Summary report - Accueil - NF Validation · Listeria monocytogenes in food products and environmental samples ... 5 CONCLUSION ... and environmental samples, before running the MDA

ACCREDITATION

N°1-0144

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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

3M

Boulevard de l’Oise

F-95029 CERGY PONTOISE CEDEX

NF VALIDATION

Validation of alternative analytical methods

Application in food microbiology

Summary report

EN ISO 16140-2:2016 validation study of

the 3MTM Molecular Detection Assay 2 -

Listeria monocytogenes for detection of Listeria monocytogenes in food products

and environmental samples

Qualitative method

This report includes 66 pages, with 5 appendixes.

Only copies including the totality of this report are authorized.

Competences of the laboratory are certified by COFRAC accreditation for the analyses

marked with symbol.

Version 0

November 14, 2016

mailto:[email protected]

http://www.adria.tm.fr/

3M

ADRIA Développement 2/66 November 14, 2016

Summary Report (Version 0)

3M MDA 2 - Listeria monocytogenes

1 AIM OF THE STUDY ____________________________________________ 4

2 METHOD PROTOCOLS _________________________________________ 4

2.1 Reference method _______________________________________________ 4

2.2 Alternative method ______________________________________________ 4

3 METHOD COMPARISON STUDY __________________________________ 5

3.1 Sensitivity study ________________________________________________ 5

3.1.1 Number and nature of samples ______________________________________ 5

3.1.2 Artificial contamination of samples ____________________________________ 6

3.1.3 Confirmation protocols _____________________________________________ 6

3.1.4 Test results ______________________________________________________ 7

3.1.5 Calculation of relative accuracy (RT), relative sensitivity (SE) and false positive

ratio (FPR) ______________________________________________________ 9

3.1.6 Analysis of discordants ___________________________________________ 11

3.1.7 Enrichment broth storage at 2 - 8°C for 72 h ___________________________ 17

3.1.8 Confirmation ____________________________________________________ 18

3.1.9 Inhibitions ______________________________________________________ 19

3.2 Relative level of detection study __________________________________ 19

3.2.1 Experimental design ______________________________________________ 19

3.2.2 Calculation and interpretation of the RLOD ____________________________ 20

3.3 Inclusivity / exclusivity __________________________________________ 21

3.3.1 Test protocols ___________________________________________________ 21

3.3.2 Results ________________________________________________________ 21

3.4 Practicability __________________________________________________ 22

4 INTER-LABORATORY STUDY ORGANISATION AND RESULTS _______ 23

4.1 Study organisation _____________________________________________ 23

4.2 Experimental parameters controls _________________________________ 24

4.2.1 Sample stability _________________________________________________ 24

4.2.2 Logistic conditions _______________________________________________ 26

4.3 Calculation and summary of data _________________________________ 27

4.3.1 Results obtained by the expert Lab. _________________________________ 27

4.3.2 Results obtained by the collaborators ________________________________ 27

4.4 Interpretation __________________________________________________ 32

5 CONCLUSION ________________________________________________ 34

Appendix 1 – Flow diagram of the EN ISO 11290-1/A1 (2004) method: Horizontal method

for the detection and enumeration of Listeria monocytogenes - Part 1: detection of Listeria monocytogenes

in foods ___________________________________________________________________________ 36

Appendix 2 – Flow diagram of the 3MTM Molecular Detection Assay 2 - Listeria monocytogenes ______ 37

Appendix 3 – Relative level of detection study: raw data _____________________________________ 40

Appendix 4 – Inclusivity and exclusivity study: raw data _____________________________________ 46

Appendix 5 – Results obtained by the collaborators and the expert laboratory____________________ 51

3M




Quality Assurance documents related to this study can be consulted upon

request from 3M.

The technical protocol and the result interpretation were realised according to

the EN ISO 16140-2:2016 and the AFNOR technical rules.

Company: 3M

Boulevard de l’Oise

F-95029 CERGY PONTOISE CEDEX

Expert Laboratory: ADRIA Développement

ZA Creac’h Gwen

F-29196 QUIMPER Cedex

Studied method: 3MTM Molecular Detection Assay 2 - Listeria

monocytogenes

Validation standard : EN ISO 16140-2:2016 - Microbiology of the food

chain - Method validation

Part 1: Vocabulary

Part 2: Protocol for the validation of alternative

(proprietary) methods against a reference method

Standard method: EN ISO 11290-1/A1 (2004): Microbiology of food and

animal feeding stuffs - Horizontal method for the

detection and enumeration of Listeria

monocytogenes - Part 1: detection of Listeria

monocytogenes in foods

Scope: Food products and Environmental samples

(excluding samples from primary production)

Certification body: AFNOR Certification

Analyses performed according to the COFRAC accreditation

3M




1 AIM OF THE STUDY

The 3MTM Molecular Detection Assay 2 - Listeria monocytogenes for the

detection of Listeria monocytogenes was validated for human food

products and environmental samples (excluding samples from primary

production) was validated in September 2016 (Certificate number:

3M 01/15 - 09/16) according to the EN ISO 16140-2 (2016).

2 METHOD PROTOCOLS

2.1 Reference method

The reference method correspond to the ISO 11290-1/A1 (2004) standard:

Microbiology of food and animal feeding stuffs - Horizontal method for the

detection and enumeration of Listeria monocytogenes – Part 1: detection

method (See Appendix 1).

2.2 Alternative method

Principle

The 3MTM Molecular Detection Assay 2 - Listeria monocytogenes Test Kit

uses isothermal amplification of unique DNA target sequences, the amplified

sequences are detected by bioluminescence.

Protocol (see Appendix 2)

- Enrichment step in Half Fraser broth at 37°C ± 1°C:

* Specific protocol: 25 g + 225 ml Half Fraser for raw dairy, raw meat

and raw seafood products. Incubation for 22 h ± 2 h at 37°C.

Subculture in Fraser broth for 22 h ± 2 h at 37°C.

* General protocol: 25 g + 225 ml Half Fraser for all other products

(food and environmental samples). Incubation for 27 h ± 3 h at 37°C

- Lysis step

- Amplification and real-time detection

Analysis performed according to the COFRAC accreditation

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- Confirmation step by streaking 100 µl of enriched Half Fraser broth onto

O&A and Palcam plates (General protocol), and by streaking 10 µl of

Fraser broth (Specific protocol).

It is possible to store the enrichment broths for 72 h at 2 - 8°C for all the

categories and to store the lysates for 72 h at 2 - 8°C for all the categories,

except for Dairy products and environmental samples, before running the

MDA test.

The flow diagram is given in Appendix 2.

3 METHOD COMPARISON STUDY

As the enrichment procedure is different between the alternative and

the reference methods, this corresponds to an UNPAIRED DATA

STUDY DESIGN.

3.1 Sensitivity study

The sensitivity (SE) is the ability of the method to detect the analyte by either the reference or

alternative method.

3.1.1 Number and nature of samples

400 samples were analyzed.

The distribution per tested category and type is given in Table 1.

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Table 1 – Distribution per tested category and type

Categories TypesPositive

sample

Negative

samplesTotal

a Ready-to-eat 11 10 21

b Ready-to-reheat 10 10 20

c Pastries and egg products 9 12 21

Total 30 32 62

a Meat products (raw, frozen, seasoned) 17 10 27

b Ready-to-eat and processed meat products 7 14 21

c Delicatessen 10 13 23

Total 34 37 71

a Raw dairy products 13 10 23

b Pasteurized milk cheeses 9 11 20

c Ice cream, milk (pasteurized), flavoured milk

(pasteurized) 8 12 20

Total 30 33 63

a Raw vegetable products (fresh and frozen) 10 16 26

b Mapped vegetables and heat processed vegetables

14 6 20

c Vegetables based preparations, processed vegetables

7 15 22

Total 31 37 68

a Raw products (fresh, frozen) 10 10 20

b Smoked, marinated 10 10 20

c Ready-to-eat or ready-to-reheat 10 12 22

Total 30 32 62

a Process water 7 14 21

b Dusts 14 12 26

c Wipes 11 16 27

Total 32 42 74

187 213 400

SEAFOOD AND

FISHERY PRODUCTS

ENVIRONMENTAL

SAMPLES

TOTAL

COMPOSITE FOODS/

READY-TO-EAT AND

READY-TO-REHEAT

MEAT PRODUCTS

DAIRY PRODUCTS

VEGETABLES

3.1.2 Artificial contamination of samples

Artificial contaminations were done by seeding protocol.

87 samples were artificially contaminated, using 37 different strains. 62 gave

a positive result. All the samples were inoculated at level 3 CFU.

53.5 % of the samples were naturally contaminated.

3.1.3 Confirmation protocols

The positive tests were confirmed by streaking 100 µl of the enrichment broth

onto O&A. During the validation study, the typical colonies were identified by

the tests described in the reference method.

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3.1.4 Test results

The results are given in the following tables.

Table 2 – Summary of results obtained with the reference

and the alternative method: All the samples

Response Reference method

positive (R+)

Reference method

negative (R-)

Alternative method

positive (A+)

Positive agreement (A+/R+)

PA = 119

Positive deviation (R-/A+)

PD = 35

Alternative method

negative (A-)

Negative deviation (A-/R+)

ND = 33 (PPND = 1)

Negative agreement (A-/R-)

NA = 213 (PPNA = 2)

PP: positive presumptive non confirmed samples

PD = positive deviation (R-/A+) ND = negative deviation (A-/R+)

Results per category of samples


and the alternative method:

composite foods / ready-to-eat and ready-to-reheat


positive (R+)

Reference method

negative (R-)

Alternative method

positive (A+)


PA = 22


PD = 5

Alternative method

negative (A-)


ND = 3


NA = 32


and the alternative method: Meat products


positive (R+)

Reference method

negative (R-)

Alternative method

positive (A+)


PA = 17


PD = 8

Alternative method

negative (A-)


ND = 9


NA = 37

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and the alternative method: Dairy products


positive (R+)

Reference method

negative (R-)

Alternative method

positive (A+)


PA = 18


PD = 8

Alternative method

negative (A-)


ND = 4


NA = 33


and the alternative method: Vegetables


positive (R+)

Reference method

negative (R-)

Alternative method

positive (A+)


PA = 19


PD = 6

Alternative method

negative (A-)


ND = 6 (PPND = 1)


NA = 37


and the alternative method: Seafood and fishery products


positive (R+)

Reference method

negative (R-)

Alternative method

positive (A+)


PA = 22


PD = 4

Alternative method

negative (A-)


ND = 4


NA = 32


and the alternative method: Environmental samples


positive (R+)

Reference method

negative (R-)

Alternative method

positive (A+)


PA = 21


PD = 4

Alternative method

negative (A-)


ND = 7


NA = 42 (PPNA = 2)

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The summary of results per protocol is given tables 10 and 11.


and the alternative method: General protocol


positive (R+)

Reference method

negative (R-)

Alternative method

positive (A+)


PA = 91


PD = 28

Alternative method

negative (A-)


ND = 28 (PPND = 1)


NA = 183 (PPNA = 2)


and the alternative method: Specific protocol


positive (R+)

Reference method

negative (R-)

Alternative method

positive (A+)


PA = 28


PD = 7

Alternative method

negative (A-)


ND = 5


NA = 30

3.1.5 Calculation of relative accuracy (RT), relative sensitivity (SE) and

false positive ratio (FPR)

The calculations are presented in table 11.

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Table 11 – Calculation of the relative accuracy (RT), the relative sensitivity (SE) and the false positive ratio (FPR)

Category Type PA NA PD ND PPND PPNA SE alt % SE ref % RT % FPR %

1 Composite foods/

Ready to eat/ Ready to reheat

a Ready to eat 7 10 3 1 0 0 90.9 72.7 81.0 0.0

b Ready to reheat 7 10 2 1 0 0 90.0 80.0 85.0 0.0

c Pastries, desserts, omelettes 8 12 0 1 0 0 88.9 100.0 95.2 0.0

Total 22 32 5 3 0 0 90.0 83.3 87.1 0.0

2 Meat products

a Raw, frozen, seasoned 10 10 3 4 0 0 76.5 82.4 74.1 0.0

b Ready to eat and processed meat products 3 14 2 2 0 0 71.4 71.4 81.0 0.0

c Delicatessen 4 13 3 3 0 0 70.0 70.0 73.9 0.0

Total 17 37 8 9 0 0 73.5 76.5 76.1 0.0

3 Dairy products

a Raw dairy products 10 10 3 0 0 0 100.0 76.9 87.0 0.0

b Pasteurized milk cheese 3 11 3 3 0 0 66.7 66.7 70.0 0.0

c Ice cream, milk (pasteurized), flavored milk 5 12 2 1 0 0 87.5 75.0 85.0 0.0

Total 18 33 8 4 0 0 86.7 73.3 81.0 0.0

4 Vegetables

a Raw vegetable products (fresh, frozen) 8 16 1 1 0 0 90.0 90.0 92.3 0.0

b Mapped vegetables and heat processed vegetables 7 6 4 2 1 0 78.6 71.4 65.0 16.7

c Vegetables based preparations, processed vegetables 4 15 1 2 0 0 71.4 85.7 86.4 0.0

Total 19 37 6 5 1 0 80.6 80.6 82.4 2.7

5 Seafood and fishery products

a Raw products (fresh, frozen) 8 10 1 1 0 0 90.0 90.0 90.0 0.0

b Smoked, marinated 7 10 3 0 0 0 100.0 70.0 85.0 0.0

c Ready to eat or ready to reheat 7 12 0 3 0 0 70.0 100.0 86.4 0.0

Total 22 32 4 4 0 0 86.7 86.7 87.1 0.0

6 Environmental samples

a Process water 7 14 0 0 0 0 100.0 100.0 100.0 0.0

b Dusts 5 11 4 5 0 1 64.3 71.4 65.4 9.1

c Wipes 9 15 0 2 0 1 81.8 100.0 92.6 6.7

Total 21 40 4 7 0 2 78.1 87.5 85.1 4.8

All categories 119 211 35 32 1 2 82.4 81.3 83.0 1.4

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The following results are observed:

Table 12

Sensitivity for the alternative method

%100

)(

PDNDPA

PDPASEalt 82.4 %

Sensitivity for the reference method

%100

)(

PDNDPA

NDPASEref 81.3 %

Relative trueness %100)(

N

NAPART 83.0 %

False positive ratio for the alternative method*

FP = PPNA + PPND

%100)(

NA

FPFPR

1.4 %

* With ND = ND + PPND NA = NA + PPNA

3.1.6 Analysis of discordants

Among the 33 negative deviations (see table 13), 13 concern artificially

contaminated samples and 20 naturally contaminated samples.

The presence of Listeria monocytogenes was confirmed for 7 samples by the

tests of the alternative method (streaking onto O&A plates) and one

additional sample (No 2565) was confirmed by applying the whole protocol of

the reference method (subculture in Fraser for 48 h at 37°C and streaking

onto O&A).

For 25 samples, the negative results were probably due to sampling

heterogeneity in this unpaired data study when dealing with low

contamination levels.

Among the 35 positive deviations (see table 14), 9 concern artificially

contaminated samples and 24 concern naturally contaminated samples.

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Table 13 - Negative deviations

Category Type Sample

N° Product Inoculated strain

Inoculation level

(cfu/sample)

MDA result

Confirmation result Final result Alternative method

Reference method (Fraser 48h 37°C)

Composite foods / Ready to eat and ready to reheat

b 2761 RTRH composite food -/-/- L.monocytogenes H+ -

c 3202 Tortilla L.monocytogenes Ad1757 1.8 - / / -

a 3748 Sandwich (ham, cheese) L.monocytogenes Ad1197 0.8 - / - -

Meat products

c 1097 Rillettes - L.grayi - -

c 4451 Cooked ham - / - -

c 4664 Smoked sliced bacon - / - -

a 6178 Raw chicken meat -/-/- L.monocytogenes / -

a 6179 Beef meat -/-/- L.monocytogenes / -

a 6186 Ground poultry - / - -

a 6365 Gizzards - / - -

b 7553 Ready to reheat meal L.monocytogenes A00C052 1.4 - / - -

b 7558 Ready to reheat meal (beef) L.monocytogenes Ad266 0.8 - / - -

Dairy products

c 3189 Pasteurized half skimmed milk L.monocytogenes A00L107 0.8 - / / -

b 3405 Pasteurized cow milk cheese L.monocytogenes A00L106 1.2 -/-/- L.monocytogenes + -

b 3752 Pasteurized milk cheese L.monocytogenes Ad1201 2.4 -/-/- L.monocytogenes + -

b 7561 Pasteurized ewe milk cheese L.monocytogenes Ad1201 0.8 - / - -

Vegetables

b 4443 Heat processed vegetables - / - -

a 4445 Sprouts - / - -

b 4447 Zucchini + L.innocua - -

b 4454 Frozen broccoli - / - -

c 7372 Frozen vegetables mix - L.welshimeri/

L.innocua - -

c 7570 Processed vegetables mix L.monocytogenes 1011/1410

1.0 - / - -

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Inoculation level

(cfu/sample)

MDA result

Confirmation result Final result Alternative method

Reference method (Fraser 48h 37°C)

Seafood and

fishery products

c 1100 RTRH salmon - L.welshimeri - -

c 2565 RTRH salmon balls - / H+(L.mono) -

c 2938 Cooked shrimps i/+* L.monocytogenes / -

a 6200 Raw fish - / - -

Environmental

samples

c 4142 Swab (dairy environment) L.monocytogenes Ad629 1.6 - / - -

b 4384 Wastes (Fish industry) - L.innocua - -

b 6869 Wastes (Meat industry) - L.welshimeri - -

c 6873 Wipe (Dairy industry) -/i/+ L.monocytogenes/

L.innocua / -

b 6877 Wastes (meat) L.monocytogenes Ad1265 0.4 -/+/+ L.innocua - -

b 6878 Wastes (meat) L.monocytogenes Ad1265 0.4 -/+/+ L.welshimeri - -

b 6881 Wastes (meat) L.monocytogenes Ad1255 L.innocua Ad1251

1.4 - L.innocua - -

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Table 14 - Positive deviations



Inoculation level

(cfu/sample)

MDA result

Confirmation result Final result

Composite foods /

Ready to eat /

Ready to reheat

b 1076 Deli salad + L.monocytogenes +

a 1079 RTE + L.monocytogenes +

a 2760 Sandwich (goat cheese, vegetables) + L.monocytogenes +

a 2933 Chinese pasta + L.monocytogenes +

b 3150 RTRH composite food + L.monocytogenes +

Meat products

b 2952 Sliced cooked chicken + L.monocytogenes +

c 3158 Terrine + L.monocytogenes +

c 3165 Sausages with aromatic herbs + L.monocytogenes/

L.innocua +

c 4441 Cooked sausage +/-/- L.monocytogenes /L.innocua +

a 6180 Sheep meat + L.monocytogenes (72h) +

a 6183 Chicken leg + L.monocytogenes +

a 6190 Chicken leg + L.monocytogenes +

b 7557 Ready to reheat meal (beef) L.monocytogenes Ad266 0.8 + L.monocytogenes +

Dairy products

c 3190 Pasteurized half skimmed milk L.monocytogenes 910 1.8 + L.monocytogenes +

c 3192 Cocoa flavored milk L.monocytogenes A00L098 1.2 + L.monocytogenes +

b 3404 Pasteurized cow milk cheese L.monocytogenes A00L106 1.2 + L.monocytogenes +

b 3749 Pasteurized cow milk cheese L.monocytogenes Ad1784 1.0 + L.monocytogenes +

a 4149 Raw milk cheese L.monocytogenes Ad665 2 + L.monocytogenes +

a 4703 Raw milk cheese + L.monocytogenes +

a 6246 Raw ewe milk cheese + L.monocytogenes +

b 7563 Pasteurized ewe milk cheese L.monocytogenes Ad523 0.8 + L.monocytogenes +

Vegetables

b 3539 Frozen mushrooms + L.monocytogenes +

b 4458 Frozen precooked onions + L.monocytogenes +

b 4459 Frozen zucchini + L.monocytogenes (+72h)/L.innocua +

b 4461 Vegetables for couscous + L.monocytogenes +

a 4655 Pieces of chestnuts + L.monocytogenes +

c 7569 Processed vegetables mix L.monocytogenes 1011/1410 1.0 + L.monocytogenes +

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Inoculation level

(cfu/sample)

MDA result

Confirmation result Final result

Seafood and

fishery products

b 3528 Smoked salmon + L.monocytogenes +



a 6194 Raw fish + L.monocytogenes (Rapid'L.mono)

+

Environmental

samples

b 6870 Wastes (meat) + L.monocytogenes +


1.4 + L.monocytogenes +


1.4 + L.monocytogenes (Fraser 1) /

L.welshimeri +

b 7932 Dusts L.monocytogenes Ad634 1.8 + L.monocytogenes +

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The analyses of discordant results according to the EN ISO 16140-2: 2016 is

the following (See table 15):

Table 15

Category Type (ND+PPND)-

PD AL

1 Composite foods/Ready to eat/Ready to reheat

a Ready to eat -2

b Ready to reheat -1

c Pastries, desserts, omelettes 1

Total -2 3

2 Meat products

a Raw, frozen, seasoned 1

b Ready to eat and processed meat products 0

c Delicatessen 0

Total 1 3

3 Dairy products

a Raw dairy products -3

b Pasteurized milk cheese 0

c Ice cream, milk (pasteurized), flavored milk -1

Total -4 3

4 Vegetables

a Raw vegetable products (fresh, frozen) 0

b Mapped vegetables and heat processed vegetables -1

c Vegetables based preparations, processed vegetables 1

Total 0 3

5 Seafood and fishery

products

a Raw products (fresh, frozen) 0

b Smoked, marinated -3

c Ready to eat or ready to reheat 3

Total 0 3

6 Environmental samples

a Process water 0

b Dusts 1

c Wipes 2

Total 3 3

All categories -2 6

The observed values for ((ND + PPND) - PD) are below or equal to the

acceptability limit for each category and for all the categories.

Additional Listeria monocytogenes strains were recovered from five samples

with negative agreement results (See table 16). Three samples were

artificially contaminated; the other one were naturally contaminated samples.

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Table 16 - Strains recovered from samples with NA results

Category Sample

No Product

MDA test

MDA confirmation tests Reference method *

Type O&A Identification

Meat products 7554 Ready-to-reheat

meat - / + H+ L. monocytogenes + b

Vegetables

3146 Vegetable based

preparation - H+ L. monocytogenes + c

3538 Precooked frozen

onions - / - / + H+ L. monocytogenes / b

Dairy products 3195 Pasteurized milk

cheese - / - / - H+ L. monocytogenes + b

Environmental

samples 4144 Wastes (fish) - H+ L. monocytogenes + b

* Subculture in Fraser broth from the Half Fraser of the alternative method

3.1.7 Enrichment broth storage at 2 - 8°C for 72 h

224 samples were tested again after enrichments and lysates storage for

72 h at 2 - 8°C. The following changes are observed (See table 17).

Table 17

Sample No Before storage After storage

Enrichment Lysate

2761 ND PA ND

3150 PD PD NA

3146 NA PD PD

4441 PD PD NA

6178 ND PA PA

3527 PA PA ND

3538 NA PD NA

6179 ND ND PA

3749 PD PD NA

4382 PA PA ND

3405 ND PA PA

3752 ND PA ND

4155 PA PA ND

6248 PA PA ND

6189 PA PA ND

6201 PA PA ND

6203 PA PA ND

6873 ND PA PA

7574 NA PD NA

6881 ND PA PA

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The analysis of discordant becomes (See table 18).

Table 18

Category Enrichment storage for 72h at 2-8°C Lysate storage for 72h at 2-8°C

PD ND+PPND (ND+PPND)-PD AL PD ND+PPND (ND+PPND)-PD AL

Composite foods /Ready to eat / Ready to reheat

5 2 -3 3 4 3 -1 3

Meat products 9 8 -1 3 7 8 1 3

Dairy products 8 2 -6 3 7 4 -3 3

Vegetables 8 5 -3 3 7 6 -1 3

Seafood and fishery products

4 4 0 3 4 7 3 3

Environmental samples 4 5 1 3 4 6 2 3

Total 38 26 -12 6 33 34 1 6

The observed values for ((ND + PPND) - PD) are below or equal to the

acceptability limit for each category and for all the categories.

Based on the AFNOR technical rules, the deviations and positive samples

have to be tested for the storage protocols. Three NA were as well tested

here: the samples No 3146, 5358 and 7574. They all became positive after

storage of the broth, and one sample became positive after storage of the

lysate. Note that the inoculation level requirements were as well recently

changed in the AFNOR technical rules, corresponding now more to the limits

of detection of the methods. In that way, the probability to get positive data

from firstly screened negative samples is higher, particularly if these samples

were supposed to be naturally contaminated or inoculated.

As the AL are based on differences between ND and PD, it may be

relevant to tests as well the NA.

3.1.8 Confirmation

Confirmations of the positive MDA tests were performed by streaking the

enrichment broth (Half Fraser for General protocol and Fraser for Specific

protocol) onto O&A plates.

In 8 cases, it was necessary to apply additional confirmatory tests to confirm

the presence of Listeria monocytogenes in the enrichment broth; they are

listed in table 19.

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Table 19

Sample No Additional tests applied

4441 Subculture in Fraser

3530 Streaking onto RAPID’L.mono agar

4459 Recovered from enrichment storage for 72 h at 2 - 8°C




6870 Recovered from enrichment storage for 72 h at 2 - 8°C

6880 Subculture in Fraser

3.1.9 Inhibitions

One inhibition was observed (sample No 2938); this sample gave a positive

MDA test after lysate dilution (1/10). This sample was considered negative

for interpretation, and gave a negative deviation.

3.2 Relative level of detection study

The relative level of detection is the level of detection at P = 0.50 (LOD50) of the alternative

(proprietary) method divided by the level of detection at P = 0.50 (LOD50) of the reference

method. The RLOD is defined as the ratio of the alternative and reference methods:

3.2.1 Experimental design

Six (matrix/strain) pairs were analyzed by the reference method and by the

alternative method (See Table 20):

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Table 20 - Defined (matrix/strain) pairs for the RLOD determination

Matrix and related

category number Inoculated strain Origin

Inoculation

protocol

Analysis

protocol

- Deli-salad (Piémontaise) Listeria monocytogenes Ad494 Deli salad

Seeding

protocol

with 4 °C

storage prior

analyses

General

- Beef meat Listeria monocytogenes Ad1206 Ground beef Specific

- Raw milk Listeria monocytogenes Ad618 Cheese Specific

- Bagged raw spinach Listeria monocytogenes Ad1178 Ready-to-cook General

- Cold smoked salmon Listeria monocytogenes Ad670 Smoked salmon General

- Process water Listeria monocytogenes Ad551 Environmental

sample General

3.2.2 Calculation and interpretation of the RLOD

The raw data are given in Appendix 3.

The RLOD calculations were performed using Excel spreadsheet of the

international standard (ISO 16140), as described in the EN ISO 16140-

2:2016 standard (http://standards.iso.org/iso/16140). The RLOD are given

table 21.

Table 21 – Presentation of RLOD before and after confirmation of the

alternative method results

Name RLOD RLODL RLODU b=ln

(RLOD) sd(b)

z-Test statistic

p-value

Deli-salad (Piémontaise) / L.monocytogenes Ad494 1.0 0.4 3.0 0.0 0.5 0.1 0.9

Beef meat / L.monocytogenes Ad1206 0.7 0.3 1.6 -0.4 0.4 0.9 1.6

Raw milk / L.monocytogenes Ad618 1.0 0.4 2.2 0.0 0.4 0.0 1.0

Bagged raw spinach / L.monocytogenes Ad1178 1.2 0.5 3.0 0.1 0.5 0.3 0.8

Cold smoked salmon / L.monocytogenes Ad670 1.0 0.5 2.3 0.0 0.4 0.1 0.9

Process water / L.monocytogenes Ad551 1.0 0.5 2.2 0.0 0.4 0.0 1.0

The RLOD are below the AL fixed at 2.5 for unpaired studies for all the tested

matrix/strain pairs and for the alternative method.

http://standards.iso.org/iso/16140

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3.3 Inclusivity / exclusivity

Inclusivity is the ability of the alternative method to detect the target analyte from a wide range

of strains.

Exclusivity is the lack of interference from a relevant range of non-target strains of the

alternative method.

3.3.1 Test protocols

50 targeted strains and 30 non targeted strains were tested.

Inclusivity

50 Listeria monocytogenes strains were thawed and grown in BHI broth

overnight at 37°C ± 1°C. The cultures were diluted in peptone salt in order to

inoculate between 10 to 100 cells per 225 ml of Half Fraser. The enrichment

broths were then incubated for 24 h at 37°C ± 1°C. The protocol of the

alternative method was then applied.

Exclusivity

30 non Listeria strains were thawed and grown in BHI broth overnight at

37°C ± 1°C. The cultures were diluted in order to inoculate 105 cells/ml of an

appropriate media (BPW or MRS). The broths were then incubated for 24 h

at the appropriate incubation temperature in order to have culture to test with

the alternative method.

3.3.2 Results

Raw data are given in Appendix 4.

Inclusivity

The 50 specific inclusivity strains tested were detected.

Exclusivity

Among the 30 non target strains tested, no cross reaction was observed.

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3.4 Practicability

The alternative method practicability was evaluated according to the AFNOR

criteria relative to method comparison study.

Storage conditions, shelf-

life and modalities of

utilization after first use

The storage temperature is: 2-8°C. The shelf-life is given on the package. All the

reagents shall be stored at the temperature mentioned on the package.

Time to result Negative samples

Steps Reference

method

Alternative method

General

protocol

Specific

protocol

Sampling enrichment Day 0 Day 0 Day 0

Subculture in Fraser 1 Day 1 / /

Lysate / Day 1 Day 2

MDA test / Day 1 Day 2

Streaking onto O&A plates

(O1/P1) Day 1 / /

Second streaking (O2/P2) Day 3 / /

Reading plates (O1 – P1) Day 2 - Day 3 / /

Reading plates (O2 – P2) Day 4 - ay 5 / /

Results Day 5 Day 1 Day 2

Presumptive positive or positive results

Steps Reference

method

Alternative method

General

protocol

Specific

protocol

Sub-culture of typical colonies Day 2 - Day 5 / /

Streaking onto O&A plates / Day 1 Day 2

O&A plates reading / Day 2 - Day 3 Day 3 - Day 4

Confirmatory tests Day 3 - Day 6 / /

Results Day 4 - Day 7

Day 8 - Day 11 (1) Day 2 - Day 3 Day 3 - Day 4

(1) In the case of the rhamnose and xylose, tests are realized in tubes.

Common step with the

reference method No common step

The negative results are available in one day for the general protocol, and in

two days for the specific protocol.

The positive results are available in two or three days for the general

protocol, and in three or four days for the specific protocol.

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4 INTER-LABORATORY STUDY ORGANISATION AND

RESULTS

4.1 Study organisation

Collaborators number

Samples were sent to 13 laboratories. For 2 Labs, 2 technicians were

involved in the study: Labs K and L in order to have 15 sets of data.

Matrix and strain used

Pasteurised cheese sample (Brie: 31 % fat, 1.4 % NaCl) was inoculated with

Listeria monocytogenes 153, isolated from raw milk cheese.

Samples

Samples were prepared and inoculated on Monday 27th June 2016, as

described below:

- 24 blind coded samples for analysis of Listeria monocytogenes by the

3MTM Molecular Detection Assay 2 - Listeria monocytogenes method

(= RED LABEL)

- 24 blind coded samples for Listeria monocytogenes analysis by the

reference method (EN ISO 11290-1/A1) (= BLUE LABEL)

- 1 sample for aerobic mesophilic flora enumeration by ISO 4833-1 method,

- 1 water flask labelled “Temperature Control” with a temperature probe.

Inoculation

The targeted inoculation levels were the following:

- Level: 0 CFU/25 g,

- Level 1: 2 CFU/g, inoculation level providing as much as possible

fractional positive recovery data;

- Level 2: 8 CFU/25 g.

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Labelling and shipping

Blind coded samples were placed in isothermal boxes, which contained

cooling blocks, and express-shipped to the different laboratories.

A temperature control flask containing a sensor was added to the package in

order to register the temperature profile during the transport, the package

delivery and storage until analyses.

Samples were shipped in 24 h to 48 h to the involved laboratories. The

temperature conditions had to stay lower or equal to 8°C during transport,

and between 0°C – 8°C in the labs.

Analyses

Collaborative study laboratories and the expert laboratory carried out the

analyses on Tuesday 28th or Wednesday 29th June 2016 with the alternative

and reference methods. The analyses by the reference method and the

alternative method were performed on the same day.

Expedition conditions

The collaborative study instructions were sent on 10th June 2016.

4.2 Experimental parameters controls

4.2.1 Sample stability

Contamination levels

In order to detect the presence of Listeria monocytogenes, the reference

method was performed on five portions (25 g) before the inoculation. All the

results were negative.

The contamination levels and the confidence intervals were (See Table 22):

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Table 22

Level Samples Theoretical

target level

True level

(CFU/25 g

sample)

Low limit /

CFU/25 g sample

High limit /

CFU/25 g sample

Level 0

7 - 10 - 11 - 14 -

16 - 18 - 20 - 23 0 / / /

26 - 29 - 30 - 32 -

34 - 37 - 38 - 43

Low level

1 - 3 - 4 - 9 -

15 - 19 - 21 - 22 2 2.4 2.0 2.9

27 - 31 - 33 - 36 -

41 - 44 - 45 - 48

High level

2 - 5 - 6 -8 -

12 - 13 - 17 - 24 8 8.8 7.1 10.9

25 - 28 - 35 - 39 -

40 - 42 - 46 - 47

Strain stability during transport

Three samples inoculated at a high level (100 CFU/g) were tested for

enumeration after 24 h and 48 h storage. Three samples inoculated at a low

level were tested for detection after 24 h and 48 h storage (See Table 23).

Table 23 – Listeria monocytogenes stability in the matrix

Day of analysis Listeria monocytogenes detection Mesophilic aerobic

flora (CFU/g) CFU/g Detection/25 g

Day 0

80 +

2.0 108 110 +

100 +

Day 1

120 +

1.9 108 40 +

100 -

Day 2

110 +

3.4 108 170 +

90 +

No evolution was observed during storage for 48 h at 2 - 8°C.

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4.2.2 Logistic conditions

The temperatures measured at reception by the Labs, the temperatures

registered by the thermo-probe, and the receipt dates are given in Table 24.

Table 24 - Sample temperatures at receipt

Collaborators

Temperature

measured

at receipt (°C)

Temperature

measured

by the probe (°C)

Receipt date

and time Analysis date

A 6.0 4.0 28/06/2016 10h30 28/06/2016

B 5.8 3.5 28/06/2016 10h30 28/06/2016

C 5.5 4.5 28/06/2016 10h30 28/06/2016

D 6.4 5.5 28/06/2016 10h00 28/06/2016

E 6.9 4.5 28/06/2016 11h50 28/06/2016

F 5.5 5.0 28/06/2016 13h50 28/06/2016

G 8.0 4.5 28/06/2016 10h00 28/06/2016

H 6.8 4.0 28/06/2016 13h22 29/06/2016

I 6.5 5.0 28/06/2016 11h10 29/06/2016

J 6.5 4.5 28/06/2016 11h40 28/06/2016

K1 10.7 5.0 28/06/2016 10h10 28/06/2016

K2 10.7 4.5 28/06/2016 10h10 28/06/2016

L1 4.4 3.5 28/06/2016 11h00 29/06/2016

L 2 4.8 4.0 28/06/2016 11h00 29/06/2016

N 5.5 5.0 28/06/2016 10h50 28/06/2016

All the samples were delivered on time and in appropriated conditions.

Temperatures during shipment and at receipt were all correct.

Note that Lab K measured a temperature of 10.7°C at receipt while the

temperature measured with the probe indicated clearly a temperature of

5.0°C.

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4.3 Calculation and summary of data

The raw data are given in Appendix 7.

4.3.1 Results obtained by the expert Lab.

The results obtained by the expert Lab. are the following (See table 4).

Table 25 – Results obtained by the expert Lab.

Level Reference method Alternative method

L0 0/8 0/8

L1 8/8 7/8

L2 8/8 8/8

For Level 1, one sample (M4) gave a negative MDA test while the

confirmatory tests concluded to the presence of Listeria monocytogenes in

the enrichment broth. The lysate was tested twice again and one positive and

one negative results were observed. A second lysate was also tested and a

positive MDA result was obtained.

4.3.2 Results obtained by the collaborators

Samples were sent to 15 collaborators.

Mesophilic aerobic flora

The enumeration of the mesophilic aerobic flora varies from 6.4 107 to

4.2 109 CFU/g, which is a high level of background microflora.

Reference method

The positive results by the reference method are provided in Table 26.

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Table 26 - Positive results by the reference method (ALL the collaborators)

Collaborators Contamination level

L0 L1 L2

A 0 8 8

B 0 8 8

C 0 7 8

D 0 7 8

E 0 8 8

F 0 8 8

G 0 7 8

H 0 8 8

I 0 8 8

J 0 6 8

K1 0 8 8

K2 1 8 8

L1 0 8 8

L2 0 8 8

N 0 8 8

Total P0 = 1 P1 = 115 P2 = 120

Alternative method

The positive results of the alternative method (before and after confirmation)

are given in table 27.

Table 27 - Positive results (before and after confirmation) by the alternative

method (ALL the collaborators)

Collabo- rators

Contamination level

L0 L1 L2

Before confirmation

Confir- mation

After confirmation

Before confirmation

Confir- mation

After confirmation

Before confirmation

Confir- mation

After confirmation

A 0 0 0 8 8 8 8 8 8

B 0 0 0 5 5 5 8 8 8

C 0 0 0 8 8 8 8 8 8

D 0 0 0 8 8 8 8 8 8

E 0 0 0 8 8 8 8 8 8

F 0 4 0 8 8 8 8 8 8

G 0 0 0 6 6 6 8 8 8

H 0 0 0 8 8 8 8 8 8

I 0 0 0 8 8 8 8 8 8

J 0 0 0 7 7 7 8 8 8

K1 0 0 0 7 7 7 8 8 8

K2 0 0 0 6 6 6 8 8 8

L1 1 0 0 8 8 8 8 8 8

L2 0 0 0 7 7 7 8 8 8

N 1 3 0 7 7 7 8 8 8

Total P0 = 2 C = 7 CP0 = 0 P1 = 109 C = 109 CP1 = 109 P2 = 120 C = 120 CP2 = 120

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The following unexpected results were observed (See Appendix 7):

- Collaborator F detected the presence of Listeria monocytogenes in the

enrichment broth (O&A plates) of the alternative method for 4 unspiked

samples (F14, F16, F18 and F20) (See Appendix 7).

- Collaborator K2 found a positive result with the reference method for a

control sample (K26).

- Collaborator L obtained a positive MDA test for a control sample (L23); the

analysis was repeated twice and negative results were then observed.

- Collaborator N detected the presence of Listeria monocytogenes in the

enrichment broth of the alternative method for 3 unspiked samples (N16,

N18 and N23) (See Appendix 7)

A positive MDA test was obtained for a control sample (N14) but the

presence of Listeria monocytogenes was not confirmed in the enrichment

broth. The Collaborator proceeded to a new extraction on this sample and

a negative result was then observed.

For sample N21 (Level 1), a negative MDA test was observed while the

presence of Listeria monocytogenes was confirmed on O&A plates only.

It was asked to the Collaborators to send the strains isolated from unspiked

samples. The expert laboratory received only the plates from Collaborator N.

A fingerprinting using the Sma I restriction enzyme was run on the strains in

order to compare the profiles obtained with the profile of the inoculated strain.

The following profiles were obtained (See Figure 1).

Figure 1

These results show clearly that the strains isolated from the unspiked

samples were the same strains as that inoculated for Levels 1 and 2.

Inoculated

strain

N23

N21

N18

N16

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Two Collaborators (F and N) were not retained for interpretation due to cross

contamination at the level L0.

According to the AFNOR technical rules, it is possible to keep collaborators

with one unspiked sample providing a positive result per tested method. This

was applied in this study for Collaborators K2 and L1.

Finally, the results obtained by 13 Collaborators were kept for interpretation;

they are provided in Tables 28 and 29.

Table 28 - Positive results by the reference method

(Without Collaborators F and N)

Collaborators Contamination level

L0 L1 L2

A 0 8 8

B 0 8 8

C 0 7 8

D 0 7 8

E 0 8 8

G 0 7 8

H 0 8 8

I 0 8 8

J 0 6 8

K1 0 8 8

K2 1 8 8

L1 0 8 8

L2 0 8 8

Total P0 = 1 P1 = 99 P2 = 104

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Table 29 - Positive results (before and after confirmation)

by the alternative method (Without Collaborators F and N)

Collabo- rators

Contamination level

L0 L1 L2 Before

confirmation Confir- mation

After confirmation

Before confirmation

Confir- mation

After confirmation

Before confirmation

Confir- mation

After confirmation

A 0 0 0 8 8 8 8 8 8

B 0 0 0 5 5 5 8 8 8

C 0 0 0 8 8 8 8 8 8

D 0 0 0 8 8 8 8 8 8

E 0 0 0 8 8 8 8 8 8

G 0 0 0 6 6 6 8 8 8

H 0 0 0 8 8 8 8 8 8

I 0 0 0 8 8 8 8 8 8

J 0 0 0 7 7 7 8 8 8

K1 0 0 0 7 7 7 8 8 8

K2 0 0 0 6 6 6 8 8 8

L1 1 0 0 8 8 8 8 8 8

L2 0 0 0 7 7 7 8 8 8

Total P0 = 1 C =0 CP0 = 0 P1 = 94 C = 94 CP1 = 94 P2 = 104 C = 104 CP2 = 104

The percentage specificities (SP) of the reference method and of the

alternative method, using the data after confirmation, based on the results of

level L0 are the following:

Specificity for the reference method:

Specificity for the alternative method:

Taking into account the results obtained for Level 1 (level for which fractional

recoveries was observed), the summary is the following (See table 309).

Table 30 - Summary of the obtained results with the reference method

and the alternative method for Level 1


positive (R+)

Reference method

negative (R-)

Alternative method

positive (A+)


PA = 90


PD = 4

Alternative method

negative (A-)


ND = 9


NA = 1

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Based on the data summarised in Table 30, the values of sensitivity of the

alternative and reference methods, as well as the relative trueness and false

positive ratio for the alternative method taking account the confirmations, are

the following (See Table 31):

Table 31

Sensitivity for the

alternative method: SEalt =

91.3 %

Sensitivity for the

reference method: SEref =

96.1 %

Relative trueness AC RT =

87.5 %

False positive ratio for

the alternative method FPR =

0 %

4.4 Interpretation

For Level 1, the negative deviations are listed in Table 32 and the positive

deviations in Table 33.

Table 32 - Negative deviations

Collaborator Sample No MDA 2 test Confirmation

B

B15 - -

B21 - -

B22 - -

G G9 - -

G15 - -

K

K3 - -

K27 - -

K31 - -

L L36 - -

Note that for all the samples, the presence of Listeria monocytogenes was

not confirmed in the enrichment broths of the alternative method.

Table 33 - Positive deviations

Collaborator Sample No

C C19

D D21

G G19

J J19

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For an unpaired study, the difference between (ND – PD) is calculated for

the level(s) where fractional recovery is obtained (so L1 and possibly L2). The

observed value found for (ND – PD) shall not be higher than the AL. The AL

is defined as [(ND – PD)max] and calculated per level where fractional

recovery is obtained as described below using the following three

parameters:

refx

x

Pp

N

where

Px = number of samples with a positive result obtained with the reference method

at level x (L1 or L2) for all the laboratories

Nx = number of samples tested at level x (L1 or L2) with the reference method by

all the laboratories.

altx

x

CPp

N

where

CPx = number of samples with a confirmed positive result obtained with the

alternative method at level x (L1 or L2) for all the laboratories;

Nx = number of samples tested at level x (L1 or L2) with the alternative method by

all the laboratories.

max ref alt ref altND-PD 3 2xN p p p p

where

Nx = the total number of samples tested for level x (L1 or L2) with the

reference method by all the laboratories.

The AL is not met when the observed value is higher than the AL.

In this study, fractional recovery was observed at Level 1. According to the

EN ISO 16140-2:2016, the calculations are the following (See table 34).

Table 34

NX 104

(p+)ref 1.0

(p+)alt 0.9

AL = (ND - PD) max 6.49

ND - PD 5

Conclusion ND - PD < AL

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The EN ISO 16140-2:2016 requirements are fulfilled as (ND - PD) is lower

than the AL for Level 1.

The RLOD calculations were performed using Excel spreadsheet of the

international standard (ISO 16140), as described in the EN ISO 16140-

2:2016 standard (http://standards.iso.org/iso/16140). The results are used

only for information (see table 35).

Table 35

RLOD RLODL RLODV b=ln(RLOD) sd(b) z Test

statistic p.value

1.3 0.9 1.9 0.3 0.2 1.4 0.2

5 CONCLUSION

The method comparison study conclusions are:

The method comparison study scheme corresponds to an UNPAIRED

STUDY design as the alternative and reference methods do have split

enrichment procedures.

In the sensitivity study, 6 categories were tested: 5 food categories

and the environmental samples. The protocol of the alternative

method shows 35 positive deviations (PD) and 33 negative deviations

(ND) for the overall categories. The values for ((ND+ PPND) - PD) are

below the acceptability limits (AL) whatever the categories, and as well

for the 6 tested categories.

The Relative Levels of Detection (RLOD) are all below the AL fixed at

2.5 for the unpaired data study whatever the matrix/strain pairs.

The inclusivity and exclusivity testing did give the expected results for

the 50 target strains and the 30 non target strains.

It is possible to store the primary enrichment broth for all the

categories and the lysates for 72 h at 2-8°C for all the categories,

except dairy products and environmental samples.

http://standards.iso.org/iso/16140

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The alternative method allows a one-day screening of the negative

samples for the general protocol, and two-days for the specific

protocol.

The alternative method fulfils all the EN ISO 16140-2:2016 and

AFNOR technical rules requirements.

The inter-laboratory study conclusions are:

The data and interpretations comply with the EN ISO 16140-2:2016

requirements. The 3MTM Molecular Detection Assay 2 -

Listeria monocytogenes is considered equivalent to the ISO

standard.

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Appendix 1 – Flow diagram of the EN ISO 11290-1/A1 (2004) method:

Horizontal method for the detection and enumeration of Listeria

monocytogenes - Part 1: detection of Listeria monocytogenes in foods

25 g or ml of the sample

1/10 dilution in ½ Fraser enrichment broth

Incubation 30°C 1°C, 24 h 3 h

0,1 ml ½ Fraser broth in 10 ml

Fraser 1 enrichment broth

Streaking on Palcam and O&A

Incubation 37°C 1°C,

48 3h

Streaking on Palcam

and OAA

Incubation 37°C 1°C, 24 h 3 h

Presence of characteristic colonies

of Listeria spp.

Yes No

Confirmation test Incubation 18 - 24 h added

37°C 1°C

Presence of characteristic

colonies of Listeria spp.

L. monocytogenes Other than

L. monocytogenes

Yes

Confirmation test

No

Presence of

L. monocytogenes

x g or ml

Absence of

L. monocytogenes

x g or ml

L. monocytogenes Other than

L. monocytogenes

Absence of

L. monocytogenes

x g or ml

Presence of

L. monocytogenes

x g or ml

Absence of

L. monocytogenes

x g or ml

Confirmation tests: Gram, Catalase, Haemolysis, Camp Test, API Listeria Gallery

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Appendix 2 – Flow diagram of the 3MTM Molecular Detection Assay 2 -

Listeria monocytogenes

Specific protocol General protocol

Raw dairy products

Raw Seafood and

Raw meat products

Food products

(excluding raw meats and raw

seafood and raw milk based

products)

Environmental samples

25 g + 225 ml

Half Fraser

25 g + 225 ml

Half Fraser

Incubation for

22 h ± 2 h

at 37°C

Subculture in

Fraser broth

Incubation for

22 h ± 2 h

at 37°C

Incubation for

27 h ± 3 h

at 37°C ± 1°C

Possibility

to store 72 h

at 2-8°C

Lysis

Possibility

to store the

lysates for

72 h at 2-8°C

except for dairy

products and

environmental

samples

Amplification and detection by the Listeria monocytogenes kit

Confirmation:

- by streaking 100 µl of Half Fraser broth onto Palcam and O&A plates

(General protocol)

- by streaking 10 µl of Fraser broth onto Palcam and O&A plates

(Specific protocol)

3M




Specific protocol 1 Raw dairy, Raw meat, Raw seafood

products

25 g + 225 ml Half Fraser (d 1/10)

Incubation for 22 h 2 h

at 37°C 1°C

Lysis on 10 µl enrichment broth

MDA 2 test on 20 µl lysate (Listeria monocytogenes kit)

-

Confirmatory tests: streak 10 µl Fraser

onto O&A and Palcam

Storage for 72 h at 2-8°C

Lysis

MDA 2 test

Confirmatory tests:

streak 10 µl Fraser onto O&A and Palcam

STOP

* This is done during the validation study in order to confirm that the typical colonies observed on the plates are really Listeria strains.

+

- +

Subculture in Fraser broth


at 37°C 1°C

3M




General protocol

Food products

(excluding raw meats and raw seafood and

raw milk based products)

Environmental samples

25 g + 225 ml Half Fraser (d 1/10)


at 37°C 1°C

Lysis on 20 µl enrichment broth

MDA 2 test on 20 µl lysate (Listeria monocytogenes kit)

-

Confirmatory tests: streak 100 µl Half Fraser

onto O&A and Palcam



(except for Dairy products and environmental

samples)

Lysis

MDA 2 test

Confirmatory tests:

streak 100 µl Half Fraser

onto O&A and Palcam

STOP

+

- +

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Appendix 3 – Relative level of detection study: raw data

Matrix : Deli-salad (Piémontaise) Strain : Listeria monocytogenes Ad494 Aerobic mesophilic flora :210 CFU/g

N° sample Level Contamination level- (cfu/sample)-MPN

determination

Reference method: ISO 11290-1/A1 Number positive samples/Total

Alternative method: 3M MDA 2 - Listeria monocytogenes Number positive

samples/Total Half Fraser Fraser Final result O&A Palcam O&A Palcam MDA test

Confirmation result

Final result

3369

0 /

st - - - -

0/5

- - -

0/5 3370 st - - - - - - - 3371 st - - - - - - - 3372 st st - - - - - - 3373 st - - - - - - - 3374

Low 0,6

st - - - -

4 /20

- - -

6/20

3375 st - - - - + + + 3376 st st st st - + + + 3377 H+ + / / + + + + 3378 st - - - - - - - 3379 st - - - - + + + 3380 st - - - - - - - 3381 st st - - - + + + 3382 st st st st - - - - 3383 st st - - - - - - 3384 st - - - - - - - 3385 st - - - - - - - 3386 H+ + / / + - - - 3387 st st - - - - - - 3388 st - - - - - - - 3389 H+ + / / + - - - 3390 H+ + / / + - - - 3391 st - - - - - - - 3392 st - - - - + + + 3393 st st - - - - - - 3394

High 1,7

H+ + / / +

3/5

- - -

1/5 3395 H+ + / / + - - - 3396 H+ + / / + + + + 3397 st - - - - - - - 3398 st - - - - - - -


3M




Matrix : Beef meat Strain : Listeria monocytogenes Ad1206

Aerobic mesophilic flora : 5,0.105 CFU/g


determination


Alternative method: 3M MDA 2 - Listeria monocytogenes

Number positive samples/Total Half Fraser Fraser

Final result O&A Palcam O&A Palcam MDA test Confirmation result Final result

7950

0 /

st st st st -

0/5

- - -

0/5 7951 st st st st - - - - 7952 st st st - - - - - 7953 st st st - - - - - 7954 H- (L.welshimeri) + H- + - - - - 8026

Low 0,7

H- + H- + -

8/20

- - -

8/20

8027 H+/H- + H+/H- + + - - - 8028 H+/H- + H+/H- + + - - - 8029 H+/H- + H+/H- + + - - - 8030 H- + H- + - + + + 8031 H- + H- + - - - - 8032 H+/H- + H+/H- + + - - - 8033 H- + H- + - - - - 8034 H+/H- + H+/H- + + + + + 8035 H- + H- + - - - - 8036 H- + H- + - + + + 8037 H- + H- + - + + + 8038 H+/H- + H+/H- + + - - - 8039 H- + H- + - + + + 8040 H+/H- + H+/H- + + - - - 8041 H+/H- + H+/H- + + - - - 8042 H- + H- + - + + + 8043 H- + H- + - + + + 8044 H- + H- + - - - - 8045 H- + H- + - + + + 8046

High 2

H+/H- + H+/H- + +

3/5

+ + +

5/5 8047 H+/H- + H+/H- + + + + + 8048 H+/H- + H+/H- + + + + + 8049 H- + H- + - + + + 8050 H- + H- + - + + +


3M




Matrix : Raw milk

Strain : Listeria monocytogenes Ad618 Aerobic mesophilic flora : 6,4.105 CFU/g


determination




Confirmation result

Final result

5481

0 /

St St - - -

0/5

- - -

0/5 5482 St St St - - - - - 5483 St - St - - - - - 5484 St - - - - - - - 5485 St St - - - - - - 5486

Low 0,5

St St - - -

11/20

- - -

11/20

5487 - - - - - - - - 5488 H+ + H+ + + - - - 5489 H+ + H+ + + -/+/+ + - 5490 H+ + H+ + + - - - 5491 H+ + H+ + + + + + 5492 - - St St - + + + 5493 H+ + H+ + + + + + 5494 H+ + H+ + + + + + 5495 H+ + H+ + + + + + 5496 - - St - - - - - 5497 St St - - - + + + 5498 St - - - - + + + 5499 H+ + H+ + + - - - 5500 St - St - - - - - 5501 - - - - - + + + 5502 H+ + H+ + + + + + 5503 H+ + H+ + + + + + 5504 St - - - - + + + 5505 St - H+ + + - - - 5506

High 1,7

H+ + H+ + +

5/5

+ + +

5/5 5507 H+ + H+ + + + + + 5508 H+ + H+ + + + + + 5509 H+ + H+ + + + + + 5510 H+ + H+ + + + + +


3M




Matrix : Bagged raw spinach

Strain : Listeria monocytogenes Ad1178 Aerobic mesophilic flora : 8.0 106 CFU/g


determination




Confirmation result

Final result

2369

0 /

- - - - -

0/5

- - -

0/5 2370 - - - - - - - - 2371 - - - - - - - - 2372 - - - - - - - - 2373 - - st st - - - - 2374

Low 1,2

H+ + H+ + +

10 /20

+ - + (subculture in Fraser)

9/20

2375 - - - - - - + - 2376 - - - - - + + + 2377 - - - - - + - + (subculture in Fraser) 2378 H+ + H+ + + - - - 2379 - - st st - - - - 2380 H+ + H+ + + + + 2381 H+ + H+ + + - - - 2382 - - - - - + + + 2383 - - - - - + + + 2384 - - - - - - - - 2385 H+ + H+ + + + + + 2386 - - - - - - - - 2387 H+ + H+ + + + + + 2388 H+ + H+ + + - - - 2389 H+ + H+ + + - - - 2390 H+ + H+ + + - - - 2391 H+ + H+ + + + + + 2392 - - st st - - - - 2393 - - - - - - - - 2394

High 2,9

H+ + H+ + +

5/5

+ + +

5/5 2395 H+ + H+ + + + + + 2396 H+ + H+ + + + + + 2397 H+ + H+ + + + + + 2398 H+ + H+ + + + + +


3M




Matrix : Cold smoked salmon

Strain : Listeria monocytogenes Ad670 Aerobic mesophilic flora : 400 CFU/g

N° sample Level Contamination level-

(cfu/sample)- MPN determination




Confirmation result

Final result

1999

0 /

- - st st -

0/5

- - -

/5 2000 st st st st - - - - 2001 - - st st - - - - 2002 st st st st - - - - 2003 st st st st - - - - 2004

Low 0,4

H+ + / / +

9/20

- - -

10 /20

2005 H+ + / / + + + + 2006 st st st st - - - - 2007 st st st st - + + + 2008 st st st st - - - - 2009 H+ + / / + - - - 2010 H- + H- + - + + + 2011 st st st st - - - - 2012 H+ + / / + - - - 2013 H+ + / / + - - - 2014 st st st st - - - - 2015 H+ + / / + + + + 2016 H- + H- + - + + + 2017 H+ + / / + + + + 2018 st - - st - + + + 2019 H+ + / / + - - - 2020 st st st st - + + + 2021 H+ + / / + - - - 2022 st st st st - + + + 2023 st st st st - + + + 2024

High 2,7

st st st st -

4/5

+ + +

3/5 2025 H+ + / / + - - - 2026 H+ + / / + - - - 2027 H+ + / / + + + + 2028 H+ + / / + + + +


3M




Matrix : Process water

Strain : Listeria monocytogenes Ad551 Aerobic mesophilic flora : 9,7.103CFU/g

N° sample Level Contamination level-

(cfu/sample)- MPN determination




Confirmation result

Final result

4135

0 /

st st - - -

0/5

- - -

0/5 4136 st st st st - - - - 4137 st st st - - - - - 4138 st st st st - - - - 4139 st st st st - - - - 4353

Low 0,6

st st st st -

11 /20

- - -

11/20

4354 H+ + / / + - - - 4355 st st st st - - - - 4356 st st st st - + + + 4357 H+ + / / + + + + 4358 H+ + / / + - - - 4359 H+ + / / + - - - 4360 st st st st - - - - 4361 H+ + / / + + + + 4362 st st st st - + + + 4363 H+ + / / + + + + 4364 H+ + / / + + + + 4365 st st st st - + + + 4366 st st st st - + + + 4367 H+ + / / + + + + 4368 H+ + / / + + + + 4369 H+ + / / + - - - 4370 st st st st - + + + 4371 H+ + / / + - - - 4372 st st st st - - - - 4373

High 1,7

st st st st -

2/5

+ + +

2/5 4374 st st st st - - - - 4375 st st st st - - - - 4376 H+ + / / + - - - 4377 H+ + / / + + + +


3M




Appendix 4 – Inclusivity and exclusivity study: raw data

INCLUSIVITY

Strain Species Reference Origin Inoculation level

(cfu/225ml)

3M Molecular Detection Assay 2 Half Fraser broth 24h at 37°C

MDA results Confirmation 100 µl

MDA Listeria monocytogenes ALOA Palcam

1 Listeria monocytogenes 153 Soft cheese (Munster) 39 + H+ +

2 Listeria monocytogenes 1011/1410 Frozen broccoli 28 + H+ +

3 Listeria monocytogenes 1972/2399 Puff pastry with mushrooms 38 + H+ +

4 Listeria monocytogenes 1973/2400 Puff pastry egg and ham (Quiche-lorraine) 36 + H+ +

5 Listeria monocytogenes 2407/3139 Tripes with tomatoes 31 + H+ +

6 Listeria monocytogenes 2760/3145 Raw bacon 44 + H+ +

7 Listeria monocytogenes 32.183 Croque-Monsieur 31 + H+ +

8 Listeria monocytogenes 38/181 Toulouse sausages 33 + H+ +

9 Listeria monocytogenes 5721/6179 Smoked bacon 49 + H+ +

10 Listeria monocytogenes 7111/7516 Pâté (Rillettes) 64 + H+ +

11 Listeria monocytogenes 850/109 RTE food (deli salad with seafood) 34 + H+ +

12 Listeria monocytogenes 877/113 Environmental sample (pastry) 28 + H+ +

13 Listeria monocytogenes 913/1048 Black pudding 39 + H+ +

14 Listeria monocytogenes A00C014 Sausage 37 + H+ +

15 Listeria monocytogenes A00C022 Merguez 33 + H+ +


17 Listeria monocytogenes A00C036 Poultry (guinea) 41 + H+ +

18 Listeria monocytogenes A00C039 Sausages 20 + H+ +

19 Listeria monocytogenes A00C040 Cooked delicatessen (Museau) 36 + H+ +

3M




INCLUSIVITY


(cfu/225ml)





21 Listeria monocytogenes A00C042 Raw sausage 32 + H+ +

22 Listeria monocytogenes A00C043 Smoked Bacon 50 + H+ +

23 Listeria monocytogenes A00C044 Poultry (duck) 34 + H+ +

24 Listeria monocytogenes A00C052 RTE food (Osso bucco with turkey) 34 + H+ +

25 Listeria monocytogenes A00C053 Gizzards 30 + H+ +

26 Listeria monocytogenes A00C054 Beef hart 30 + H+ +

27 Listeria monocytogenes A00C055 Raw sausages 43 + H+ +

28 Listeria monocytogenes A00E008 Environmental sample 27 + H+ +

29 Listeria monocytogenes A00E049 Environmental sample (smoked salmon) 41 + H+ +

30 Listeria monocytogenes A00E082 Environmental sample (smoked salmon) 33 + H+ +

31 Listeria monocytogenes A00L097 Milk 60 + H+ +

32 Listeria monocytogenes A00M009 Smoked salmon 40 + H+ +




36 Listeria monocytogenes Ad235 Poultry 45 + H+ +

37 Listeria monocytogenes Ad253 Hard cheese 58 + H+ +

38 Listeria monocytogenes Ad260 Semi hard cheese 27 + H+ +

39 Listeria monocytogenes Ad265 Tong 62 + H+ +

40 Listeria monocytogenes Ad266 Poultry 26 + H+ +

3M




INCLUSIVITY


(cfu/225ml)




41 Listeria monocytogenes Ad267 Dry sausage 26 + H+ +

42 Listeria monocytogenes Ad268 Cured ham 35 + H+ +

43 Listeria monocytogenes Ad270 Fermented sausage 42 + H+ +

44 Listeria monocytogenes Ad272 Fermented sausage 56 + H+ +

45 Listeria monocytogenes Ad273 Cured delicatessen 27 + H+ +

46 Listeria monocytogenes Ad274 Ready-to-eat food (Asiatic meal) 40 + H+ +

47 Listeria monocytogenes Ad534 Fruits 54 + H+ +

48 Listeria monocytogenes Ad544 Onion 43 + H+ +

49 Listeria monocytogenes Ad546 Flour 83 + H+ +

50 Listeria monocytogenes Ad623 Bread crumbs 50 + H+ +

3M




EXCLUSIVITY


(cfu/ml)

3M Molecular Detection Assay 2

MDA results

MDA Listeria monocytogenes MC

1 Listeria grayi Ad 1198 Smoked salmon 3.2 105 - +

2 Listeria grayi Ad 1443 Pork meat sausages 4.9 105 - +

3 Listeria innocua 1 Smoked salmon 4.9 105 - +

4 Listeria innocua Ad 658 Gorgonzola 5.9 105 - +

5 Listeria ivanovii Ad 466 Raw veal meat 3.3 105 - +

6 Listeria ivanovii Ad 662 Environment (dairy industry) 3.6 105 - +

7 Listeria seeligeri Ad 649 Cheese 5.4 105 - +

8 Listeria seeligeri BR1 Trout 5.1 105 - +

9 Listeria welshimeri Ad1276 Environment (Slaughterhouse) 5.2 105 - +

10 Listeria welshimeri Ad1175 Ready-to-eat-food 6.4 105 - +

11 Bacillus cereus Ad 465 Salmon Terrine 7.2 104 - +

12 Bacillus circulans Ad 760 Vegetables 2.0 104 - +

13 Bacillus coagulans Ad 731 Dairy product <2.0 103 (opacity +) - +

14 Bacillus licheniformis Ad 978 Dairy product 1.6 104 - +

15 Bacillus pumilus Ad 284 Ready-to-eat 1.7 105 - +

16 Brochrotrix campestris CIP 102920T

Environment <2.0 103 (opacity +) - +

17 Carnobacterium piscicola Ad 369 Raw milk <2.0 103 (opacity +) - +

18 Enterococcus durans Ad 149 Ham <2.0 103 (opacity +) - +

19 Enterococcus faecalis 89L326 Soft cheese (Vacherin) 1.6 105 - +

3M




EXCLUSIVITY


(cfu/ml)

3M Molecular Detection Assay 2

MDA results

MDA Listeria monocytogenes MC

20 Lactobacillus brevis 86L126 Ham 1.4 105 - +

21 Lactobacillus curvatus Ad 380 Delicatessen <2.0 103 (opacity +) - +

22 Lactobacillus sakei Ad 473 Ham <2.0 103 (opacity +) - +

23 Leuconostoc carnosum Ad 411 Ham <2.0 103 (opacity +) - +

24 Leuconostoc citreum Ad 396 Ham 4.9 105 - +

25 Micrococcus luteus Ad 432 Cocktail <2.0 103 (opacity +) - +

26 Staphylococcus aureus Ad 165 Smoked delicatessen <2.0 103 (opacity +) - +

27 Staphylococcus epidermis Ad 931 Fruits 2.0 103 - +

28 Staphylococcus haemoliticus Ad 989 Dairy product 2.8 104 - +

29 Streptococcus bovis 92L622 Cheese 2.0 103 - +

30 Streptococcus salivarus Ad 441 Dairy product <2.0 103 (opacity +) - +

3M




Appendix 5 - Results obtained by the collaborators and the expert laboratory

Collaborator A Aerobic mesophilic flora: 2,0.109/g

N°Sample

Reference method: ISO 11290-1 3MTM Molecular Detection Assay 2 -Listeria

for detection of Listeria monocytogenes Agreement

Half Fraser Fraser Final result MDA test result O&A Palcam Final result

O&A Palcam O&A Palcam

A7 - - - - - - - - - NA

A10 - - - - - - - - - NA

A11 - - - - - - - - - NA

A14 - - - - - - - - - NA

A16 - - - - - - - - - NA

A18 - - - - - - - - - NA

A20 - - - - - - - - - NA

A23 - - - - - - - - - NA

A1 + + + + + + + + + PA

A3 + + + + + + + + + PA

A4 + + + + + + + + + PA

A9 + + + + + + + + + PA

A15 + + + + + + + + + PA

A19 + + + + + + + + + PA

A21 + + + + + + + + + PA

A22 + + + + + + + + + PA

A2 + + + + + + + + + PA

A5 + + + + + + + + + PA

A6 + + + + + + + + + PA

A8 + + + + + + + + + PA

A12 + + + + + + + + + PA

A13 + + + + + + + + + PA

A17 + + + + + + + + + PA

A24 + + + + + + + + + PA

3M




Collaborator B Aerobic mesophilic flora: 9,6 108 /g

N°Sample





B7 - - - - - - - - - NA

B10 - - - - - - - - - NA

B11 - - - - - - - - - NA

B14 - - - - - - - - - NA

B16 - - - - - - - - - NA

B18 - - - - - - - - - NA

B20 - - - - - - - - - NA

B23 - - - - - - - - - NA

B1 + + + + + + + + + PA

B3 + + + + + + + + + PA

B4 + + + + + + + + + PA

B9 + + + + + + + + + PA

B15 + + + + + - - - - ND

B19 + + + + + + + + + PA

B21 + + + + + - - - - ND

B22 + + + + + - - - - ND

B2 + + + + + + + + + PA

B5 + + + + + + + + + PA

B6 + + + + + + + + + PA

B8 + + + + + + + + + PA

B12 + + + + + + + + + PA

B13 + + + + + + + + + PA

B17 + + + + + + + + + PA

B24 + + + + + + + + + PA

3M




Collaborator C Aerobic mesophilic flora: 4,2 109/g

N°Sample





C7 - - - - - - - - - NA

C10 - - - - - - - - - NA

C11 - - - - - - - - - NA

C14 - - - - - - - - - NA

C16 - - - - - - - - - NA

C18 - - - - - - - - - NA

C20 - - - - - - - - - NA

C23 - - - - - - - - - NA

C1 + + + + + + + + + PA

C3 + + + + + + + + + PA

C4 + + + + + + + + + PA

C9 + + + + + + + + + PA

C15 + + + + + + + + + PA

C19 - - - - - + + + + PD

C21 + + + + + + + + + PA

C22 + + + + + + + + + PA

C2 + + + + + + + + + PA

C5 + + + + + + + + + PA

C6 + + + + + + + + + PA

C8 + + + + + + + + + PA

C12 + + + + + + + + + PA

C13 + + + + + + + + + PA

C17 + + + + + + + + + PA

C24 + + + + + + + + + PA

3M




Collaborator D Aerobic mesophilic flora: 2,0 109/g

N°Sample





D7 - - - - - - - - - NA

D10 - - - - - - - - - NA

D11 - - - - - - - - - NA

D14 - - - - - - - - - NA

D16 - - - - - - - - - NA

D18 - - - - - - - - - NA

D20 - - - - - - - - - NA

D23 - - - - - - - - - NA

D1 + + + + + + + + + PA

D3 + + + + + + + + + PA

D4 + + + + + + + + + PA

D9 + + + + + + + + + PA

D15 + + + + + + + + + PA

D19 + + + + + + + + + PA

D21 - - - - - + + + + PD

D22 + + + + + + + + + PA

D2 + + + + + + + + + PA

D5 + + + + + + + + + PA

D6 + + + + + + + + + PA

D8 + + + + + + + + + PA

D12 + + + + + + + + + PA

D13 + + + + + + + + + PA

D17 + + + + + + + + + PA

D24 + + + + + + + + + PA

3M




Collaborator E

Aerobic mesophilic flora: 1,2 109/g

N°Sample


for detection of Listeria monocytogenes Agreement Half Fraser Fraser

Final result MDA test result O&A Palcam Final result O&A Palcam O&A Palcam

E7 - - - - - - - - - NA

E10 - - - - - - - - - NA

E11 - - - - - - - - - NA

E14 - - - - - - - - - NA

E16 - - - - - - - - - NA

E18 - - - - - - - - - NA

E20 - - - - - - - - - NA

E23 - - - - - - - - - NA

E1 + + + + + + + + + PA

E3 + + + + + + + + + PA

E4 + + + + + + + + + PA

E9 + + + + + + + + + PA

E15 + + + + + + + + + PA

E19 + + + + + + + + + PA

E21 + + + + + + + + + PA

E22 + + + + + + + + + PA

E2 + + + + + + + + + PA

E5 + + + + + + + + + PA

E6 + + + + + + + + + PA

E8 + + + + + + + + + PA

E12 + + + + + + + + + PA

E13 + + + + + + + + + PA

E17 + + + + + + + + + PA

E24 + + + + + + + + + PA

3M




Collaborator F Aerobic mesophilic flora: 4,2 108/g

N°Sample





F7 - - - - - - - - - NA

F10 - - - - - - - - - NA

F11 - - - - - - - - - NA

F14 - - - - - - + - - NA

F16 - - - - - - + - - NA

F18 - - - - - - + - - NA

F20 - - - - - - + - - NA

F23 - - - - - - - - - NA

F1 + + + + + + + + + PA

F3 + + + + + + + + + PA

F4 + + + + + + + + + PA

F9 + + + + + + + + + PA

F15 + + + + + + + + + PA

F19 + + + + + + + + + PA

F21 + + + + + + + + + PA

F22 + + + + + + + + + PA

F2 + + + + + + + + + PA

F5 + + + + + + + + + PA

F6 + + + + + + + + + PA

F8 + + + + + + + + + PA

F12 + + + + + + + + + PA

F13 + + + + + + + + + PA

F17 + + + + + + + + + PA

F24 + + + + + + + + + PA

3M




Collaborator G Aerobic mesophilic flora: 1,6 109/g

N°Sample





G7 - - - - - - - - - NA

G10 - - - - - - - - - NA

G11 - - - - - - - - - NA

G14 - - - - - - - - - NA

G16 - - - - - - - - - NA

G18 - - - - - - - - - NA

G20 - - - - - - - - - NA

G23 - - - - - - - - - NA

G1 + + + + + + + + + PA

G3 + + + + + + + + + PA

G4 + + + + + + + + + PA

G9 + + + + + - - - - ND

G15 + + + + + - - - - ND

G19 - - - - - + + + + PD

G21 + + + + + + + + + PA

G22 + + + + + + + + + PA

G2 + + + + + + + + + PA

G5 + + + + + + + + + PA

G6 + + + + + + + + + PA

G8 + + + + + + + + + PA

G12 + + + + + + + + + PA

G13 + + + + + + + + + PA

G17 + + + + + + + + + PA

G24 + + + + + + + + + PA

3M




Collaborator H Aerobic mesophilic flora: 1,7 109/g

N°Sample





H7 - - - - - - - - - NA

H10 - - - - - - - - - NA

H11 - - - - - - - - - NA

H14 - - - - - - - - - NA

H16 - - - - - - - - - NA

H18 - - - - - - - - - NA

H20 - - - - - - - - - NA

H23 - - - - - - - - - NA

H1 + + + + + + + + + PA

H3 + + + + + + + + + PA

H4 + + + + + + + + + PA

H9 + + + + + + + + + PA

H15 + + + + + + + + + PA

H19 + + + + + + + + + PA

H21 + + + + + + + + + PA

H22 + + + + + + + + + PA

H2 + + + + + + + + + PA

H5 + + + + + + + + + PA

H6 + + + + + + + + + PA

H8 + + + + + + + + + PA

H12 + + + + + + + + + PA

H13 + + + + + + + + + PA

H17 + + + + + + + + + PA

H24 + + + + + + + + + PA

3M




Collaborator I


N°Sample





I7 - - - - - - - - - NA

I10 - - - - - - - - - NA

I11 - - - - - - - - - NA

I14 - - - - - - - - - NA

I16 - - - - - - - - - NA

I18 - - - - - - - - - NA

I20 - - - - - - - - - NA

I23 - - - - - - - - - NA

I1 + + + + + + + + + PA

I3 + + + + + + + + + PA

I4 + + + + + + + + + PA

I9 + + + + + + + + + PA

I15 + + + + + + + + + PA

I19 + + + + + + + + + PA

I21 + + + + + + + + + PA

I22 + + + + + + + + + PA

I2 + + + + + + + + + PA

I5 + + + + + + + + + PA

I6 + + + + + + + + + PA

I8 + + + + + + + + + PA

I12 + + + + + + + + + PA

I13 + + + + + + + + + PA

I17 + + + + + + + + + PA

I24 + + + + + + + + + PA

3M




Collaborator J Aerobic mesophilic flora: 9,2 108/g

N°Sample





J7 - - - - - - - - - NA

J10 - - - - - - - - - NA

J11 - - - - - - - - - NA

J14 - - - - - - - - - NA

J16 - - - - - - - - - NA

J18 - - - - - - - - - NA

J20 - - - - - - - - - NA

J23 - - - - - - - - - NA

J1 + + + + + + + + + PA

J3 + + + + + + + + + PA

J4 + + + + + + + + + PA

J9 + + + + + + + + + PA

J15 + + + + + + + + + PA

J19 - - - - - + + + + PD

J21 - - - - - - - - - NA

J22 + + + + + + + + + PA

J2 + + + + + + + + + PA

J5 + + + + + + + + + PA

J6 + + + + + + + + + PA

J8 + + + + + + + + + PA

J12 + + + + + + + + + PA

J13 + + + + + + + + + PA

J17 + + + + + + + + + PA

J24 + + + + + + + + + PA

3M




Collaborator K1

TECHNICIAN 1 Aerobic mesophilic flora: 2,1 109/g

N°Sample





K7 - - - - - - - - - NA

K10 - - - - - - - - - NA

K11 - - - - - - - - - NA

K14 - - - - - - - - - NA

K16 - - - - - - - - - NA

K18 - - - - - - - - - NA

K20 - - - - - - - - - NA

K23 - - - - - - - - - NA

K1 + + + + + + + + + PA

K3 + + + + + - - - - ND

K4 + + + + + + + + + PA

K9 + + + + + + + + + PA

K15 + + + + + + + + + PA

K19 + + + + + + + + + PA

K21 + + + + + + + + + PA

K22 + + + + + + + + + PA

K2 + + + + + + + + + PA

K5 + + + + + + + + + PA

K6 + + + + + + + + + PA

K8 + + + + + + + + + PA

K12 + + + + + + + + + PA

K13 + + + + + + + + + PA

K17 + + + + + + + + + PA

K24 + + + + + + + + + PA

3M




Collaborator K2

TECHNICIAN 2 Aerobic mesophilic flora: 3,4.109 /g

N°Sample





K26 + + - - + - - - - ND

K29 - - - - - - - - - NA

K30 - - - - - - - - - NA

K32 - - - - - - - - - NA

K34 - - - - - - - - - NA

K37 - - - - - - - - - NA

K38 - - - - - - - - - NA

K43 - - - - - - - - - NA

K27 + + + + + - - - - ND

K31 + + + + + - - - - ND

K33 + + + + + + + + + PA

K36 + + + + + + + + + PA

K41 + + + + + + + + + PA

K44 + + + + + + + + + PA

K45 + + + + + + + + + PA

K48 + + + + + + + + + PA

K25 + + + + + + + + + PA

K28 + + + + + + + + + PA

K35 + + + + + + + + + PA

K39 + + + + + + + + + PA

K40 + + + + + + + + + PA

K42 + + + + + + + + + PA

K46 + + + + + + + + + PA

K47 + + + + + + + + + PA

3M




Collaborator L1

TECHNICIAN 1 Aerobic mesophilic flora: 6,4 107/g

N°Sample





L7 - - - - - - - - - NA

L10 - - - - - - - - - NA

L11 - - - - - - - - - NA

L14 - - - - - - - - - NA

L16 - - - - - - - - - NA

L18 - - - - - - - - - NA

L20 - - - - - - - - - NA

L23 - - - - - +/-/-(1) - - - PPNA

L1 + + + + + + + + + PA

L3 + + + + + + + + + PA

L4 + + + + + + + + + PA

L9 + + + + + + + + + PA

L15 + + + + + + + + + PA

L19 + + + + + + + + + PA

L21 + + + + + + + + + PA

L22 + + + + + + + + + PA

L2 + + + + + + + + + PA

L5 + + + + + + + + + PA

L6 + + + + + + + + + PA

L8 + + + + + + + + + PA

L12 + + + + + + + + + PA

L13 + + + + + + + + + PA

L17 + + + + + + + + + PA

L24 + + + + + + + + + PA

(1): MDA tests run on the 5 July /2016

3M




Collaborator L2

TECHNICIAN 2 Aerobic mesophilic flora: 7,0 108 /g

N°Sample





L26 - - - - - - - - - NA

L29 - - - - - - - - - NA

L30 - - - - - - - - - NA

L32 - - - - - - - - - NA

L34 - - - - - - - - - NA

L37 - - - - - - - - - NA

L38 - - - - - - - - - NA

L43 - - - - - - - - - NA

L27 + + + + + + + + + PA

L31 + + + + + + + + + PA

L33 + + + + + + + + + PA

L36 + + + + + - - - - ND

L41 + + + + + + + + + PA

L44 + + + + + + + + + PA

L45 + + + + + + + + + PA

L48 + + + + + + + + + PA

L25 + + + + + + + + + PA

L28 + + + + + + + + + PA

L35 + + + + + + + + + PA

L39 + + + + + + + + + PA

L40 + + + + + + + + + PA

L42 + + + + + + + + + PA

L46 + + + + + + + + + PA

L47 + + + + + + + + + PA

3M




Collaborator N


N°Sample


for detection of Listeria monocytogenes Agreement Half Fraser Fraser

Final result MDA test result O&A Palcam Final result O&A Palcam O&A Palcam

N7 - - - - - - - - - NA

N10 - - - - - - - - - NA

N11 - - - - - - - - - NA

N14 - - - - - +/+ /-* -/- * -/-* - PPNA

N16 - - - - - - + - - NA

N18 - - - - - - + - - NA

N20 - - - - - - - - - NA

N23 - - - - - - + + - NA

N1 + + + + + + + + + PA

N3 + + + + + + + + + PA

N4 + + + + + + + + + PA

N9 + + + + + + + + + PA

N15 + + + + + + + + + PA

N19 + + + + + + + + + PA

N21 + + + + + - + - - ND

N22 + + + + + + + + + PA

N2 + + + + + + + + + PA

N5 + + + + + + + + + PA

N6 + + + + + + + + + PA

N8 + + + + + + + + + PA

N12 + + + + + + + + + PA

N13 + + + + + + + + + PA

N17 + + + + + + + + + PA

N24 + + + + + + + + + PA

* new extraction

3M




Expert Laboratory M (ADRIA) Aerobic mesophilic flora: 3,4.108/g

N°Sample





M7 st st st st - - st - - NA







M23 st st - st - - st - - NA

M1 + + + + + + H+ + + PA

M3 + + + + + + H+ + + PA

M4 + + + + + -/+(1)/-(1)/+ (2) H+ + - ND

M9 + + + + + + H+ + + PA

M15 + + + + + + H+ + + PA

M19 + + + + + + H+ + + PA

M21 + + + + + + H+ + + PA

M22 + + + + + + H+ + + PA

M2 + + + + + + H+ + + PA

M5 + + + + + + H+ + + PA

M6 + + + + + + H+ + + PA

M8 + + + + + + H+ + + PA

M12 + + + + + + H+ + + PA

M13 + + + + + + H+ + + PA

M17 + + + + + + H+ + + PA

M24 + + + + + + H+ + + PA (1): same lysate (2): second extraction


Documents

Summary report - Accueil - NF Validation · Listeria monocytogenes in food products and environmental samples ... 5 CONCLUSION ... and environmental samples, before running the MDA