176

Sr(eacutecart-type de reacutepeacutetabiliteacute) 02607 Sr 01900

RSDr(coefficient de variation de la reacutepeacutetabiliteacute)981 RSDr 732

r(repeatability limit)) 0730 r 0532

SL(eacutecart-type interlaboratoire) -002464 SLlt0 SL 000205

SR(eacutecart-type de reproductibiliteacute) 02607 SR 01953

RSDR(coefficient de variation de la reproductibiliteacute)0098 RSDR 0075

R(limite de reproductibiliteacute)) 072996 R(limite de reproductibiliteacute))054686

Laboratoires

Niveau 2 bis

Moyenne si Laboratoires

Niveau 2 bis

DRef Alt Moyenne si Deviation h Deviation h k k

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3838 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Level 3

Nombre de laboratoires (p) 10

A 3690 3663 3676 002 0014 -0014 -0311 0513 A 3690 3708 3699 001 -0009 0009 0354 0257 0022

B 3613 3672 3642 004 -0030 0030 -1851 1109 B 3708 3568 3638 010 0070 -0070 -0844 2061 -0005

C 3663 3732 3698 005 -0035 0035 0645 1302 C 3663 3699 3681 003 -0018 0018 0000 0536 -0017

D 3690 3690 3690 000 0000 0000 0311 0000 D 3643 3663 3653 001 -0010 0010 -0545 0286 -0037

E 3732 3653 3693 006 0040 -0040 0429 1480 E 3613 3732 3673 008 -0060 0060 -0162 1769 -0020

F 3643 3672 3658 002 -0014 0014 -1157 0536 F 3690 3771 3731 006 -0040 0040 0975 1193 0073

G 3699 3653 3676 003 0023 -0023 -0328 0856 G 3602 3643 3623 003 -0021 0021 -1141 0612 -0053

H 3623 3672 3648 003 -0024 0024 -1615 0913 H 3748 3681 3715 005 0033 -0033 0664 0990 0067

L 3724 3663 3694 004 0031 -0031 0461 1150 L 3748 3771 3760 002 -0011 0011 1544 0335 0066

M 3690 3690 3690 000 0000 0000 0311 0000 M 3806 3681 3744 009 0062 -0062 1234 1848 0054

m 368334 m 368086 mediane D 00089

Cn(2p) 1867 Cn(2p) 1867 Cp 16101

Cn(p) 16101 Cn(p) 16101 Qn(D) 00325359

n=2p n 20 n=2p n 20 Qdiff 00523852

f 11 f 11 t 043

l 55 l 55

n=p n 10 n=p n 10

f 6 f 6

l 15 l 15

Qn(2p) 0014323 Qn(2p) 001811

Qn(p) 0013719 Qn(p) 003164

Qintra 002674 Qintra 003381

Qinter 002209 Qinter 005094

Sr(eacutecart-type de reacutepeacutetabiliteacute) 00378 Sr 00478

RSDr(coefficient de variation de la reacutepeacutetabiliteacute)103 RSDr 130

r(repeatability limit)) 0106 r 0134

SL(eacutecart-type interlaboratoire) -000023 SLlt0 SL 000145

SR(eacutecart-type de reproductibiliteacute) 00378 SR 00611

RSDR(coefficient de variation de la reproductibiliteacute)0010 RSDR 0017

R(limite de reproductibiliteacute)) 0105894 R(limite de reproductibiliteacute))017119

Ref Alt DLaboratoires

Niveau 3

Moyenne si Deviation h k Laboratoires

Niveau 3

Moyenne si Deviation h k

Page 29: ÞÀ¿¸7 ú Y5zñLjÜ< /u +æü t¬Á ... - NF Validation · NF VALIDATION Validation of alternative analytical methods Application in food microbiology ... 500 – 1 000 1 000 –

Thermo Fisher Scientific

ADRIA Deacuteveloppement 2938 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

214 Conclusion

Method comparison study

The Listeria PrecisTM enumeration method shows satisfying linearity and

relative accuracy

The alternative method shows satisfying inclusivity and exclusivity results

Two Listeria monocytogenes strains gave negative results with the

alternative method These strains grew weakly on the reference method

media (OampA Palcam and Oxford)

The limits of repeatability of the alternative method are similar to those of the

reference method

The bias between both methods are low between - 0133 Log CFUg and

- 0008 Log CFUg

The Listeria PrecisTM enumeration method offers the saving for confirmatory

tests

Positive results are obtained with the alternative method in 2-3 days (with

OBIS Mono test) or in 4-7 days (by the classical tests described in the

standardized method) against 4-7 days for the reference method

Inter-laboratory study

The bias between the reference and the alternative methods shows satisfying

and correct values

- level 1 (100 - 1 000 CFUg) bias = + 007 log CFUg

- level 2 (1 000 - 10 000 CFUg) bias = 002 log CFUg

- level 2 bis (1 000 - 10 000 CFUg) bias = - 004 log CFUg

- level 3 (10 000 - 100 000 CFUg) bias = - 004 log CFUg

The repeatability and the reproducibility of the alternative method look similar

to those of the reference method

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3038 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

22 Extension study (realised by ASEPT 2007)

An extension study was performed in 2007 for a new confirmatory test OBIS

MONO

150 Listeria monocytogenes strains and 100 non-target strains were isolated

onto TSAYE and BrillianceTM Listeria Agar plates All the Listeria

monocytogenes and the non-target strains gave the expected results

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3138 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Appendix 1 ndash Flow diagram of the Listeria PrecisTM method

x g sample + 9 x ml buffered peptone water

Revivification step for 1 h plusmn 5 min at 20degC plusmn 1degC

Streaking 1 ml onto 3 Brilliance Listeria Agar plates (mother suspension)

or 01 ml onto 1 Brilliance Listeria Agar plates (mother suspension and decimal dilutions)

Incubation for 48 h plusmn 3 h at 37degC plusmn 1degC

Confirmatory tests on at least one typical colony

- by the tests described in the ISO method

- by applying the OBIS mono test on an isolated colony

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3238 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Appendix 2 ndash ISO 11290-2 (1998) amp ISO 11290-2A1 (2004) Horizontal

method for the detection and enumeration of Listeria monocytogenes -

Part 2 enumeration of Listeria monocytogenes in foods

10 g cheese + 90 ml buffered peptone water or Half Fraser Broth (without supplements) (110 dilution)

Stomach 1 min 30 seconds

Keep at 20degC plusmn 2degC for 1 h plusmn 5 min

Decimal dilutions in 9 ml peptone salt tubes

Dilution 110

Dilution 1100

Dilution 11000

1 ml on 3 Chromogenic Listeria medium (ISO) plates (x 2) (-1)

01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-2)

01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-3)

01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-4)

Incubation at 37degC plusmn 1degC

24 h - 48 h

Numerate typical colonies at 24 h

(bluegreen surrounded by an opaque halo)

Numerate at 48 h

Streak typical colonies on TSYEA

(5 coloniesplate 2 successive dilutions)

Confirmation tests

Catalase

Gram stain

For confirmation of L monocytogenes

Haemolysis

Carbohydrate (rhamnose and xylose) utilisation

CAMP Test

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3338 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Appendix 3 ndash Mandelrsquos graphics

Laboratoire h Ref

Niveau 1 h Ref

Niveau 2 h Ref

Niveau 3 h Alt

Niveau 1 h Alt

Niveau 2 h Alt

Niveau 3 h5 h1 h5 h1

A -050 -015 083 062 -212 -039 198 299 -198 -299

B -261 -270 021 -077 -058 173 198 299 -198 -299

C -151 -031 -014 -015 -004 -005 198 299 -198 -299

D 038 040 -137 000 072 207 198 299 -198 -299

E -001 090 155 -052 056 -101 198 299 -198 -299

F -112 -321 -153 070 -325 023 198 299 -198 -299

G 042 -071 040 147 -009 176 198 299 -198 -299

H 169 046 -169 192 018 -083 198 299 -198 -299

L 009 -120 021 -077 -231 005 198 299 -198 -299

M 001 031 014 -032 004 -011 198 299 -198 -299

-300

-200

-100

000

100

200

300

A B C D E F G H L M

Vale

urs

h d

e M

an

del(

rob

uste

s)

Laboratoires

Meacutethode de reacutefeacuterence analyse robuste

h Ref Niveau 1

h Ref Niveau 2

h Ref Niveau 3

h 5=198

h 1=299

-300

-200

-100

000

100

200

300

A B C D E F G H L M

Vale

urs

de h

de M

an

del(

rob

uste

s)

Laboratoires

Meacutethode alternative analyse robuste

h Alt Niveau 1

h Alt Niveau 2

h Alt Niveau 2

h 1=299

h 5=198

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3438 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Laboratoire k Ref

Niveau 1 k Ref

Niveau 2 k Ref

Niveau 3 k Alt

Niveau 1 k Alt

Niveau 2 k Alt

Niveau 3 k1 k5

A 000 089 142 084 162 046 259 181

B 125 087 073 104 075 046 259 181

C 117 013 091 188 022 083 259 181

D 052 036 069 000 035 083 259 181

E 028 034 037 138 019 112 259 181

F 140 222 018 027 281 112 259 181

G 088 068 024 131 145 000 259 181

H 129 094 173 016 061 000 259 181

L 069 000 073 104 270 224 259 181

M 000 048 038 043 099 241 259 181

000

050

100

150

200

250

300

350

400

A B C D E F G H L M

Vale

urs

k d

e M

an

del(

rob

uste

s)

Laboratoires

Meacutethode de reacutefeacuterence analyse robuste

k Ref Niveau 1k Ref Niveau 2k Ref Niveau 3

k 1=259

k 5=181

000

050

100

150

200

250

300

A B C D E F G H L M

Vale

urs

k d

e M

an

del

(ro

bu

ste

s)

Laboratoires

Meacutethode alternative analyse robuste

k Alt Niveau 3

k Alt Niveau 1

k Alt Niveau 2

k 1=259

k 5=181

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3538 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Appendix 4 ndash Statistical results

Level 1

Nombre de laboratoires (p) 10

A 1602 1602 1602 000 0000 0000 -0495 0000 A 1845 1602 1724 017 0122 -0122 0621 0840 0122

B 1602 1176 1389 030 0213 -0213 -2613 1247 B 1602 1301 1452 021 0151 -0151 -0769 1041 0062

C 1699 1301 1500 028 0199 -0199 -1510 1165 C 1301 1845 1573 038 -0272 0272 -0148 1881 0073

D 1602 1778 1690 012 -0088 0088 0380 0516 D 1602 1602 1602 000 0000 0000 0000 0000 -0088

E 1699 1602 1651 007 0048 -0048 -0013 0284 E 1699 1301 1500 028 0199 -0199 -0522 1376 -0151

F 1778 1301 1540 034 0239 -0239 -1116 1397 F 1778 1699 1739 006 0040 -0040 0698 0274 0199

G 1845 1544 1695 021 0151 -0151 0425 0881 G 2079 1699 1889 027 0190 -0190 1467 1315 0194

H 2041 1602 1822 031 0220 -0220 1689 1286 H 2000 1954 1977 003 0023 -0023 1917 0158 0155

L 1544 1778 1661 017 -0117 0117 0092 0685 L 1301 1602 1452 021 -0151 0151 -0769 1041 -0210

M 1653 1653 1653 000 0000 0000 0013 0000 M 1477 1602 1540 009 -0062 0062 -0319 0432 -0114

m 165186 m 160206 mediane D 00678

Cn(2p) 1867 Cn(2p) 1867 Cp 16101

Cn(p) 16101 Cn(p) 16101 Qn(D) 0092926

n=2p n 20 n=2p n 20 Qdiff 01496169

f 11 f 11 t 114

l 55 l 55

n=p n 10 n=p n 10

f 6 f 6

l 15 l 15

Qn(2p) 0091465 Qn(2p) 007745

Qn(p) 0062469 Qn(p) 0121519

Qintra 017078 Qintra 014461

Qinter 010058 Qinter 019565

Sr(eacutecart-type de reacutepeacutetabiliteacute) 02415 Sr 02045

RSDr(coefficient de variation de la reacutepeacutetabiliteacute)1462 RSDr 1277

r(repeatability limit)) 0676 r 0573

SL(eacutecart-type interlaboratoire) -001905 SLlt0 SL 001737

SR(eacutecart-type de reproductibiliteacute) 02415 SR 02433

RSDR(coefficient de variation de la reproductibiliteacute)0146 RSDR 0152

R(limite de reproductibiliteacute)) 06762 R(limite de reproductibiliteacute))068123

h k h MoyenneLaboratoires

Niveau 1

si LaboratoiresDeviationMoyenne Dk DeviationsiAlt

Niveau 1

Ref

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3638 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Level 2

Nombre de laboratoires (p) 10

A 2681 2613 2647 005 0034 -0034 -0151 0891 A 2322 2531 2427 015 -0105 0105 -2119 1620 -0220

B 2544 2477 2511 005 0033 -0033 -2699 0872 B 2556 2653 2605 007 -0048 0048 -0581 0750 0094

C 2643 2633 2638 001 0005 -0005 -0311 0130 C 2681 2653 2667 002 0014 -0014 -0040 0217 0029

D 2690 2663 2676 002 0014 -0014 0400 0357 D 2778 2732 2755 003 0023 -0023 0721 0354 0079

E 2690 2716 2703 002 -0013 0013 0897 0336 E 2724 2748 2736 002 -0012 0012 0556 0185 0033

F 2568 2398 2483 012 0085 -0085 -3214 2217 F 2477 2114 2296 026 0182 -0182 -3255 2811 -0188

G 2643 2591 2617 004 0026 -0026 -0707 0682 G 2756 2568 2662 013 0094 -0094 -0085 1453 0045

H 2716 2643 2680 005 0036 -0036 0460 0945 H 2653 2732 2693 006 -0040 0040 0181 0613 0013

L 2591 2591 2591 000 0000 0000 -1196 0000 L 2230 2580 2405 025 -0175 0175 -2307 2704 -0186

M 2653 2690 2672 003 -0018 0018 0311 0481 M 2740 2613 2677 009 0064 -0064 0040 0987 0005

m 2655082 m 26719001 mediane D 00209

Cn(2p) 1867 Cn(2p) 1867 Cp 16101

Cn(p) 16101 Cn(p) 16101 Qn(D) 0039911

n=2p n 20 n=2p n 20 Qdiff 00642591

f 11 f 11 t 082

l 55 l 55

n=p n 10 n=p n 10

f 6 f 6

l 15 l 15

Qn(2p) 0020570 Qn(2p) 00345985

Qn(p) 00332437 Qn(p) 00718158

Qintra 003841 Qintra 006460

Qinter 005352 Qinter 011563

Sr(eacutecart-type de reacutepeacutetabiliteacute) 00543 Sr 00914

RSDr(coefficient de variation de la reacutepeacutetabiliteacute)205 RSDr 342

r(repeatability limit)) 0152 r 0256

SL(eacutecart-type interlaboratoire) 000139 SL 000920

SR(eacutecart-type de reproductibiliteacute) 00659 SR 01325

RSDR(coefficient de variation de la reproductibiliteacute)0025 RSDR 0050

R(limite de reproductibiliteacute)) 018446 R(limite de reproductibiliteacute))03708618

Laboratoires

Niveau 2

Moyenne si Laboratoires

Niveau 2

DRef Alt Moyenne si Deviation h Deviation h k k

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3738 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Level 2 bis

Nombre de laboratoires (p) 10

A 3000 2477 2739 037 0261 -0261 0832 1418 A 2602 2477 2540 009 0062 -0062 -0391 0465 -0199

B 2544 2813 2678 019 -0134 0134 0211 0729 B 2903 2778 2841 009 0062 -0062 1732 0465 0162

C 2477 2813 2645 024 -0168 0168 -0135 0911 C 2477 2699 2588 016 -0111 0111 -0049 0826 -0057

D 2398 2653 2526 018 -0128 0128 -1370 0692 D 2778 3000 2889 016 -0111 0111 2074 0826 0363

E 2875 2740 2808 010 0067 -0067 1547 0365 E 2602 2301 2452 021 0151 -0151 -1012 1120 -0356

F 2477 2544 2511 005 -0033 0033 -1525 0182 F 2778 2477 2628 021 0151 -0151 0230 1120 0117

G 2653 2740 2697 006 -0044 0044 0400 0236 G 2845 2845 2845 000 0000 0000 1764 0000 0148

H 2176 2813 2495 045 -0318 0318 -1692 1727 H 2477 2477 2477 000 0000 0000 -0832 0000 -0017

L 2813 2544 2678 019 0134 -0134 0211 0729 L 2903 2301 2602 043 0301 -0301 0049 2241 -0076

M 2602 2740 2671 010 -0069 0069 0135 0375 M 2903 2255 2579 046 0324 -0324 -0112 2411 -0092

m 2658114 m 259505 mediane D -00372

Cn(2p) 1867 Cn(2p) 1867 Cp 16101

Cn(p) 16101 Cn(p) 16101 Qn(D) 01656915

n=2p n 20 n=2p n 20 Qdiff 02667753

f 11 f 11 t 035

l 55 l 55

n=p n 10 n=p n 10

f 6 f 6

l 15 l 15

Qn(2p) 0098745 Qn(2p) 007195

Qn(p) 006007 Qn(p) 008805

Qintra 018437 Qintra 013435

Qinter 009672 Qinter 014176

Sr(eacutecart-type de reacutepeacutetabiliteacute) 02607 Sr 01900

RSDr(coefficient de variation de la reacutepeacutetabiliteacute)981 RSDr 732

r(repeatability limit)) 0730 r 0532

SL(eacutecart-type interlaboratoire) -002464 SLlt0 SL 000205

SR(eacutecart-type de reproductibiliteacute) 02607 SR 01953

RSDR(coefficient de variation de la reproductibiliteacute)0098 RSDR 0075

R(limite de reproductibiliteacute)) 072996 R(limite de reproductibiliteacute))054686

Laboratoires

Niveau 2 bis

Moyenne si Laboratoires

Niveau 2 bis

DRef Alt Moyenne si Deviation h Deviation h k k

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3838 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Level 3

Nombre de laboratoires (p) 10

A 3690 3663 3676 002 0014 -0014 -0311 0513 A 3690 3708 3699 001 -0009 0009 0354 0257 0022

B 3613 3672 3642 004 -0030 0030 -1851 1109 B 3708 3568 3638 010 0070 -0070 -0844 2061 -0005

C 3663 3732 3698 005 -0035 0035 0645 1302 C 3663 3699 3681 003 -0018 0018 0000 0536 -0017

D 3690 3690 3690 000 0000 0000 0311 0000 D 3643 3663 3653 001 -0010 0010 -0545 0286 -0037

E 3732 3653 3693 006 0040 -0040 0429 1480 E 3613 3732 3673 008 -0060 0060 -0162 1769 -0020

F 3643 3672 3658 002 -0014 0014 -1157 0536 F 3690 3771 3731 006 -0040 0040 0975 1193 0073

G 3699 3653 3676 003 0023 -0023 -0328 0856 G 3602 3643 3623 003 -0021 0021 -1141 0612 -0053

H 3623 3672 3648 003 -0024 0024 -1615 0913 H 3748 3681 3715 005 0033 -0033 0664 0990 0067

L 3724 3663 3694 004 0031 -0031 0461 1150 L 3748 3771 3760 002 -0011 0011 1544 0335 0066

M 3690 3690 3690 000 0000 0000 0311 0000 M 3806 3681 3744 009 0062 -0062 1234 1848 0054

m 368334 m 368086 mediane D 00089

Cn(2p) 1867 Cn(2p) 1867 Cp 16101

Cn(p) 16101 Cn(p) 16101 Qn(D) 00325359

n=2p n 20 n=2p n 20 Qdiff 00523852

f 11 f 11 t 043

l 55 l 55

n=p n 10 n=p n 10

f 6 f 6

l 15 l 15

Qn(2p) 0014323 Qn(2p) 001811

Qn(p) 0013719 Qn(p) 003164

Qintra 002674 Qintra 003381

Qinter 002209 Qinter 005094

Sr(eacutecart-type de reacutepeacutetabiliteacute) 00378 Sr 00478

RSDr(coefficient de variation de la reacutepeacutetabiliteacute)103 RSDr 130

r(repeatability limit)) 0106 r 0134

SL(eacutecart-type interlaboratoire) -000023 SLlt0 SL 000145

SR(eacutecart-type de reproductibiliteacute) 00378 SR 00611

RSDR(coefficient de variation de la reproductibiliteacute)0010 RSDR 0017

R(limite de reproductibiliteacute)) 0105894 R(limite de reproductibiliteacute))017119

Ref Alt DLaboratoires

Niveau 3

Moyenne si Deviation h k Laboratoires

Niveau 3

Moyenne si Deviation h k

Page 30: ÞÀ¿¸7 ú Y5zñLjÜ< /u +æü t¬Á ... - NF Validation · NF VALIDATION Validation of alternative analytical methods Application in food microbiology ... 500 – 1 000 1 000 –

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3038 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

22 Extension study (realised by ASEPT 2007)

An extension study was performed in 2007 for a new confirmatory test OBIS

MONO

150 Listeria monocytogenes strains and 100 non-target strains were isolated

onto TSAYE and BrillianceTM Listeria Agar plates All the Listeria

monocytogenes and the non-target strains gave the expected results

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3138 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Appendix 1 ndash Flow diagram of the Listeria PrecisTM method

x g sample + 9 x ml buffered peptone water

Revivification step for 1 h plusmn 5 min at 20degC plusmn 1degC

Streaking 1 ml onto 3 Brilliance Listeria Agar plates (mother suspension)

or 01 ml onto 1 Brilliance Listeria Agar plates (mother suspension and decimal dilutions)

Incubation for 48 h plusmn 3 h at 37degC plusmn 1degC

Confirmatory tests on at least one typical colony

- by the tests described in the ISO method

- by applying the OBIS mono test on an isolated colony

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3238 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Appendix 2 ndash ISO 11290-2 (1998) amp ISO 11290-2A1 (2004) Horizontal

method for the detection and enumeration of Listeria monocytogenes -

Part 2 enumeration of Listeria monocytogenes in foods

10 g cheese + 90 ml buffered peptone water or Half Fraser Broth (without supplements) (110 dilution)

Stomach 1 min 30 seconds

Keep at 20degC plusmn 2degC for 1 h plusmn 5 min

Decimal dilutions in 9 ml peptone salt tubes

Dilution 110

Dilution 1100

Dilution 11000

1 ml on 3 Chromogenic Listeria medium (ISO) plates (x 2) (-1)

01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-2)

01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-3)

01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-4)

Incubation at 37degC plusmn 1degC

24 h - 48 h

Numerate typical colonies at 24 h

(bluegreen surrounded by an opaque halo)

Numerate at 48 h

Streak typical colonies on TSYEA

(5 coloniesplate 2 successive dilutions)

Confirmation tests

Catalase

Gram stain

For confirmation of L monocytogenes

Haemolysis

Carbohydrate (rhamnose and xylose) utilisation

CAMP Test

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3338 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Appendix 3 ndash Mandelrsquos graphics

Laboratoire h Ref

Niveau 1 h Ref

Niveau 2 h Ref

Niveau 3 h Alt

Niveau 1 h Alt

Niveau 2 h Alt

Niveau 3 h5 h1 h5 h1

A -050 -015 083 062 -212 -039 198 299 -198 -299

B -261 -270 021 -077 -058 173 198 299 -198 -299

C -151 -031 -014 -015 -004 -005 198 299 -198 -299

D 038 040 -137 000 072 207 198 299 -198 -299

E -001 090 155 -052 056 -101 198 299 -198 -299

F -112 -321 -153 070 -325 023 198 299 -198 -299

G 042 -071 040 147 -009 176 198 299 -198 -299

H 169 046 -169 192 018 -083 198 299 -198 -299

L 009 -120 021 -077 -231 005 198 299 -198 -299

M 001 031 014 -032 004 -011 198 299 -198 -299

-300

-200

-100

000

100

200

300

A B C D E F G H L M

Vale

urs

h d

e M

an

del(

rob

uste

s)

Laboratoires

Meacutethode de reacutefeacuterence analyse robuste

h Ref Niveau 1

h Ref Niveau 2

h Ref Niveau 3

h 5=198

h 1=299

-300

-200

-100

000

100

200

300

A B C D E F G H L M

Vale

urs

de h

de M

an

del(

rob

uste

s)

Laboratoires

Meacutethode alternative analyse robuste

h Alt Niveau 1

h Alt Niveau 2

h Alt Niveau 2

h 1=299

h 5=198

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3438 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Laboratoire k Ref

Niveau 1 k Ref

Niveau 2 k Ref

Niveau 3 k Alt

Niveau 1 k Alt

Niveau 2 k Alt

Niveau 3 k1 k5

A 000 089 142 084 162 046 259 181

B 125 087 073 104 075 046 259 181

C 117 013 091 188 022 083 259 181

D 052 036 069 000 035 083 259 181

E 028 034 037 138 019 112 259 181

F 140 222 018 027 281 112 259 181

G 088 068 024 131 145 000 259 181

H 129 094 173 016 061 000 259 181

L 069 000 073 104 270 224 259 181

M 000 048 038 043 099 241 259 181

000

050

100

150

200

250

300

350

400

A B C D E F G H L M

Vale

urs

k d

e M

an

del(

rob

uste

s)

Laboratoires

Meacutethode de reacutefeacuterence analyse robuste

k Ref Niveau 1k Ref Niveau 2k Ref Niveau 3

k 1=259

k 5=181

000

050

100

150

200

250

300

A B C D E F G H L M

Vale

urs

k d

e M

an

del

(ro

bu

ste

s)

Laboratoires

Meacutethode alternative analyse robuste

k Alt Niveau 3

k Alt Niveau 1

k Alt Niveau 2

k 1=259

k 5=181

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3538 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Appendix 4 ndash Statistical results

Level 1

Nombre de laboratoires (p) 10

A 1602 1602 1602 000 0000 0000 -0495 0000 A 1845 1602 1724 017 0122 -0122 0621 0840 0122

B 1602 1176 1389 030 0213 -0213 -2613 1247 B 1602 1301 1452 021 0151 -0151 -0769 1041 0062

C 1699 1301 1500 028 0199 -0199 -1510 1165 C 1301 1845 1573 038 -0272 0272 -0148 1881 0073

D 1602 1778 1690 012 -0088 0088 0380 0516 D 1602 1602 1602 000 0000 0000 0000 0000 -0088

E 1699 1602 1651 007 0048 -0048 -0013 0284 E 1699 1301 1500 028 0199 -0199 -0522 1376 -0151

F 1778 1301 1540 034 0239 -0239 -1116 1397 F 1778 1699 1739 006 0040 -0040 0698 0274 0199

G 1845 1544 1695 021 0151 -0151 0425 0881 G 2079 1699 1889 027 0190 -0190 1467 1315 0194

H 2041 1602 1822 031 0220 -0220 1689 1286 H 2000 1954 1977 003 0023 -0023 1917 0158 0155

L 1544 1778 1661 017 -0117 0117 0092 0685 L 1301 1602 1452 021 -0151 0151 -0769 1041 -0210

M 1653 1653 1653 000 0000 0000 0013 0000 M 1477 1602 1540 009 -0062 0062 -0319 0432 -0114

m 165186 m 160206 mediane D 00678

Cn(2p) 1867 Cn(2p) 1867 Cp 16101

Cn(p) 16101 Cn(p) 16101 Qn(D) 0092926

n=2p n 20 n=2p n 20 Qdiff 01496169

f 11 f 11 t 114

l 55 l 55

n=p n 10 n=p n 10

f 6 f 6

l 15 l 15

Qn(2p) 0091465 Qn(2p) 007745

Qn(p) 0062469 Qn(p) 0121519

Qintra 017078 Qintra 014461

Qinter 010058 Qinter 019565

Sr(eacutecart-type de reacutepeacutetabiliteacute) 02415 Sr 02045

RSDr(coefficient de variation de la reacutepeacutetabiliteacute)1462 RSDr 1277

r(repeatability limit)) 0676 r 0573

SL(eacutecart-type interlaboratoire) -001905 SLlt0 SL 001737

SR(eacutecart-type de reproductibiliteacute) 02415 SR 02433

RSDR(coefficient de variation de la reproductibiliteacute)0146 RSDR 0152

R(limite de reproductibiliteacute)) 06762 R(limite de reproductibiliteacute))068123

h k h MoyenneLaboratoires

Niveau 1

si LaboratoiresDeviationMoyenne Dk DeviationsiAlt

Niveau 1

Ref

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3638 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Level 2

Nombre de laboratoires (p) 10

A 2681 2613 2647 005 0034 -0034 -0151 0891 A 2322 2531 2427 015 -0105 0105 -2119 1620 -0220

B 2544 2477 2511 005 0033 -0033 -2699 0872 B 2556 2653 2605 007 -0048 0048 -0581 0750 0094

C 2643 2633 2638 001 0005 -0005 -0311 0130 C 2681 2653 2667 002 0014 -0014 -0040 0217 0029

D 2690 2663 2676 002 0014 -0014 0400 0357 D 2778 2732 2755 003 0023 -0023 0721 0354 0079

E 2690 2716 2703 002 -0013 0013 0897 0336 E 2724 2748 2736 002 -0012 0012 0556 0185 0033

F 2568 2398 2483 012 0085 -0085 -3214 2217 F 2477 2114 2296 026 0182 -0182 -3255 2811 -0188

G 2643 2591 2617 004 0026 -0026 -0707 0682 G 2756 2568 2662 013 0094 -0094 -0085 1453 0045

H 2716 2643 2680 005 0036 -0036 0460 0945 H 2653 2732 2693 006 -0040 0040 0181 0613 0013

L 2591 2591 2591 000 0000 0000 -1196 0000 L 2230 2580 2405 025 -0175 0175 -2307 2704 -0186

M 2653 2690 2672 003 -0018 0018 0311 0481 M 2740 2613 2677 009 0064 -0064 0040 0987 0005

m 2655082 m 26719001 mediane D 00209

Cn(2p) 1867 Cn(2p) 1867 Cp 16101

Cn(p) 16101 Cn(p) 16101 Qn(D) 0039911

n=2p n 20 n=2p n 20 Qdiff 00642591

f 11 f 11 t 082

l 55 l 55

n=p n 10 n=p n 10

f 6 f 6

l 15 l 15

Qn(2p) 0020570 Qn(2p) 00345985

Qn(p) 00332437 Qn(p) 00718158

Qintra 003841 Qintra 006460

Qinter 005352 Qinter 011563

Sr(eacutecart-type de reacutepeacutetabiliteacute) 00543 Sr 00914

RSDr(coefficient de variation de la reacutepeacutetabiliteacute)205 RSDr 342

r(repeatability limit)) 0152 r 0256

SL(eacutecart-type interlaboratoire) 000139 SL 000920

SR(eacutecart-type de reproductibiliteacute) 00659 SR 01325

RSDR(coefficient de variation de la reproductibiliteacute)0025 RSDR 0050

R(limite de reproductibiliteacute)) 018446 R(limite de reproductibiliteacute))03708618

Laboratoires

Niveau 2

Moyenne si Laboratoires

Niveau 2

DRef Alt Moyenne si Deviation h Deviation h k k

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3738 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Level 2 bis

Nombre de laboratoires (p) 10

A 3000 2477 2739 037 0261 -0261 0832 1418 A 2602 2477 2540 009 0062 -0062 -0391 0465 -0199

B 2544 2813 2678 019 -0134 0134 0211 0729 B 2903 2778 2841 009 0062 -0062 1732 0465 0162

C 2477 2813 2645 024 -0168 0168 -0135 0911 C 2477 2699 2588 016 -0111 0111 -0049 0826 -0057

D 2398 2653 2526 018 -0128 0128 -1370 0692 D 2778 3000 2889 016 -0111 0111 2074 0826 0363

E 2875 2740 2808 010 0067 -0067 1547 0365 E 2602 2301 2452 021 0151 -0151 -1012 1120 -0356

F 2477 2544 2511 005 -0033 0033 -1525 0182 F 2778 2477 2628 021 0151 -0151 0230 1120 0117

G 2653 2740 2697 006 -0044 0044 0400 0236 G 2845 2845 2845 000 0000 0000 1764 0000 0148

H 2176 2813 2495 045 -0318 0318 -1692 1727 H 2477 2477 2477 000 0000 0000 -0832 0000 -0017

L 2813 2544 2678 019 0134 -0134 0211 0729 L 2903 2301 2602 043 0301 -0301 0049 2241 -0076

M 2602 2740 2671 010 -0069 0069 0135 0375 M 2903 2255 2579 046 0324 -0324 -0112 2411 -0092

m 2658114 m 259505 mediane D -00372

Cn(2p) 1867 Cn(2p) 1867 Cp 16101

Cn(p) 16101 Cn(p) 16101 Qn(D) 01656915

n=2p n 20 n=2p n 20 Qdiff 02667753

f 11 f 11 t 035

l 55 l 55

n=p n 10 n=p n 10

f 6 f 6

l 15 l 15

Qn(2p) 0098745 Qn(2p) 007195

Qn(p) 006007 Qn(p) 008805

Qintra 018437 Qintra 013435

Qinter 009672 Qinter 014176

Sr(eacutecart-type de reacutepeacutetabiliteacute) 02607 Sr 01900

RSDr(coefficient de variation de la reacutepeacutetabiliteacute)981 RSDr 732

r(repeatability limit)) 0730 r 0532

SL(eacutecart-type interlaboratoire) -002464 SLlt0 SL 000205

SR(eacutecart-type de reproductibiliteacute) 02607 SR 01953

RSDR(coefficient de variation de la reproductibiliteacute)0098 RSDR 0075

R(limite de reproductibiliteacute)) 072996 R(limite de reproductibiliteacute))054686

Laboratoires

Niveau 2 bis

Moyenne si Laboratoires

Niveau 2 bis

DRef Alt Moyenne si Deviation h Deviation h k k

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3838 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Level 3

Nombre de laboratoires (p) 10

A 3690 3663 3676 002 0014 -0014 -0311 0513 A 3690 3708 3699 001 -0009 0009 0354 0257 0022

B 3613 3672 3642 004 -0030 0030 -1851 1109 B 3708 3568 3638 010 0070 -0070 -0844 2061 -0005

C 3663 3732 3698 005 -0035 0035 0645 1302 C 3663 3699 3681 003 -0018 0018 0000 0536 -0017

D 3690 3690 3690 000 0000 0000 0311 0000 D 3643 3663 3653 001 -0010 0010 -0545 0286 -0037

E 3732 3653 3693 006 0040 -0040 0429 1480 E 3613 3732 3673 008 -0060 0060 -0162 1769 -0020

F 3643 3672 3658 002 -0014 0014 -1157 0536 F 3690 3771 3731 006 -0040 0040 0975 1193 0073

G 3699 3653 3676 003 0023 -0023 -0328 0856 G 3602 3643 3623 003 -0021 0021 -1141 0612 -0053

H 3623 3672 3648 003 -0024 0024 -1615 0913 H 3748 3681 3715 005 0033 -0033 0664 0990 0067

L 3724 3663 3694 004 0031 -0031 0461 1150 L 3748 3771 3760 002 -0011 0011 1544 0335 0066

M 3690 3690 3690 000 0000 0000 0311 0000 M 3806 3681 3744 009 0062 -0062 1234 1848 0054

m 368334 m 368086 mediane D 00089

Cn(2p) 1867 Cn(2p) 1867 Cp 16101

Cn(p) 16101 Cn(p) 16101 Qn(D) 00325359

n=2p n 20 n=2p n 20 Qdiff 00523852

f 11 f 11 t 043

l 55 l 55

n=p n 10 n=p n 10

f 6 f 6

l 15 l 15

Qn(2p) 0014323 Qn(2p) 001811

Qn(p) 0013719 Qn(p) 003164

Qintra 002674 Qintra 003381

Qinter 002209 Qinter 005094

Sr(eacutecart-type de reacutepeacutetabiliteacute) 00378 Sr 00478

RSDr(coefficient de variation de la reacutepeacutetabiliteacute)103 RSDr 130

r(repeatability limit)) 0106 r 0134

SL(eacutecart-type interlaboratoire) -000023 SLlt0 SL 000145

SR(eacutecart-type de reproductibiliteacute) 00378 SR 00611

RSDR(coefficient de variation de la reproductibiliteacute)0010 RSDR 0017

R(limite de reproductibiliteacute)) 0105894 R(limite de reproductibiliteacute))017119

Ref Alt DLaboratoires

Niveau 3

Moyenne si Deviation h k Laboratoires

Niveau 3

Moyenne si Deviation h k

Page 31: ÞÀ¿¸7 ú Y5zñLjÜ< /u +æü t¬Á ... - NF Validation · NF VALIDATION Validation of alternative analytical methods Application in food microbiology ... 500 – 1 000 1 000 –

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3138 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Appendix 1 ndash Flow diagram of the Listeria PrecisTM method

x g sample + 9 x ml buffered peptone water

Revivification step for 1 h plusmn 5 min at 20degC plusmn 1degC

Streaking 1 ml onto 3 Brilliance Listeria Agar plates (mother suspension)

or 01 ml onto 1 Brilliance Listeria Agar plates (mother suspension and decimal dilutions)

Incubation for 48 h plusmn 3 h at 37degC plusmn 1degC

Confirmatory tests on at least one typical colony

- by the tests described in the ISO method

- by applying the OBIS mono test on an isolated colony

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3238 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Appendix 2 ndash ISO 11290-2 (1998) amp ISO 11290-2A1 (2004) Horizontal

method for the detection and enumeration of Listeria monocytogenes -

Part 2 enumeration of Listeria monocytogenes in foods

10 g cheese + 90 ml buffered peptone water or Half Fraser Broth (without supplements) (110 dilution)

Stomach 1 min 30 seconds

Keep at 20degC plusmn 2degC for 1 h plusmn 5 min

Decimal dilutions in 9 ml peptone salt tubes

Dilution 110

Dilution 1100

Dilution 11000

1 ml on 3 Chromogenic Listeria medium (ISO) plates (x 2) (-1)

01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-2)

01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-3)

01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-4)

Incubation at 37degC plusmn 1degC

24 h - 48 h

Numerate typical colonies at 24 h

(bluegreen surrounded by an opaque halo)

Numerate at 48 h

Streak typical colonies on TSYEA

(5 coloniesplate 2 successive dilutions)

Confirmation tests

Catalase

Gram stain

For confirmation of L monocytogenes

Haemolysis

Carbohydrate (rhamnose and xylose) utilisation

CAMP Test

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3338 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Appendix 3 ndash Mandelrsquos graphics

Laboratoire h Ref

Niveau 1 h Ref

Niveau 2 h Ref

Niveau 3 h Alt

Niveau 1 h Alt

Niveau 2 h Alt

Niveau 3 h5 h1 h5 h1

A -050 -015 083 062 -212 -039 198 299 -198 -299

B -261 -270 021 -077 -058 173 198 299 -198 -299

C -151 -031 -014 -015 -004 -005 198 299 -198 -299

D 038 040 -137 000 072 207 198 299 -198 -299

E -001 090 155 -052 056 -101 198 299 -198 -299

F -112 -321 -153 070 -325 023 198 299 -198 -299

G 042 -071 040 147 -009 176 198 299 -198 -299

H 169 046 -169 192 018 -083 198 299 -198 -299

L 009 -120 021 -077 -231 005 198 299 -198 -299

M 001 031 014 -032 004 -011 198 299 -198 -299

-300

-200

-100

000

100

200

300

A B C D E F G H L M

Vale

urs

h d

e M

an

del(

rob

uste

s)

Laboratoires

Meacutethode de reacutefeacuterence analyse robuste

h Ref Niveau 1

h Ref Niveau 2

h Ref Niveau 3

h 5=198

h 1=299

-300

-200

-100

000

100

200

300

A B C D E F G H L M

Vale

urs

de h

de M

an

del(

rob

uste

s)

Laboratoires

Meacutethode alternative analyse robuste

h Alt Niveau 1

h Alt Niveau 2

h Alt Niveau 2

h 1=299

h 5=198

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3438 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Laboratoire k Ref

Niveau 1 k Ref

Niveau 2 k Ref

Niveau 3 k Alt

Niveau 1 k Alt

Niveau 2 k Alt

Niveau 3 k1 k5

A 000 089 142 084 162 046 259 181

B 125 087 073 104 075 046 259 181

C 117 013 091 188 022 083 259 181

D 052 036 069 000 035 083 259 181

E 028 034 037 138 019 112 259 181

F 140 222 018 027 281 112 259 181

G 088 068 024 131 145 000 259 181

H 129 094 173 016 061 000 259 181

L 069 000 073 104 270 224 259 181

M 000 048 038 043 099 241 259 181

000

050

100

150

200

250

300

350

400

A B C D E F G H L M

Vale

urs

k d

e M

an

del(

rob

uste

s)

Laboratoires

Meacutethode de reacutefeacuterence analyse robuste

k Ref Niveau 1k Ref Niveau 2k Ref Niveau 3

k 1=259

k 5=181

000

050

100

150

200

250

300

A B C D E F G H L M

Vale

urs

k d

e M

an

del

(ro

bu

ste

s)

Laboratoires

Meacutethode alternative analyse robuste

k Alt Niveau 3

k Alt Niveau 1

k Alt Niveau 2

k 1=259

k 5=181

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3538 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Appendix 4 ndash Statistical results

Level 1

Nombre de laboratoires (p) 10

A 1602 1602 1602 000 0000 0000 -0495 0000 A 1845 1602 1724 017 0122 -0122 0621 0840 0122

B 1602 1176 1389 030 0213 -0213 -2613 1247 B 1602 1301 1452 021 0151 -0151 -0769 1041 0062

C 1699 1301 1500 028 0199 -0199 -1510 1165 C 1301 1845 1573 038 -0272 0272 -0148 1881 0073

D 1602 1778 1690 012 -0088 0088 0380 0516 D 1602 1602 1602 000 0000 0000 0000 0000 -0088

E 1699 1602 1651 007 0048 -0048 -0013 0284 E 1699 1301 1500 028 0199 -0199 -0522 1376 -0151

F 1778 1301 1540 034 0239 -0239 -1116 1397 F 1778 1699 1739 006 0040 -0040 0698 0274 0199

G 1845 1544 1695 021 0151 -0151 0425 0881 G 2079 1699 1889 027 0190 -0190 1467 1315 0194

H 2041 1602 1822 031 0220 -0220 1689 1286 H 2000 1954 1977 003 0023 -0023 1917 0158 0155

L 1544 1778 1661 017 -0117 0117 0092 0685 L 1301 1602 1452 021 -0151 0151 -0769 1041 -0210

M 1653 1653 1653 000 0000 0000 0013 0000 M 1477 1602 1540 009 -0062 0062 -0319 0432 -0114

m 165186 m 160206 mediane D 00678

Cn(2p) 1867 Cn(2p) 1867 Cp 16101

Cn(p) 16101 Cn(p) 16101 Qn(D) 0092926

n=2p n 20 n=2p n 20 Qdiff 01496169

f 11 f 11 t 114

l 55 l 55

n=p n 10 n=p n 10

f 6 f 6

l 15 l 15

Qn(2p) 0091465 Qn(2p) 007745

Qn(p) 0062469 Qn(p) 0121519

Qintra 017078 Qintra 014461

Qinter 010058 Qinter 019565

Sr(eacutecart-type de reacutepeacutetabiliteacute) 02415 Sr 02045

RSDr(coefficient de variation de la reacutepeacutetabiliteacute)1462 RSDr 1277

r(repeatability limit)) 0676 r 0573

SL(eacutecart-type interlaboratoire) -001905 SLlt0 SL 001737

SR(eacutecart-type de reproductibiliteacute) 02415 SR 02433

RSDR(coefficient de variation de la reproductibiliteacute)0146 RSDR 0152

R(limite de reproductibiliteacute)) 06762 R(limite de reproductibiliteacute))068123

h k h MoyenneLaboratoires

Niveau 1

si LaboratoiresDeviationMoyenne Dk DeviationsiAlt

Niveau 1

Ref

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3638 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Level 2

Nombre de laboratoires (p) 10

A 2681 2613 2647 005 0034 -0034 -0151 0891 A 2322 2531 2427 015 -0105 0105 -2119 1620 -0220

B 2544 2477 2511 005 0033 -0033 -2699 0872 B 2556 2653 2605 007 -0048 0048 -0581 0750 0094

C 2643 2633 2638 001 0005 -0005 -0311 0130 C 2681 2653 2667 002 0014 -0014 -0040 0217 0029

D 2690 2663 2676 002 0014 -0014 0400 0357 D 2778 2732 2755 003 0023 -0023 0721 0354 0079

E 2690 2716 2703 002 -0013 0013 0897 0336 E 2724 2748 2736 002 -0012 0012 0556 0185 0033

F 2568 2398 2483 012 0085 -0085 -3214 2217 F 2477 2114 2296 026 0182 -0182 -3255 2811 -0188

G 2643 2591 2617 004 0026 -0026 -0707 0682 G 2756 2568 2662 013 0094 -0094 -0085 1453 0045

H 2716 2643 2680 005 0036 -0036 0460 0945 H 2653 2732 2693 006 -0040 0040 0181 0613 0013

L 2591 2591 2591 000 0000 0000 -1196 0000 L 2230 2580 2405 025 -0175 0175 -2307 2704 -0186

M 2653 2690 2672 003 -0018 0018 0311 0481 M 2740 2613 2677 009 0064 -0064 0040 0987 0005

m 2655082 m 26719001 mediane D 00209

Cn(2p) 1867 Cn(2p) 1867 Cp 16101

Cn(p) 16101 Cn(p) 16101 Qn(D) 0039911

n=2p n 20 n=2p n 20 Qdiff 00642591

f 11 f 11 t 082

l 55 l 55

n=p n 10 n=p n 10

f 6 f 6

l 15 l 15

Qn(2p) 0020570 Qn(2p) 00345985

Qn(p) 00332437 Qn(p) 00718158

Qintra 003841 Qintra 006460

Qinter 005352 Qinter 011563

Sr(eacutecart-type de reacutepeacutetabiliteacute) 00543 Sr 00914

RSDr(coefficient de variation de la reacutepeacutetabiliteacute)205 RSDr 342

r(repeatability limit)) 0152 r 0256

SL(eacutecart-type interlaboratoire) 000139 SL 000920

SR(eacutecart-type de reproductibiliteacute) 00659 SR 01325

RSDR(coefficient de variation de la reproductibiliteacute)0025 RSDR 0050

R(limite de reproductibiliteacute)) 018446 R(limite de reproductibiliteacute))03708618

Laboratoires

Niveau 2

Moyenne si Laboratoires

Niveau 2

DRef Alt Moyenne si Deviation h Deviation h k k

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3738 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Level 2 bis

Nombre de laboratoires (p) 10

A 3000 2477 2739 037 0261 -0261 0832 1418 A 2602 2477 2540 009 0062 -0062 -0391 0465 -0199

B 2544 2813 2678 019 -0134 0134 0211 0729 B 2903 2778 2841 009 0062 -0062 1732 0465 0162

C 2477 2813 2645 024 -0168 0168 -0135 0911 C 2477 2699 2588 016 -0111 0111 -0049 0826 -0057

D 2398 2653 2526 018 -0128 0128 -1370 0692 D 2778 3000 2889 016 -0111 0111 2074 0826 0363

E 2875 2740 2808 010 0067 -0067 1547 0365 E 2602 2301 2452 021 0151 -0151 -1012 1120 -0356

F 2477 2544 2511 005 -0033 0033 -1525 0182 F 2778 2477 2628 021 0151 -0151 0230 1120 0117

G 2653 2740 2697 006 -0044 0044 0400 0236 G 2845 2845 2845 000 0000 0000 1764 0000 0148

H 2176 2813 2495 045 -0318 0318 -1692 1727 H 2477 2477 2477 000 0000 0000 -0832 0000 -0017

L 2813 2544 2678 019 0134 -0134 0211 0729 L 2903 2301 2602 043 0301 -0301 0049 2241 -0076

M 2602 2740 2671 010 -0069 0069 0135 0375 M 2903 2255 2579 046 0324 -0324 -0112 2411 -0092

m 2658114 m 259505 mediane D -00372

Cn(2p) 1867 Cn(2p) 1867 Cp 16101

Cn(p) 16101 Cn(p) 16101 Qn(D) 01656915

n=2p n 20 n=2p n 20 Qdiff 02667753

f 11 f 11 t 035

l 55 l 55

n=p n 10 n=p n 10

f 6 f 6

l 15 l 15

Qn(2p) 0098745 Qn(2p) 007195

Qn(p) 006007 Qn(p) 008805

Qintra 018437 Qintra 013435

Qinter 009672 Qinter 014176

Sr(eacutecart-type de reacutepeacutetabiliteacute) 02607 Sr 01900

RSDr(coefficient de variation de la reacutepeacutetabiliteacute)981 RSDr 732

r(repeatability limit)) 0730 r 0532

SL(eacutecart-type interlaboratoire) -002464 SLlt0 SL 000205

SR(eacutecart-type de reproductibiliteacute) 02607 SR 01953

RSDR(coefficient de variation de la reproductibiliteacute)0098 RSDR 0075

R(limite de reproductibiliteacute)) 072996 R(limite de reproductibiliteacute))054686

Laboratoires

Niveau 2 bis

Moyenne si Laboratoires

Niveau 2 bis

DRef Alt Moyenne si Deviation h Deviation h k k

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3838 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Level 3

Nombre de laboratoires (p) 10

A 3690 3663 3676 002 0014 -0014 -0311 0513 A 3690 3708 3699 001 -0009 0009 0354 0257 0022

B 3613 3672 3642 004 -0030 0030 -1851 1109 B 3708 3568 3638 010 0070 -0070 -0844 2061 -0005

C 3663 3732 3698 005 -0035 0035 0645 1302 C 3663 3699 3681 003 -0018 0018 0000 0536 -0017

D 3690 3690 3690 000 0000 0000 0311 0000 D 3643 3663 3653 001 -0010 0010 -0545 0286 -0037

E 3732 3653 3693 006 0040 -0040 0429 1480 E 3613 3732 3673 008 -0060 0060 -0162 1769 -0020

F 3643 3672 3658 002 -0014 0014 -1157 0536 F 3690 3771 3731 006 -0040 0040 0975 1193 0073

G 3699 3653 3676 003 0023 -0023 -0328 0856 G 3602 3643 3623 003 -0021 0021 -1141 0612 -0053

H 3623 3672 3648 003 -0024 0024 -1615 0913 H 3748 3681 3715 005 0033 -0033 0664 0990 0067

L 3724 3663 3694 004 0031 -0031 0461 1150 L 3748 3771 3760 002 -0011 0011 1544 0335 0066

M 3690 3690 3690 000 0000 0000 0311 0000 M 3806 3681 3744 009 0062 -0062 1234 1848 0054

m 368334 m 368086 mediane D 00089

Cn(2p) 1867 Cn(2p) 1867 Cp 16101

Cn(p) 16101 Cn(p) 16101 Qn(D) 00325359

n=2p n 20 n=2p n 20 Qdiff 00523852

f 11 f 11 t 043

l 55 l 55

n=p n 10 n=p n 10

f 6 f 6

l 15 l 15

Qn(2p) 0014323 Qn(2p) 001811

Qn(p) 0013719 Qn(p) 003164

Qintra 002674 Qintra 003381

Qinter 002209 Qinter 005094

Sr(eacutecart-type de reacutepeacutetabiliteacute) 00378 Sr 00478

RSDr(coefficient de variation de la reacutepeacutetabiliteacute)103 RSDr 130

r(repeatability limit)) 0106 r 0134

SL(eacutecart-type interlaboratoire) -000023 SLlt0 SL 000145

SR(eacutecart-type de reproductibiliteacute) 00378 SR 00611

RSDR(coefficient de variation de la reproductibiliteacute)0010 RSDR 0017

R(limite de reproductibiliteacute)) 0105894 R(limite de reproductibiliteacute))017119

Ref Alt DLaboratoires

Niveau 3

Moyenne si Deviation h k Laboratoires

Niveau 3

Moyenne si Deviation h k

Page 32: ÞÀ¿¸7 ú Y5zñLjÜ< /u +æü t¬Á ... - NF Validation · NF VALIDATION Validation of alternative analytical methods Application in food microbiology ... 500 – 1 000 1 000 –

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3238 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Appendix 2 ndash ISO 11290-2 (1998) amp ISO 11290-2A1 (2004) Horizontal

method for the detection and enumeration of Listeria monocytogenes -

Part 2 enumeration of Listeria monocytogenes in foods

10 g cheese + 90 ml buffered peptone water or Half Fraser Broth (without supplements) (110 dilution)

Stomach 1 min 30 seconds

Keep at 20degC plusmn 2degC for 1 h plusmn 5 min

Decimal dilutions in 9 ml peptone salt tubes

Dilution 110

Dilution 1100

Dilution 11000

1 ml on 3 Chromogenic Listeria medium (ISO) plates (x 2) (-1)

01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-2)

01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-3)

01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-4)

Incubation at 37degC plusmn 1degC

24 h - 48 h

Numerate typical colonies at 24 h

(bluegreen surrounded by an opaque halo)

Numerate at 48 h

Streak typical colonies on TSYEA

(5 coloniesplate 2 successive dilutions)

Confirmation tests

Catalase

Gram stain

For confirmation of L monocytogenes

Haemolysis

Carbohydrate (rhamnose and xylose) utilisation

CAMP Test

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3338 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Appendix 3 ndash Mandelrsquos graphics

Laboratoire h Ref

Niveau 1 h Ref

Niveau 2 h Ref

Niveau 3 h Alt

Niveau 1 h Alt

Niveau 2 h Alt

Niveau 3 h5 h1 h5 h1

A -050 -015 083 062 -212 -039 198 299 -198 -299

B -261 -270 021 -077 -058 173 198 299 -198 -299

C -151 -031 -014 -015 -004 -005 198 299 -198 -299

D 038 040 -137 000 072 207 198 299 -198 -299

E -001 090 155 -052 056 -101 198 299 -198 -299

F -112 -321 -153 070 -325 023 198 299 -198 -299

G 042 -071 040 147 -009 176 198 299 -198 -299

H 169 046 -169 192 018 -083 198 299 -198 -299

L 009 -120 021 -077 -231 005 198 299 -198 -299

M 001 031 014 -032 004 -011 198 299 -198 -299

-300

-200

-100

000

100

200

300

A B C D E F G H L M

Vale

urs

h d

e M

an

del(

rob

uste

s)

Laboratoires

Meacutethode de reacutefeacuterence analyse robuste

h Ref Niveau 1

h Ref Niveau 2

h Ref Niveau 3

h 5=198

h 1=299

-300

-200

-100

000

100

200

300

A B C D E F G H L M

Vale

urs

de h

de M

an

del(

rob

uste

s)

Laboratoires

Meacutethode alternative analyse robuste

h Alt Niveau 1

h Alt Niveau 2

h Alt Niveau 2

h 1=299

h 5=198

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3438 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Laboratoire k Ref

Niveau 1 k Ref

Niveau 2 k Ref

Niveau 3 k Alt

Niveau 1 k Alt

Niveau 2 k Alt

Niveau 3 k1 k5

A 000 089 142 084 162 046 259 181

B 125 087 073 104 075 046 259 181

C 117 013 091 188 022 083 259 181

D 052 036 069 000 035 083 259 181

E 028 034 037 138 019 112 259 181

F 140 222 018 027 281 112 259 181

G 088 068 024 131 145 000 259 181

H 129 094 173 016 061 000 259 181

L 069 000 073 104 270 224 259 181

M 000 048 038 043 099 241 259 181

000

050

100

150

200

250

300

350

400

A B C D E F G H L M

Vale

urs

k d

e M

an

del(

rob

uste

s)

Laboratoires

Meacutethode de reacutefeacuterence analyse robuste

k Ref Niveau 1k Ref Niveau 2k Ref Niveau 3

k 1=259

k 5=181

000

050

100

150

200

250

300

A B C D E F G H L M

Vale

urs

k d

e M

an

del

(ro

bu

ste

s)

Laboratoires

Meacutethode alternative analyse robuste

k Alt Niveau 3

k Alt Niveau 1

k Alt Niveau 2

k 1=259

k 5=181

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3538 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Appendix 4 ndash Statistical results

Level 1

Nombre de laboratoires (p) 10

A 1602 1602 1602 000 0000 0000 -0495 0000 A 1845 1602 1724 017 0122 -0122 0621 0840 0122

B 1602 1176 1389 030 0213 -0213 -2613 1247 B 1602 1301 1452 021 0151 -0151 -0769 1041 0062

C 1699 1301 1500 028 0199 -0199 -1510 1165 C 1301 1845 1573 038 -0272 0272 -0148 1881 0073

D 1602 1778 1690 012 -0088 0088 0380 0516 D 1602 1602 1602 000 0000 0000 0000 0000 -0088

E 1699 1602 1651 007 0048 -0048 -0013 0284 E 1699 1301 1500 028 0199 -0199 -0522 1376 -0151

F 1778 1301 1540 034 0239 -0239 -1116 1397 F 1778 1699 1739 006 0040 -0040 0698 0274 0199

G 1845 1544 1695 021 0151 -0151 0425 0881 G 2079 1699 1889 027 0190 -0190 1467 1315 0194

H 2041 1602 1822 031 0220 -0220 1689 1286 H 2000 1954 1977 003 0023 -0023 1917 0158 0155

L 1544 1778 1661 017 -0117 0117 0092 0685 L 1301 1602 1452 021 -0151 0151 -0769 1041 -0210

M 1653 1653 1653 000 0000 0000 0013 0000 M 1477 1602 1540 009 -0062 0062 -0319 0432 -0114

m 165186 m 160206 mediane D 00678

Cn(2p) 1867 Cn(2p) 1867 Cp 16101

Cn(p) 16101 Cn(p) 16101 Qn(D) 0092926

n=2p n 20 n=2p n 20 Qdiff 01496169

f 11 f 11 t 114

l 55 l 55

n=p n 10 n=p n 10

f 6 f 6

l 15 l 15

Qn(2p) 0091465 Qn(2p) 007745

Qn(p) 0062469 Qn(p) 0121519

Qintra 017078 Qintra 014461

Qinter 010058 Qinter 019565

Sr(eacutecart-type de reacutepeacutetabiliteacute) 02415 Sr 02045

RSDr(coefficient de variation de la reacutepeacutetabiliteacute)1462 RSDr 1277

r(repeatability limit)) 0676 r 0573

SL(eacutecart-type interlaboratoire) -001905 SLlt0 SL 001737

SR(eacutecart-type de reproductibiliteacute) 02415 SR 02433

RSDR(coefficient de variation de la reproductibiliteacute)0146 RSDR 0152

R(limite de reproductibiliteacute)) 06762 R(limite de reproductibiliteacute))068123

h k h MoyenneLaboratoires

Niveau 1

si LaboratoiresDeviationMoyenne Dk DeviationsiAlt

Niveau 1

Ref

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3638 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Level 2

Nombre de laboratoires (p) 10

A 2681 2613 2647 005 0034 -0034 -0151 0891 A 2322 2531 2427 015 -0105 0105 -2119 1620 -0220

B 2544 2477 2511 005 0033 -0033 -2699 0872 B 2556 2653 2605 007 -0048 0048 -0581 0750 0094

C 2643 2633 2638 001 0005 -0005 -0311 0130 C 2681 2653 2667 002 0014 -0014 -0040 0217 0029

D 2690 2663 2676 002 0014 -0014 0400 0357 D 2778 2732 2755 003 0023 -0023 0721 0354 0079

E 2690 2716 2703 002 -0013 0013 0897 0336 E 2724 2748 2736 002 -0012 0012 0556 0185 0033

F 2568 2398 2483 012 0085 -0085 -3214 2217 F 2477 2114 2296 026 0182 -0182 -3255 2811 -0188

G 2643 2591 2617 004 0026 -0026 -0707 0682 G 2756 2568 2662 013 0094 -0094 -0085 1453 0045

H 2716 2643 2680 005 0036 -0036 0460 0945 H 2653 2732 2693 006 -0040 0040 0181 0613 0013

L 2591 2591 2591 000 0000 0000 -1196 0000 L 2230 2580 2405 025 -0175 0175 -2307 2704 -0186

M 2653 2690 2672 003 -0018 0018 0311 0481 M 2740 2613 2677 009 0064 -0064 0040 0987 0005

m 2655082 m 26719001 mediane D 00209

Cn(2p) 1867 Cn(2p) 1867 Cp 16101

Cn(p) 16101 Cn(p) 16101 Qn(D) 0039911

n=2p n 20 n=2p n 20 Qdiff 00642591

f 11 f 11 t 082

l 55 l 55

n=p n 10 n=p n 10

f 6 f 6

l 15 l 15

Qn(2p) 0020570 Qn(2p) 00345985

Qn(p) 00332437 Qn(p) 00718158

Qintra 003841 Qintra 006460

Qinter 005352 Qinter 011563

Sr(eacutecart-type de reacutepeacutetabiliteacute) 00543 Sr 00914

RSDr(coefficient de variation de la reacutepeacutetabiliteacute)205 RSDr 342

r(repeatability limit)) 0152 r 0256

SL(eacutecart-type interlaboratoire) 000139 SL 000920

SR(eacutecart-type de reproductibiliteacute) 00659 SR 01325

RSDR(coefficient de variation de la reproductibiliteacute)0025 RSDR 0050

R(limite de reproductibiliteacute)) 018446 R(limite de reproductibiliteacute))03708618

Laboratoires

Niveau 2

Moyenne si Laboratoires

Niveau 2

DRef Alt Moyenne si Deviation h Deviation h k k

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3738 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Level 2 bis

Nombre de laboratoires (p) 10

A 3000 2477 2739 037 0261 -0261 0832 1418 A 2602 2477 2540 009 0062 -0062 -0391 0465 -0199

B 2544 2813 2678 019 -0134 0134 0211 0729 B 2903 2778 2841 009 0062 -0062 1732 0465 0162

C 2477 2813 2645 024 -0168 0168 -0135 0911 C 2477 2699 2588 016 -0111 0111 -0049 0826 -0057

D 2398 2653 2526 018 -0128 0128 -1370 0692 D 2778 3000 2889 016 -0111 0111 2074 0826 0363

E 2875 2740 2808 010 0067 -0067 1547 0365 E 2602 2301 2452 021 0151 -0151 -1012 1120 -0356

F 2477 2544 2511 005 -0033 0033 -1525 0182 F 2778 2477 2628 021 0151 -0151 0230 1120 0117

G 2653 2740 2697 006 -0044 0044 0400 0236 G 2845 2845 2845 000 0000 0000 1764 0000 0148

H 2176 2813 2495 045 -0318 0318 -1692 1727 H 2477 2477 2477 000 0000 0000 -0832 0000 -0017

L 2813 2544 2678 019 0134 -0134 0211 0729 L 2903 2301 2602 043 0301 -0301 0049 2241 -0076

M 2602 2740 2671 010 -0069 0069 0135 0375 M 2903 2255 2579 046 0324 -0324 -0112 2411 -0092

m 2658114 m 259505 mediane D -00372

Cn(2p) 1867 Cn(2p) 1867 Cp 16101

Cn(p) 16101 Cn(p) 16101 Qn(D) 01656915

n=2p n 20 n=2p n 20 Qdiff 02667753

f 11 f 11 t 035

l 55 l 55

n=p n 10 n=p n 10

f 6 f 6

l 15 l 15

Qn(2p) 0098745 Qn(2p) 007195

Qn(p) 006007 Qn(p) 008805

Qintra 018437 Qintra 013435

Qinter 009672 Qinter 014176

Sr(eacutecart-type de reacutepeacutetabiliteacute) 02607 Sr 01900

RSDr(coefficient de variation de la reacutepeacutetabiliteacute)981 RSDr 732

r(repeatability limit)) 0730 r 0532

SL(eacutecart-type interlaboratoire) -002464 SLlt0 SL 000205

SR(eacutecart-type de reproductibiliteacute) 02607 SR 01953

RSDR(coefficient de variation de la reproductibiliteacute)0098 RSDR 0075

R(limite de reproductibiliteacute)) 072996 R(limite de reproductibiliteacute))054686

Laboratoires

Niveau 2 bis

Moyenne si Laboratoires

Niveau 2 bis

DRef Alt Moyenne si Deviation h Deviation h k k

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3838 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Level 3

Nombre de laboratoires (p) 10

A 3690 3663 3676 002 0014 -0014 -0311 0513 A 3690 3708 3699 001 -0009 0009 0354 0257 0022

B 3613 3672 3642 004 -0030 0030 -1851 1109 B 3708 3568 3638 010 0070 -0070 -0844 2061 -0005

C 3663 3732 3698 005 -0035 0035 0645 1302 C 3663 3699 3681 003 -0018 0018 0000 0536 -0017

D 3690 3690 3690 000 0000 0000 0311 0000 D 3643 3663 3653 001 -0010 0010 -0545 0286 -0037

E 3732 3653 3693 006 0040 -0040 0429 1480 E 3613 3732 3673 008 -0060 0060 -0162 1769 -0020

F 3643 3672 3658 002 -0014 0014 -1157 0536 F 3690 3771 3731 006 -0040 0040 0975 1193 0073

G 3699 3653 3676 003 0023 -0023 -0328 0856 G 3602 3643 3623 003 -0021 0021 -1141 0612 -0053

H 3623 3672 3648 003 -0024 0024 -1615 0913 H 3748 3681 3715 005 0033 -0033 0664 0990 0067

L 3724 3663 3694 004 0031 -0031 0461 1150 L 3748 3771 3760 002 -0011 0011 1544 0335 0066

M 3690 3690 3690 000 0000 0000 0311 0000 M 3806 3681 3744 009 0062 -0062 1234 1848 0054

m 368334 m 368086 mediane D 00089

Cn(2p) 1867 Cn(2p) 1867 Cp 16101

Cn(p) 16101 Cn(p) 16101 Qn(D) 00325359

n=2p n 20 n=2p n 20 Qdiff 00523852

f 11 f 11 t 043

l 55 l 55

n=p n 10 n=p n 10

f 6 f 6

l 15 l 15

Qn(2p) 0014323 Qn(2p) 001811

Qn(p) 0013719 Qn(p) 003164

Qintra 002674 Qintra 003381

Qinter 002209 Qinter 005094

Sr(eacutecart-type de reacutepeacutetabiliteacute) 00378 Sr 00478

RSDr(coefficient de variation de la reacutepeacutetabiliteacute)103 RSDr 130

r(repeatability limit)) 0106 r 0134

SL(eacutecart-type interlaboratoire) -000023 SLlt0 SL 000145

SR(eacutecart-type de reproductibiliteacute) 00378 SR 00611

RSDR(coefficient de variation de la reproductibiliteacute)0010 RSDR 0017

R(limite de reproductibiliteacute)) 0105894 R(limite de reproductibiliteacute))017119

Ref Alt DLaboratoires

Niveau 3

Moyenne si Deviation h k Laboratoires

Niveau 3

Moyenne si Deviation h k

Page 33: ÞÀ¿¸7 ú Y5zñLjÜ< /u +æü t¬Á ... - NF Validation · NF VALIDATION Validation of alternative analytical methods Application in food microbiology ... 500 – 1 000 1 000 –

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3338 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Appendix 3 ndash Mandelrsquos graphics

Laboratoire h Ref

Niveau 1 h Ref

Niveau 2 h Ref

Niveau 3 h Alt

Niveau 1 h Alt

Niveau 2 h Alt

Niveau 3 h5 h1 h5 h1

A -050 -015 083 062 -212 -039 198 299 -198 -299

B -261 -270 021 -077 -058 173 198 299 -198 -299

C -151 -031 -014 -015 -004 -005 198 299 -198 -299

D 038 040 -137 000 072 207 198 299 -198 -299

E -001 090 155 -052 056 -101 198 299 -198 -299

F -112 -321 -153 070 -325 023 198 299 -198 -299

G 042 -071 040 147 -009 176 198 299 -198 -299

H 169 046 -169 192 018 -083 198 299 -198 -299

L 009 -120 021 -077 -231 005 198 299 -198 -299

M 001 031 014 -032 004 -011 198 299 -198 -299

-300

-200

-100

000

100

200

300

A B C D E F G H L M

Vale

urs

h d

e M

an

del(

rob

uste

s)

Laboratoires

Meacutethode de reacutefeacuterence analyse robuste

h Ref Niveau 1

h Ref Niveau 2

h Ref Niveau 3

h 5=198

h 1=299

-300

-200

-100

000

100

200

300

A B C D E F G H L M

Vale

urs

de h

de M

an

del(

rob

uste

s)

Laboratoires

Meacutethode alternative analyse robuste

h Alt Niveau 1

h Alt Niveau 2

h Alt Niveau 2

h 1=299

h 5=198

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3438 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Laboratoire k Ref

Niveau 1 k Ref

Niveau 2 k Ref

Niveau 3 k Alt

Niveau 1 k Alt

Niveau 2 k Alt

Niveau 3 k1 k5

A 000 089 142 084 162 046 259 181

B 125 087 073 104 075 046 259 181

C 117 013 091 188 022 083 259 181

D 052 036 069 000 035 083 259 181

E 028 034 037 138 019 112 259 181

F 140 222 018 027 281 112 259 181

G 088 068 024 131 145 000 259 181

H 129 094 173 016 061 000 259 181

L 069 000 073 104 270 224 259 181

M 000 048 038 043 099 241 259 181

000

050

100

150

200

250

300

350

400

A B C D E F G H L M

Vale

urs

k d

e M

an

del(

rob

uste

s)

Laboratoires

Meacutethode de reacutefeacuterence analyse robuste

k Ref Niveau 1k Ref Niveau 2k Ref Niveau 3

k 1=259

k 5=181

000

050

100

150

200

250

300

A B C D E F G H L M

Vale

urs

k d

e M

an

del

(ro

bu

ste

s)

Laboratoires

Meacutethode alternative analyse robuste

k Alt Niveau 3

k Alt Niveau 1

k Alt Niveau 2

k 1=259

k 5=181

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3538 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Appendix 4 ndash Statistical results

Level 1

Nombre de laboratoires (p) 10

A 1602 1602 1602 000 0000 0000 -0495 0000 A 1845 1602 1724 017 0122 -0122 0621 0840 0122

B 1602 1176 1389 030 0213 -0213 -2613 1247 B 1602 1301 1452 021 0151 -0151 -0769 1041 0062

C 1699 1301 1500 028 0199 -0199 -1510 1165 C 1301 1845 1573 038 -0272 0272 -0148 1881 0073

D 1602 1778 1690 012 -0088 0088 0380 0516 D 1602 1602 1602 000 0000 0000 0000 0000 -0088

E 1699 1602 1651 007 0048 -0048 -0013 0284 E 1699 1301 1500 028 0199 -0199 -0522 1376 -0151

F 1778 1301 1540 034 0239 -0239 -1116 1397 F 1778 1699 1739 006 0040 -0040 0698 0274 0199

G 1845 1544 1695 021 0151 -0151 0425 0881 G 2079 1699 1889 027 0190 -0190 1467 1315 0194

H 2041 1602 1822 031 0220 -0220 1689 1286 H 2000 1954 1977 003 0023 -0023 1917 0158 0155

L 1544 1778 1661 017 -0117 0117 0092 0685 L 1301 1602 1452 021 -0151 0151 -0769 1041 -0210

M 1653 1653 1653 000 0000 0000 0013 0000 M 1477 1602 1540 009 -0062 0062 -0319 0432 -0114

m 165186 m 160206 mediane D 00678

Cn(2p) 1867 Cn(2p) 1867 Cp 16101

Cn(p) 16101 Cn(p) 16101 Qn(D) 0092926

n=2p n 20 n=2p n 20 Qdiff 01496169

f 11 f 11 t 114

l 55 l 55

n=p n 10 n=p n 10

f 6 f 6

l 15 l 15

Qn(2p) 0091465 Qn(2p) 007745

Qn(p) 0062469 Qn(p) 0121519

Qintra 017078 Qintra 014461

Qinter 010058 Qinter 019565

Sr(eacutecart-type de reacutepeacutetabiliteacute) 02415 Sr 02045

RSDr(coefficient de variation de la reacutepeacutetabiliteacute)1462 RSDr 1277

r(repeatability limit)) 0676 r 0573

SL(eacutecart-type interlaboratoire) -001905 SLlt0 SL 001737

SR(eacutecart-type de reproductibiliteacute) 02415 SR 02433

RSDR(coefficient de variation de la reproductibiliteacute)0146 RSDR 0152

R(limite de reproductibiliteacute)) 06762 R(limite de reproductibiliteacute))068123

h k h MoyenneLaboratoires

Niveau 1

si LaboratoiresDeviationMoyenne Dk DeviationsiAlt

Niveau 1

Ref

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3638 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Level 2

Nombre de laboratoires (p) 10

A 2681 2613 2647 005 0034 -0034 -0151 0891 A 2322 2531 2427 015 -0105 0105 -2119 1620 -0220

B 2544 2477 2511 005 0033 -0033 -2699 0872 B 2556 2653 2605 007 -0048 0048 -0581 0750 0094

C 2643 2633 2638 001 0005 -0005 -0311 0130 C 2681 2653 2667 002 0014 -0014 -0040 0217 0029

D 2690 2663 2676 002 0014 -0014 0400 0357 D 2778 2732 2755 003 0023 -0023 0721 0354 0079

E 2690 2716 2703 002 -0013 0013 0897 0336 E 2724 2748 2736 002 -0012 0012 0556 0185 0033

F 2568 2398 2483 012 0085 -0085 -3214 2217 F 2477 2114 2296 026 0182 -0182 -3255 2811 -0188

G 2643 2591 2617 004 0026 -0026 -0707 0682 G 2756 2568 2662 013 0094 -0094 -0085 1453 0045

H 2716 2643 2680 005 0036 -0036 0460 0945 H 2653 2732 2693 006 -0040 0040 0181 0613 0013

L 2591 2591 2591 000 0000 0000 -1196 0000 L 2230 2580 2405 025 -0175 0175 -2307 2704 -0186

M 2653 2690 2672 003 -0018 0018 0311 0481 M 2740 2613 2677 009 0064 -0064 0040 0987 0005

m 2655082 m 26719001 mediane D 00209

Cn(2p) 1867 Cn(2p) 1867 Cp 16101

Cn(p) 16101 Cn(p) 16101 Qn(D) 0039911

n=2p n 20 n=2p n 20 Qdiff 00642591

f 11 f 11 t 082

l 55 l 55

n=p n 10 n=p n 10

f 6 f 6

l 15 l 15

Qn(2p) 0020570 Qn(2p) 00345985

Qn(p) 00332437 Qn(p) 00718158

Qintra 003841 Qintra 006460

Qinter 005352 Qinter 011563

Sr(eacutecart-type de reacutepeacutetabiliteacute) 00543 Sr 00914

RSDr(coefficient de variation de la reacutepeacutetabiliteacute)205 RSDr 342

r(repeatability limit)) 0152 r 0256

SL(eacutecart-type interlaboratoire) 000139 SL 000920

SR(eacutecart-type de reproductibiliteacute) 00659 SR 01325

RSDR(coefficient de variation de la reproductibiliteacute)0025 RSDR 0050

R(limite de reproductibiliteacute)) 018446 R(limite de reproductibiliteacute))03708618

Laboratoires

Niveau 2

Moyenne si Laboratoires

Niveau 2

DRef Alt Moyenne si Deviation h Deviation h k k

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3738 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Level 2 bis

Nombre de laboratoires (p) 10

A 3000 2477 2739 037 0261 -0261 0832 1418 A 2602 2477 2540 009 0062 -0062 -0391 0465 -0199

B 2544 2813 2678 019 -0134 0134 0211 0729 B 2903 2778 2841 009 0062 -0062 1732 0465 0162

C 2477 2813 2645 024 -0168 0168 -0135 0911 C 2477 2699 2588 016 -0111 0111 -0049 0826 -0057

D 2398 2653 2526 018 -0128 0128 -1370 0692 D 2778 3000 2889 016 -0111 0111 2074 0826 0363

E 2875 2740 2808 010 0067 -0067 1547 0365 E 2602 2301 2452 021 0151 -0151 -1012 1120 -0356

F 2477 2544 2511 005 -0033 0033 -1525 0182 F 2778 2477 2628 021 0151 -0151 0230 1120 0117

G 2653 2740 2697 006 -0044 0044 0400 0236 G 2845 2845 2845 000 0000 0000 1764 0000 0148

H 2176 2813 2495 045 -0318 0318 -1692 1727 H 2477 2477 2477 000 0000 0000 -0832 0000 -0017

L 2813 2544 2678 019 0134 -0134 0211 0729 L 2903 2301 2602 043 0301 -0301 0049 2241 -0076

M 2602 2740 2671 010 -0069 0069 0135 0375 M 2903 2255 2579 046 0324 -0324 -0112 2411 -0092

m 2658114 m 259505 mediane D -00372

Cn(2p) 1867 Cn(2p) 1867 Cp 16101

Cn(p) 16101 Cn(p) 16101 Qn(D) 01656915

n=2p n 20 n=2p n 20 Qdiff 02667753

f 11 f 11 t 035

l 55 l 55

n=p n 10 n=p n 10

f 6 f 6

l 15 l 15

Qn(2p) 0098745 Qn(2p) 007195

Qn(p) 006007 Qn(p) 008805

Qintra 018437 Qintra 013435

Qinter 009672 Qinter 014176

Sr(eacutecart-type de reacutepeacutetabiliteacute) 02607 Sr 01900

RSDr(coefficient de variation de la reacutepeacutetabiliteacute)981 RSDr 732

r(repeatability limit)) 0730 r 0532

SL(eacutecart-type interlaboratoire) -002464 SLlt0 SL 000205

SR(eacutecart-type de reproductibiliteacute) 02607 SR 01953

RSDR(coefficient de variation de la reproductibiliteacute)0098 RSDR 0075

R(limite de reproductibiliteacute)) 072996 R(limite de reproductibiliteacute))054686

Laboratoires

Niveau 2 bis

Moyenne si Laboratoires

Niveau 2 bis

DRef Alt Moyenne si Deviation h Deviation h k k

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3838 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Level 3

Nombre de laboratoires (p) 10

A 3690 3663 3676 002 0014 -0014 -0311 0513 A 3690 3708 3699 001 -0009 0009 0354 0257 0022

B 3613 3672 3642 004 -0030 0030 -1851 1109 B 3708 3568 3638 010 0070 -0070 -0844 2061 -0005

C 3663 3732 3698 005 -0035 0035 0645 1302 C 3663 3699 3681 003 -0018 0018 0000 0536 -0017

D 3690 3690 3690 000 0000 0000 0311 0000 D 3643 3663 3653 001 -0010 0010 -0545 0286 -0037

E 3732 3653 3693 006 0040 -0040 0429 1480 E 3613 3732 3673 008 -0060 0060 -0162 1769 -0020

F 3643 3672 3658 002 -0014 0014 -1157 0536 F 3690 3771 3731 006 -0040 0040 0975 1193 0073

G 3699 3653 3676 003 0023 -0023 -0328 0856 G 3602 3643 3623 003 -0021 0021 -1141 0612 -0053

H 3623 3672 3648 003 -0024 0024 -1615 0913 H 3748 3681 3715 005 0033 -0033 0664 0990 0067

L 3724 3663 3694 004 0031 -0031 0461 1150 L 3748 3771 3760 002 -0011 0011 1544 0335 0066

M 3690 3690 3690 000 0000 0000 0311 0000 M 3806 3681 3744 009 0062 -0062 1234 1848 0054

m 368334 m 368086 mediane D 00089

Cn(2p) 1867 Cn(2p) 1867 Cp 16101

Cn(p) 16101 Cn(p) 16101 Qn(D) 00325359

n=2p n 20 n=2p n 20 Qdiff 00523852

f 11 f 11 t 043

l 55 l 55

n=p n 10 n=p n 10

f 6 f 6

l 15 l 15

Qn(2p) 0014323 Qn(2p) 001811

Qn(p) 0013719 Qn(p) 003164

Qintra 002674 Qintra 003381

Qinter 002209 Qinter 005094

Sr(eacutecart-type de reacutepeacutetabiliteacute) 00378 Sr 00478

RSDr(coefficient de variation de la reacutepeacutetabiliteacute)103 RSDr 130

r(repeatability limit)) 0106 r 0134

SL(eacutecart-type interlaboratoire) -000023 SLlt0 SL 000145

SR(eacutecart-type de reproductibiliteacute) 00378 SR 00611

RSDR(coefficient de variation de la reproductibiliteacute)0010 RSDR 0017

R(limite de reproductibiliteacute)) 0105894 R(limite de reproductibiliteacute))017119

Ref Alt DLaboratoires

Niveau 3

Moyenne si Deviation h k Laboratoires

Niveau 3

Moyenne si Deviation h k

Page 34: ÞÀ¿¸7 ú Y5zñLjÜ< /u +æü t¬Á ... - NF Validation · NF VALIDATION Validation of alternative analytical methods Application in food microbiology ... 500 – 1 000 1 000 –

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3438 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Laboratoire k Ref

Niveau 1 k Ref

Niveau 2 k Ref

Niveau 3 k Alt

Niveau 1 k Alt

Niveau 2 k Alt

Niveau 3 k1 k5

A 000 089 142 084 162 046 259 181

B 125 087 073 104 075 046 259 181

C 117 013 091 188 022 083 259 181

D 052 036 069 000 035 083 259 181

E 028 034 037 138 019 112 259 181

F 140 222 018 027 281 112 259 181

G 088 068 024 131 145 000 259 181

H 129 094 173 016 061 000 259 181

L 069 000 073 104 270 224 259 181

M 000 048 038 043 099 241 259 181

000

050

100

150

200

250

300

350

400

A B C D E F G H L M

Vale

urs

k d

e M

an

del(

rob

uste

s)

Laboratoires

Meacutethode de reacutefeacuterence analyse robuste

k Ref Niveau 1k Ref Niveau 2k Ref Niveau 3

k 1=259

k 5=181

000

050

100

150

200

250

300

A B C D E F G H L M

Vale

urs

k d

e M

an

del

(ro

bu

ste

s)

Laboratoires

Meacutethode alternative analyse robuste

k Alt Niveau 3

k Alt Niveau 1

k Alt Niveau 2

k 1=259

k 5=181

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3538 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Appendix 4 ndash Statistical results

Level 1

Nombre de laboratoires (p) 10

A 1602 1602 1602 000 0000 0000 -0495 0000 A 1845 1602 1724 017 0122 -0122 0621 0840 0122

B 1602 1176 1389 030 0213 -0213 -2613 1247 B 1602 1301 1452 021 0151 -0151 -0769 1041 0062

C 1699 1301 1500 028 0199 -0199 -1510 1165 C 1301 1845 1573 038 -0272 0272 -0148 1881 0073

D 1602 1778 1690 012 -0088 0088 0380 0516 D 1602 1602 1602 000 0000 0000 0000 0000 -0088

E 1699 1602 1651 007 0048 -0048 -0013 0284 E 1699 1301 1500 028 0199 -0199 -0522 1376 -0151

F 1778 1301 1540 034 0239 -0239 -1116 1397 F 1778 1699 1739 006 0040 -0040 0698 0274 0199

G 1845 1544 1695 021 0151 -0151 0425 0881 G 2079 1699 1889 027 0190 -0190 1467 1315 0194

H 2041 1602 1822 031 0220 -0220 1689 1286 H 2000 1954 1977 003 0023 -0023 1917 0158 0155

L 1544 1778 1661 017 -0117 0117 0092 0685 L 1301 1602 1452 021 -0151 0151 -0769 1041 -0210

M 1653 1653 1653 000 0000 0000 0013 0000 M 1477 1602 1540 009 -0062 0062 -0319 0432 -0114

m 165186 m 160206 mediane D 00678

Cn(2p) 1867 Cn(2p) 1867 Cp 16101

Cn(p) 16101 Cn(p) 16101 Qn(D) 0092926

n=2p n 20 n=2p n 20 Qdiff 01496169

f 11 f 11 t 114

l 55 l 55

n=p n 10 n=p n 10

f 6 f 6

l 15 l 15

Qn(2p) 0091465 Qn(2p) 007745

Qn(p) 0062469 Qn(p) 0121519

Qintra 017078 Qintra 014461

Qinter 010058 Qinter 019565

Sr(eacutecart-type de reacutepeacutetabiliteacute) 02415 Sr 02045

RSDr(coefficient de variation de la reacutepeacutetabiliteacute)1462 RSDr 1277

r(repeatability limit)) 0676 r 0573

SL(eacutecart-type interlaboratoire) -001905 SLlt0 SL 001737

SR(eacutecart-type de reproductibiliteacute) 02415 SR 02433

RSDR(coefficient de variation de la reproductibiliteacute)0146 RSDR 0152

R(limite de reproductibiliteacute)) 06762 R(limite de reproductibiliteacute))068123

h k h MoyenneLaboratoires

Niveau 1

si LaboratoiresDeviationMoyenne Dk DeviationsiAlt

Niveau 1

Ref

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3638 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Level 2

Nombre de laboratoires (p) 10

A 2681 2613 2647 005 0034 -0034 -0151 0891 A 2322 2531 2427 015 -0105 0105 -2119 1620 -0220

B 2544 2477 2511 005 0033 -0033 -2699 0872 B 2556 2653 2605 007 -0048 0048 -0581 0750 0094

C 2643 2633 2638 001 0005 -0005 -0311 0130 C 2681 2653 2667 002 0014 -0014 -0040 0217 0029

D 2690 2663 2676 002 0014 -0014 0400 0357 D 2778 2732 2755 003 0023 -0023 0721 0354 0079

E 2690 2716 2703 002 -0013 0013 0897 0336 E 2724 2748 2736 002 -0012 0012 0556 0185 0033

F 2568 2398 2483 012 0085 -0085 -3214 2217 F 2477 2114 2296 026 0182 -0182 -3255 2811 -0188

G 2643 2591 2617 004 0026 -0026 -0707 0682 G 2756 2568 2662 013 0094 -0094 -0085 1453 0045

H 2716 2643 2680 005 0036 -0036 0460 0945 H 2653 2732 2693 006 -0040 0040 0181 0613 0013

L 2591 2591 2591 000 0000 0000 -1196 0000 L 2230 2580 2405 025 -0175 0175 -2307 2704 -0186

M 2653 2690 2672 003 -0018 0018 0311 0481 M 2740 2613 2677 009 0064 -0064 0040 0987 0005

m 2655082 m 26719001 mediane D 00209

Cn(2p) 1867 Cn(2p) 1867 Cp 16101

Cn(p) 16101 Cn(p) 16101 Qn(D) 0039911

n=2p n 20 n=2p n 20 Qdiff 00642591

f 11 f 11 t 082

l 55 l 55

n=p n 10 n=p n 10

f 6 f 6

l 15 l 15

Qn(2p) 0020570 Qn(2p) 00345985

Qn(p) 00332437 Qn(p) 00718158

Qintra 003841 Qintra 006460

Qinter 005352 Qinter 011563

Sr(eacutecart-type de reacutepeacutetabiliteacute) 00543 Sr 00914

RSDr(coefficient de variation de la reacutepeacutetabiliteacute)205 RSDr 342

r(repeatability limit)) 0152 r 0256

SL(eacutecart-type interlaboratoire) 000139 SL 000920

SR(eacutecart-type de reproductibiliteacute) 00659 SR 01325

RSDR(coefficient de variation de la reproductibiliteacute)0025 RSDR 0050

R(limite de reproductibiliteacute)) 018446 R(limite de reproductibiliteacute))03708618

Laboratoires

Niveau 2

Moyenne si Laboratoires

Niveau 2

DRef Alt Moyenne si Deviation h Deviation h k k

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3738 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Level 2 bis

Nombre de laboratoires (p) 10

A 3000 2477 2739 037 0261 -0261 0832 1418 A 2602 2477 2540 009 0062 -0062 -0391 0465 -0199

B 2544 2813 2678 019 -0134 0134 0211 0729 B 2903 2778 2841 009 0062 -0062 1732 0465 0162

C 2477 2813 2645 024 -0168 0168 -0135 0911 C 2477 2699 2588 016 -0111 0111 -0049 0826 -0057

D 2398 2653 2526 018 -0128 0128 -1370 0692 D 2778 3000 2889 016 -0111 0111 2074 0826 0363

E 2875 2740 2808 010 0067 -0067 1547 0365 E 2602 2301 2452 021 0151 -0151 -1012 1120 -0356

F 2477 2544 2511 005 -0033 0033 -1525 0182 F 2778 2477 2628 021 0151 -0151 0230 1120 0117

G 2653 2740 2697 006 -0044 0044 0400 0236 G 2845 2845 2845 000 0000 0000 1764 0000 0148

H 2176 2813 2495 045 -0318 0318 -1692 1727 H 2477 2477 2477 000 0000 0000 -0832 0000 -0017

L 2813 2544 2678 019 0134 -0134 0211 0729 L 2903 2301 2602 043 0301 -0301 0049 2241 -0076

M 2602 2740 2671 010 -0069 0069 0135 0375 M 2903 2255 2579 046 0324 -0324 -0112 2411 -0092

m 2658114 m 259505 mediane D -00372

Cn(2p) 1867 Cn(2p) 1867 Cp 16101

Cn(p) 16101 Cn(p) 16101 Qn(D) 01656915

n=2p n 20 n=2p n 20 Qdiff 02667753

f 11 f 11 t 035

l 55 l 55

n=p n 10 n=p n 10

f 6 f 6

l 15 l 15

Qn(2p) 0098745 Qn(2p) 007195

Qn(p) 006007 Qn(p) 008805

Qintra 018437 Qintra 013435

Qinter 009672 Qinter 014176

Sr(eacutecart-type de reacutepeacutetabiliteacute) 02607 Sr 01900

RSDr(coefficient de variation de la reacutepeacutetabiliteacute)981 RSDr 732

r(repeatability limit)) 0730 r 0532

SL(eacutecart-type interlaboratoire) -002464 SLlt0 SL 000205

SR(eacutecart-type de reproductibiliteacute) 02607 SR 01953

RSDR(coefficient de variation de la reproductibiliteacute)0098 RSDR 0075

R(limite de reproductibiliteacute)) 072996 R(limite de reproductibiliteacute))054686

Laboratoires

Niveau 2 bis

Moyenne si Laboratoires

Niveau 2 bis

DRef Alt Moyenne si Deviation h Deviation h k k

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3838 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Level 3

Nombre de laboratoires (p) 10

A 3690 3663 3676 002 0014 -0014 -0311 0513 A 3690 3708 3699 001 -0009 0009 0354 0257 0022

B 3613 3672 3642 004 -0030 0030 -1851 1109 B 3708 3568 3638 010 0070 -0070 -0844 2061 -0005

C 3663 3732 3698 005 -0035 0035 0645 1302 C 3663 3699 3681 003 -0018 0018 0000 0536 -0017

D 3690 3690 3690 000 0000 0000 0311 0000 D 3643 3663 3653 001 -0010 0010 -0545 0286 -0037

E 3732 3653 3693 006 0040 -0040 0429 1480 E 3613 3732 3673 008 -0060 0060 -0162 1769 -0020

F 3643 3672 3658 002 -0014 0014 -1157 0536 F 3690 3771 3731 006 -0040 0040 0975 1193 0073

G 3699 3653 3676 003 0023 -0023 -0328 0856 G 3602 3643 3623 003 -0021 0021 -1141 0612 -0053

H 3623 3672 3648 003 -0024 0024 -1615 0913 H 3748 3681 3715 005 0033 -0033 0664 0990 0067

L 3724 3663 3694 004 0031 -0031 0461 1150 L 3748 3771 3760 002 -0011 0011 1544 0335 0066

M 3690 3690 3690 000 0000 0000 0311 0000 M 3806 3681 3744 009 0062 -0062 1234 1848 0054

m 368334 m 368086 mediane D 00089

Cn(2p) 1867 Cn(2p) 1867 Cp 16101

Cn(p) 16101 Cn(p) 16101 Qn(D) 00325359

n=2p n 20 n=2p n 20 Qdiff 00523852

f 11 f 11 t 043

l 55 l 55

n=p n 10 n=p n 10

f 6 f 6

l 15 l 15

Qn(2p) 0014323 Qn(2p) 001811

Qn(p) 0013719 Qn(p) 003164

Qintra 002674 Qintra 003381

Qinter 002209 Qinter 005094

Sr(eacutecart-type de reacutepeacutetabiliteacute) 00378 Sr 00478

RSDr(coefficient de variation de la reacutepeacutetabiliteacute)103 RSDr 130

r(repeatability limit)) 0106 r 0134

SL(eacutecart-type interlaboratoire) -000023 SLlt0 SL 000145

SR(eacutecart-type de reproductibiliteacute) 00378 SR 00611

RSDR(coefficient de variation de la reproductibiliteacute)0010 RSDR 0017

R(limite de reproductibiliteacute)) 0105894 R(limite de reproductibiliteacute))017119

Ref Alt DLaboratoires

Niveau 3

Moyenne si Deviation h k Laboratoires

Niveau 3

Moyenne si Deviation h k

Page 35: ÞÀ¿¸7 ú Y5zñLjÜ< /u +æü t¬Á ... - NF Validation · NF VALIDATION Validation of alternative analytical methods Application in food microbiology ... 500 – 1 000 1 000 –

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3538 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Appendix 4 ndash Statistical results

Level 1

Nombre de laboratoires (p) 10

A 1602 1602 1602 000 0000 0000 -0495 0000 A 1845 1602 1724 017 0122 -0122 0621 0840 0122

B 1602 1176 1389 030 0213 -0213 -2613 1247 B 1602 1301 1452 021 0151 -0151 -0769 1041 0062

C 1699 1301 1500 028 0199 -0199 -1510 1165 C 1301 1845 1573 038 -0272 0272 -0148 1881 0073

D 1602 1778 1690 012 -0088 0088 0380 0516 D 1602 1602 1602 000 0000 0000 0000 0000 -0088

E 1699 1602 1651 007 0048 -0048 -0013 0284 E 1699 1301 1500 028 0199 -0199 -0522 1376 -0151

F 1778 1301 1540 034 0239 -0239 -1116 1397 F 1778 1699 1739 006 0040 -0040 0698 0274 0199

G 1845 1544 1695 021 0151 -0151 0425 0881 G 2079 1699 1889 027 0190 -0190 1467 1315 0194

H 2041 1602 1822 031 0220 -0220 1689 1286 H 2000 1954 1977 003 0023 -0023 1917 0158 0155

L 1544 1778 1661 017 -0117 0117 0092 0685 L 1301 1602 1452 021 -0151 0151 -0769 1041 -0210

M 1653 1653 1653 000 0000 0000 0013 0000 M 1477 1602 1540 009 -0062 0062 -0319 0432 -0114

m 165186 m 160206 mediane D 00678

Cn(2p) 1867 Cn(2p) 1867 Cp 16101

Cn(p) 16101 Cn(p) 16101 Qn(D) 0092926

n=2p n 20 n=2p n 20 Qdiff 01496169

f 11 f 11 t 114

l 55 l 55

n=p n 10 n=p n 10

f 6 f 6

l 15 l 15

Qn(2p) 0091465 Qn(2p) 007745

Qn(p) 0062469 Qn(p) 0121519

Qintra 017078 Qintra 014461

Qinter 010058 Qinter 019565

Sr(eacutecart-type de reacutepeacutetabiliteacute) 02415 Sr 02045

RSDr(coefficient de variation de la reacutepeacutetabiliteacute)1462 RSDr 1277

r(repeatability limit)) 0676 r 0573

SL(eacutecart-type interlaboratoire) -001905 SLlt0 SL 001737

SR(eacutecart-type de reproductibiliteacute) 02415 SR 02433

RSDR(coefficient de variation de la reproductibiliteacute)0146 RSDR 0152

R(limite de reproductibiliteacute)) 06762 R(limite de reproductibiliteacute))068123

h k h MoyenneLaboratoires

Niveau 1

si LaboratoiresDeviationMoyenne Dk DeviationsiAlt

Niveau 1

Ref

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3638 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Level 2

Nombre de laboratoires (p) 10

A 2681 2613 2647 005 0034 -0034 -0151 0891 A 2322 2531 2427 015 -0105 0105 -2119 1620 -0220

B 2544 2477 2511 005 0033 -0033 -2699 0872 B 2556 2653 2605 007 -0048 0048 -0581 0750 0094

C 2643 2633 2638 001 0005 -0005 -0311 0130 C 2681 2653 2667 002 0014 -0014 -0040 0217 0029

D 2690 2663 2676 002 0014 -0014 0400 0357 D 2778 2732 2755 003 0023 -0023 0721 0354 0079

E 2690 2716 2703 002 -0013 0013 0897 0336 E 2724 2748 2736 002 -0012 0012 0556 0185 0033

F 2568 2398 2483 012 0085 -0085 -3214 2217 F 2477 2114 2296 026 0182 -0182 -3255 2811 -0188

G 2643 2591 2617 004 0026 -0026 -0707 0682 G 2756 2568 2662 013 0094 -0094 -0085 1453 0045

H 2716 2643 2680 005 0036 -0036 0460 0945 H 2653 2732 2693 006 -0040 0040 0181 0613 0013

L 2591 2591 2591 000 0000 0000 -1196 0000 L 2230 2580 2405 025 -0175 0175 -2307 2704 -0186

M 2653 2690 2672 003 -0018 0018 0311 0481 M 2740 2613 2677 009 0064 -0064 0040 0987 0005

m 2655082 m 26719001 mediane D 00209

Cn(2p) 1867 Cn(2p) 1867 Cp 16101

Cn(p) 16101 Cn(p) 16101 Qn(D) 0039911

n=2p n 20 n=2p n 20 Qdiff 00642591

f 11 f 11 t 082

l 55 l 55

n=p n 10 n=p n 10

f 6 f 6

l 15 l 15

Qn(2p) 0020570 Qn(2p) 00345985

Qn(p) 00332437 Qn(p) 00718158

Qintra 003841 Qintra 006460

Qinter 005352 Qinter 011563

Sr(eacutecart-type de reacutepeacutetabiliteacute) 00543 Sr 00914

RSDr(coefficient de variation de la reacutepeacutetabiliteacute)205 RSDr 342

r(repeatability limit)) 0152 r 0256

SL(eacutecart-type interlaboratoire) 000139 SL 000920

SR(eacutecart-type de reproductibiliteacute) 00659 SR 01325

RSDR(coefficient de variation de la reproductibiliteacute)0025 RSDR 0050

R(limite de reproductibiliteacute)) 018446 R(limite de reproductibiliteacute))03708618

Laboratoires

Niveau 2

Moyenne si Laboratoires

Niveau 2

DRef Alt Moyenne si Deviation h Deviation h k k

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3738 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Level 2 bis

Nombre de laboratoires (p) 10

A 3000 2477 2739 037 0261 -0261 0832 1418 A 2602 2477 2540 009 0062 -0062 -0391 0465 -0199

B 2544 2813 2678 019 -0134 0134 0211 0729 B 2903 2778 2841 009 0062 -0062 1732 0465 0162

C 2477 2813 2645 024 -0168 0168 -0135 0911 C 2477 2699 2588 016 -0111 0111 -0049 0826 -0057

D 2398 2653 2526 018 -0128 0128 -1370 0692 D 2778 3000 2889 016 -0111 0111 2074 0826 0363

E 2875 2740 2808 010 0067 -0067 1547 0365 E 2602 2301 2452 021 0151 -0151 -1012 1120 -0356

F 2477 2544 2511 005 -0033 0033 -1525 0182 F 2778 2477 2628 021 0151 -0151 0230 1120 0117

G 2653 2740 2697 006 -0044 0044 0400 0236 G 2845 2845 2845 000 0000 0000 1764 0000 0148

H 2176 2813 2495 045 -0318 0318 -1692 1727 H 2477 2477 2477 000 0000 0000 -0832 0000 -0017

L 2813 2544 2678 019 0134 -0134 0211 0729 L 2903 2301 2602 043 0301 -0301 0049 2241 -0076

M 2602 2740 2671 010 -0069 0069 0135 0375 M 2903 2255 2579 046 0324 -0324 -0112 2411 -0092

m 2658114 m 259505 mediane D -00372

Cn(2p) 1867 Cn(2p) 1867 Cp 16101

Cn(p) 16101 Cn(p) 16101 Qn(D) 01656915

n=2p n 20 n=2p n 20 Qdiff 02667753

f 11 f 11 t 035

l 55 l 55

n=p n 10 n=p n 10

f 6 f 6

l 15 l 15

Qn(2p) 0098745 Qn(2p) 007195

Qn(p) 006007 Qn(p) 008805

Qintra 018437 Qintra 013435

Qinter 009672 Qinter 014176

Sr(eacutecart-type de reacutepeacutetabiliteacute) 02607 Sr 01900

RSDr(coefficient de variation de la reacutepeacutetabiliteacute)981 RSDr 732

r(repeatability limit)) 0730 r 0532

SL(eacutecart-type interlaboratoire) -002464 SLlt0 SL 000205

SR(eacutecart-type de reproductibiliteacute) 02607 SR 01953

RSDR(coefficient de variation de la reproductibiliteacute)0098 RSDR 0075

R(limite de reproductibiliteacute)) 072996 R(limite de reproductibiliteacute))054686

Laboratoires

Niveau 2 bis

Moyenne si Laboratoires

Niveau 2 bis

DRef Alt Moyenne si Deviation h Deviation h k k

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3838 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Level 3

Nombre de laboratoires (p) 10

A 3690 3663 3676 002 0014 -0014 -0311 0513 A 3690 3708 3699 001 -0009 0009 0354 0257 0022

B 3613 3672 3642 004 -0030 0030 -1851 1109 B 3708 3568 3638 010 0070 -0070 -0844 2061 -0005

C 3663 3732 3698 005 -0035 0035 0645 1302 C 3663 3699 3681 003 -0018 0018 0000 0536 -0017

D 3690 3690 3690 000 0000 0000 0311 0000 D 3643 3663 3653 001 -0010 0010 -0545 0286 -0037

E 3732 3653 3693 006 0040 -0040 0429 1480 E 3613 3732 3673 008 -0060 0060 -0162 1769 -0020

F 3643 3672 3658 002 -0014 0014 -1157 0536 F 3690 3771 3731 006 -0040 0040 0975 1193 0073

G 3699 3653 3676 003 0023 -0023 -0328 0856 G 3602 3643 3623 003 -0021 0021 -1141 0612 -0053

H 3623 3672 3648 003 -0024 0024 -1615 0913 H 3748 3681 3715 005 0033 -0033 0664 0990 0067

L 3724 3663 3694 004 0031 -0031 0461 1150 L 3748 3771 3760 002 -0011 0011 1544 0335 0066

M 3690 3690 3690 000 0000 0000 0311 0000 M 3806 3681 3744 009 0062 -0062 1234 1848 0054

m 368334 m 368086 mediane D 00089

Cn(2p) 1867 Cn(2p) 1867 Cp 16101

Cn(p) 16101 Cn(p) 16101 Qn(D) 00325359

n=2p n 20 n=2p n 20 Qdiff 00523852

f 11 f 11 t 043

l 55 l 55

n=p n 10 n=p n 10

f 6 f 6

l 15 l 15

Qn(2p) 0014323 Qn(2p) 001811

Qn(p) 0013719 Qn(p) 003164

Qintra 002674 Qintra 003381

Qinter 002209 Qinter 005094

Sr(eacutecart-type de reacutepeacutetabiliteacute) 00378 Sr 00478

RSDr(coefficient de variation de la reacutepeacutetabiliteacute)103 RSDr 130

r(repeatability limit)) 0106 r 0134

SL(eacutecart-type interlaboratoire) -000023 SLlt0 SL 000145

SR(eacutecart-type de reproductibiliteacute) 00378 SR 00611

RSDR(coefficient de variation de la reproductibiliteacute)0010 RSDR 0017

R(limite de reproductibiliteacute)) 0105894 R(limite de reproductibiliteacute))017119

Ref Alt DLaboratoires

Niveau 3

Moyenne si Deviation h k Laboratoires

Niveau 3

Moyenne si Deviation h k

Page 36: ÞÀ¿¸7 ú Y5zñLjÜ< /u +æü t¬Á ... - NF Validation · NF VALIDATION Validation of alternative analytical methods Application in food microbiology ... 500 – 1 000 1 000 –

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3638 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Level 2

Nombre de laboratoires (p) 10

A 2681 2613 2647 005 0034 -0034 -0151 0891 A 2322 2531 2427 015 -0105 0105 -2119 1620 -0220

B 2544 2477 2511 005 0033 -0033 -2699 0872 B 2556 2653 2605 007 -0048 0048 -0581 0750 0094

C 2643 2633 2638 001 0005 -0005 -0311 0130 C 2681 2653 2667 002 0014 -0014 -0040 0217 0029

D 2690 2663 2676 002 0014 -0014 0400 0357 D 2778 2732 2755 003 0023 -0023 0721 0354 0079

E 2690 2716 2703 002 -0013 0013 0897 0336 E 2724 2748 2736 002 -0012 0012 0556 0185 0033

F 2568 2398 2483 012 0085 -0085 -3214 2217 F 2477 2114 2296 026 0182 -0182 -3255 2811 -0188

G 2643 2591 2617 004 0026 -0026 -0707 0682 G 2756 2568 2662 013 0094 -0094 -0085 1453 0045

H 2716 2643 2680 005 0036 -0036 0460 0945 H 2653 2732 2693 006 -0040 0040 0181 0613 0013

L 2591 2591 2591 000 0000 0000 -1196 0000 L 2230 2580 2405 025 -0175 0175 -2307 2704 -0186

M 2653 2690 2672 003 -0018 0018 0311 0481 M 2740 2613 2677 009 0064 -0064 0040 0987 0005

m 2655082 m 26719001 mediane D 00209

Cn(2p) 1867 Cn(2p) 1867 Cp 16101

Cn(p) 16101 Cn(p) 16101 Qn(D) 0039911

n=2p n 20 n=2p n 20 Qdiff 00642591

f 11 f 11 t 082

l 55 l 55

n=p n 10 n=p n 10

f 6 f 6

l 15 l 15

Qn(2p) 0020570 Qn(2p) 00345985

Qn(p) 00332437 Qn(p) 00718158

Qintra 003841 Qintra 006460

Qinter 005352 Qinter 011563

Sr(eacutecart-type de reacutepeacutetabiliteacute) 00543 Sr 00914

RSDr(coefficient de variation de la reacutepeacutetabiliteacute)205 RSDr 342

r(repeatability limit)) 0152 r 0256

SL(eacutecart-type interlaboratoire) 000139 SL 000920

SR(eacutecart-type de reproductibiliteacute) 00659 SR 01325

RSDR(coefficient de variation de la reproductibiliteacute)0025 RSDR 0050

R(limite de reproductibiliteacute)) 018446 R(limite de reproductibiliteacute))03708618

Laboratoires

Niveau 2

Moyenne si Laboratoires

Niveau 2

DRef Alt Moyenne si Deviation h Deviation h k k

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3738 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Level 2 bis

Nombre de laboratoires (p) 10

A 3000 2477 2739 037 0261 -0261 0832 1418 A 2602 2477 2540 009 0062 -0062 -0391 0465 -0199

B 2544 2813 2678 019 -0134 0134 0211 0729 B 2903 2778 2841 009 0062 -0062 1732 0465 0162

C 2477 2813 2645 024 -0168 0168 -0135 0911 C 2477 2699 2588 016 -0111 0111 -0049 0826 -0057

D 2398 2653 2526 018 -0128 0128 -1370 0692 D 2778 3000 2889 016 -0111 0111 2074 0826 0363

E 2875 2740 2808 010 0067 -0067 1547 0365 E 2602 2301 2452 021 0151 -0151 -1012 1120 -0356

F 2477 2544 2511 005 -0033 0033 -1525 0182 F 2778 2477 2628 021 0151 -0151 0230 1120 0117

G 2653 2740 2697 006 -0044 0044 0400 0236 G 2845 2845 2845 000 0000 0000 1764 0000 0148

H 2176 2813 2495 045 -0318 0318 -1692 1727 H 2477 2477 2477 000 0000 0000 -0832 0000 -0017

L 2813 2544 2678 019 0134 -0134 0211 0729 L 2903 2301 2602 043 0301 -0301 0049 2241 -0076

M 2602 2740 2671 010 -0069 0069 0135 0375 M 2903 2255 2579 046 0324 -0324 -0112 2411 -0092

m 2658114 m 259505 mediane D -00372

Cn(2p) 1867 Cn(2p) 1867 Cp 16101

Cn(p) 16101 Cn(p) 16101 Qn(D) 01656915

n=2p n 20 n=2p n 20 Qdiff 02667753

f 11 f 11 t 035

l 55 l 55

n=p n 10 n=p n 10

f 6 f 6

l 15 l 15

Qn(2p) 0098745 Qn(2p) 007195

Qn(p) 006007 Qn(p) 008805

Qintra 018437 Qintra 013435

Qinter 009672 Qinter 014176

Sr(eacutecart-type de reacutepeacutetabiliteacute) 02607 Sr 01900

RSDr(coefficient de variation de la reacutepeacutetabiliteacute)981 RSDr 732

r(repeatability limit)) 0730 r 0532

SL(eacutecart-type interlaboratoire) -002464 SLlt0 SL 000205

SR(eacutecart-type de reproductibiliteacute) 02607 SR 01953

RSDR(coefficient de variation de la reproductibiliteacute)0098 RSDR 0075

R(limite de reproductibiliteacute)) 072996 R(limite de reproductibiliteacute))054686

Laboratoires

Niveau 2 bis

Moyenne si Laboratoires

Niveau 2 bis

DRef Alt Moyenne si Deviation h Deviation h k k

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3838 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Level 3

Nombre de laboratoires (p) 10

A 3690 3663 3676 002 0014 -0014 -0311 0513 A 3690 3708 3699 001 -0009 0009 0354 0257 0022

B 3613 3672 3642 004 -0030 0030 -1851 1109 B 3708 3568 3638 010 0070 -0070 -0844 2061 -0005

C 3663 3732 3698 005 -0035 0035 0645 1302 C 3663 3699 3681 003 -0018 0018 0000 0536 -0017

D 3690 3690 3690 000 0000 0000 0311 0000 D 3643 3663 3653 001 -0010 0010 -0545 0286 -0037

E 3732 3653 3693 006 0040 -0040 0429 1480 E 3613 3732 3673 008 -0060 0060 -0162 1769 -0020

F 3643 3672 3658 002 -0014 0014 -1157 0536 F 3690 3771 3731 006 -0040 0040 0975 1193 0073

G 3699 3653 3676 003 0023 -0023 -0328 0856 G 3602 3643 3623 003 -0021 0021 -1141 0612 -0053

H 3623 3672 3648 003 -0024 0024 -1615 0913 H 3748 3681 3715 005 0033 -0033 0664 0990 0067

L 3724 3663 3694 004 0031 -0031 0461 1150 L 3748 3771 3760 002 -0011 0011 1544 0335 0066

M 3690 3690 3690 000 0000 0000 0311 0000 M 3806 3681 3744 009 0062 -0062 1234 1848 0054

m 368334 m 368086 mediane D 00089

Cn(2p) 1867 Cn(2p) 1867 Cp 16101

Cn(p) 16101 Cn(p) 16101 Qn(D) 00325359

n=2p n 20 n=2p n 20 Qdiff 00523852

f 11 f 11 t 043

l 55 l 55

n=p n 10 n=p n 10

f 6 f 6

l 15 l 15

Qn(2p) 0014323 Qn(2p) 001811

Qn(p) 0013719 Qn(p) 003164

Qintra 002674 Qintra 003381

Qinter 002209 Qinter 005094

Sr(eacutecart-type de reacutepeacutetabiliteacute) 00378 Sr 00478

RSDr(coefficient de variation de la reacutepeacutetabiliteacute)103 RSDr 130

r(repeatability limit)) 0106 r 0134

SL(eacutecart-type interlaboratoire) -000023 SLlt0 SL 000145

SR(eacutecart-type de reproductibiliteacute) 00378 SR 00611

RSDR(coefficient de variation de la reproductibiliteacute)0010 RSDR 0017

R(limite de reproductibiliteacute)) 0105894 R(limite de reproductibiliteacute))017119

Ref Alt DLaboratoires

Niveau 3

Moyenne si Deviation h k Laboratoires

Niveau 3

Moyenne si Deviation h k

Page 37: ÞÀ¿¸7 ú Y5zñLjÜ< /u +æü t¬Á ... - NF Validation · NF VALIDATION Validation of alternative analytical methods Application in food microbiology ... 500 – 1 000 1 000 –

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3738 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Level 2 bis

Nombre de laboratoires (p) 10

A 3000 2477 2739 037 0261 -0261 0832 1418 A 2602 2477 2540 009 0062 -0062 -0391 0465 -0199

B 2544 2813 2678 019 -0134 0134 0211 0729 B 2903 2778 2841 009 0062 -0062 1732 0465 0162

C 2477 2813 2645 024 -0168 0168 -0135 0911 C 2477 2699 2588 016 -0111 0111 -0049 0826 -0057

D 2398 2653 2526 018 -0128 0128 -1370 0692 D 2778 3000 2889 016 -0111 0111 2074 0826 0363

E 2875 2740 2808 010 0067 -0067 1547 0365 E 2602 2301 2452 021 0151 -0151 -1012 1120 -0356

F 2477 2544 2511 005 -0033 0033 -1525 0182 F 2778 2477 2628 021 0151 -0151 0230 1120 0117

G 2653 2740 2697 006 -0044 0044 0400 0236 G 2845 2845 2845 000 0000 0000 1764 0000 0148

H 2176 2813 2495 045 -0318 0318 -1692 1727 H 2477 2477 2477 000 0000 0000 -0832 0000 -0017

L 2813 2544 2678 019 0134 -0134 0211 0729 L 2903 2301 2602 043 0301 -0301 0049 2241 -0076

M 2602 2740 2671 010 -0069 0069 0135 0375 M 2903 2255 2579 046 0324 -0324 -0112 2411 -0092

m 2658114 m 259505 mediane D -00372

Cn(2p) 1867 Cn(2p) 1867 Cp 16101

Cn(p) 16101 Cn(p) 16101 Qn(D) 01656915

n=2p n 20 n=2p n 20 Qdiff 02667753

f 11 f 11 t 035

l 55 l 55

n=p n 10 n=p n 10

f 6 f 6

l 15 l 15

Qn(2p) 0098745 Qn(2p) 007195

Qn(p) 006007 Qn(p) 008805

Qintra 018437 Qintra 013435

Qinter 009672 Qinter 014176

Sr(eacutecart-type de reacutepeacutetabiliteacute) 02607 Sr 01900

RSDr(coefficient de variation de la reacutepeacutetabiliteacute)981 RSDr 732

r(repeatability limit)) 0730 r 0532

SL(eacutecart-type interlaboratoire) -002464 SLlt0 SL 000205

SR(eacutecart-type de reproductibiliteacute) 02607 SR 01953

RSDR(coefficient de variation de la reproductibiliteacute)0098 RSDR 0075

R(limite de reproductibiliteacute)) 072996 R(limite de reproductibiliteacute))054686

Laboratoires

Niveau 2 bis

Moyenne si Laboratoires

Niveau 2 bis

DRef Alt Moyenne si Deviation h Deviation h k k

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3838 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Level 3

Nombre de laboratoires (p) 10

A 3690 3663 3676 002 0014 -0014 -0311 0513 A 3690 3708 3699 001 -0009 0009 0354 0257 0022

B 3613 3672 3642 004 -0030 0030 -1851 1109 B 3708 3568 3638 010 0070 -0070 -0844 2061 -0005

C 3663 3732 3698 005 -0035 0035 0645 1302 C 3663 3699 3681 003 -0018 0018 0000 0536 -0017

D 3690 3690 3690 000 0000 0000 0311 0000 D 3643 3663 3653 001 -0010 0010 -0545 0286 -0037

E 3732 3653 3693 006 0040 -0040 0429 1480 E 3613 3732 3673 008 -0060 0060 -0162 1769 -0020

F 3643 3672 3658 002 -0014 0014 -1157 0536 F 3690 3771 3731 006 -0040 0040 0975 1193 0073

G 3699 3653 3676 003 0023 -0023 -0328 0856 G 3602 3643 3623 003 -0021 0021 -1141 0612 -0053

H 3623 3672 3648 003 -0024 0024 -1615 0913 H 3748 3681 3715 005 0033 -0033 0664 0990 0067

L 3724 3663 3694 004 0031 -0031 0461 1150 L 3748 3771 3760 002 -0011 0011 1544 0335 0066

M 3690 3690 3690 000 0000 0000 0311 0000 M 3806 3681 3744 009 0062 -0062 1234 1848 0054

m 368334 m 368086 mediane D 00089

Cn(2p) 1867 Cn(2p) 1867 Cp 16101

Cn(p) 16101 Cn(p) 16101 Qn(D) 00325359

n=2p n 20 n=2p n 20 Qdiff 00523852

f 11 f 11 t 043

l 55 l 55

n=p n 10 n=p n 10

f 6 f 6

l 15 l 15

Qn(2p) 0014323 Qn(2p) 001811

Qn(p) 0013719 Qn(p) 003164

Qintra 002674 Qintra 003381

Qinter 002209 Qinter 005094

Sr(eacutecart-type de reacutepeacutetabiliteacute) 00378 Sr 00478

RSDr(coefficient de variation de la reacutepeacutetabiliteacute)103 RSDr 130

r(repeatability limit)) 0106 r 0134

SL(eacutecart-type interlaboratoire) -000023 SLlt0 SL 000145

SR(eacutecart-type de reproductibiliteacute) 00378 SR 00611

RSDR(coefficient de variation de la reproductibiliteacute)0010 RSDR 0017

R(limite de reproductibiliteacute)) 0105894 R(limite de reproductibiliteacute))017119

Ref Alt DLaboratoires

Niveau 3

Moyenne si Deviation h k Laboratoires

Niveau 3

Moyenne si Deviation h k

Page 38: ÞÀ¿¸7 ú Y5zñLjÜ< /u +æü t¬Á ... - NF Validation · NF VALIDATION Validation of alternative analytical methods Application in food microbiology ... 500 – 1 000 1 000 –

Thermo Fisher Scientific

ADRIA Deacuteveloppement 3838 November 19 2014

Summary Report (Version 1)

Listeria Precis Enumeration

Level 3

Nombre de laboratoires (p) 10

A 3690 3663 3676 002 0014 -0014 -0311 0513 A 3690 3708 3699 001 -0009 0009 0354 0257 0022

B 3613 3672 3642 004 -0030 0030 -1851 1109 B 3708 3568 3638 010 0070 -0070 -0844 2061 -0005

C 3663 3732 3698 005 -0035 0035 0645 1302 C 3663 3699 3681 003 -0018 0018 0000 0536 -0017

D 3690 3690 3690 000 0000 0000 0311 0000 D 3643 3663 3653 001 -0010 0010 -0545 0286 -0037

E 3732 3653 3693 006 0040 -0040 0429 1480 E 3613 3732 3673 008 -0060 0060 -0162 1769 -0020

F 3643 3672 3658 002 -0014 0014 -1157 0536 F 3690 3771 3731 006 -0040 0040 0975 1193 0073

G 3699 3653 3676 003 0023 -0023 -0328 0856 G 3602 3643 3623 003 -0021 0021 -1141 0612 -0053

H 3623 3672 3648 003 -0024 0024 -1615 0913 H 3748 3681 3715 005 0033 -0033 0664 0990 0067

L 3724 3663 3694 004 0031 -0031 0461 1150 L 3748 3771 3760 002 -0011 0011 1544 0335 0066

M 3690 3690 3690 000 0000 0000 0311 0000 M 3806 3681 3744 009 0062 -0062 1234 1848 0054

m 368334 m 368086 mediane D 00089

Cn(2p) 1867 Cn(2p) 1867 Cp 16101

Cn(p) 16101 Cn(p) 16101 Qn(D) 00325359

n=2p n 20 n=2p n 20 Qdiff 00523852

f 11 f 11 t 043

l 55 l 55

n=p n 10 n=p n 10

f 6 f 6

l 15 l 15

Qn(2p) 0014323 Qn(2p) 001811

Qn(p) 0013719 Qn(p) 003164

Qintra 002674 Qintra 003381

Qinter 002209 Qinter 005094

Sr(eacutecart-type de reacutepeacutetabiliteacute) 00378 Sr 00478

RSDr(coefficient de variation de la reacutepeacutetabiliteacute)103 RSDr 130

r(repeatability limit)) 0106 r 0134

SL(eacutecart-type interlaboratoire) -000023 SLlt0 SL 000145

SR(eacutecart-type de reproductibiliteacute) 00378 SR 00611

RSDR(coefficient de variation de la reproductibiliteacute)0010 RSDR 0017

R(limite de reproductibiliteacute)) 0105894 R(limite de reproductibiliteacute))017119

Ref Alt DLaboratoires

Niveau 3

Moyenne si Deviation h k Laboratoires

Niveau 3

Moyenne si Deviation h k