Legionella pneumophila for the detection and ... · Legionella spp. and/or Legionella pneumophila by concentration and genic amplification by quantitative polymerase chain reaction

ADGENE LABORATOIRE 1 rue des conquérants -14 220 THURY-HARCOURT www.adgene.fr –[email protected] – 02 31 15 62 80

Validation study of DI-Check Legionella pneumophila for

the detection and quantification of Legionella

pneumophila in all types of water

Summary report

January, 2020

Qualitative and quantitative method

Attestation n°DTV41/01-12/19

DIATHEVA

Via Sant'Anna

131/135, 61030 Cartoceto PU

Italy

This report includes 40 pages, including 06 Annexes. Reproduction of this report is only

permitted in its full form.

http://www.adgene.fr/

mailto:info.adgene@

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DI-CHECK L.pneumophila

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Table of contents

INTRODUCTION 4

REFERENCE METHODS 4

PRESENTATION OF THE METHOD 4

PRINCIPLE OF THE ALTERNATIVE METHOD 5

OBJECTIVE OF THE STUDY 6

SUMMARY OF RESULTS 7

CONNECTION OF THE STANDARD CURVE AND THE REFERENCE MATERIAL TO THE PRIMARY STANDARD 7

METHODOLOGY 7

ROTORGENE RESULTS 7

CFX RESULTS 9

GENERAL CONCLUSION: 10

CALIBRATION FUNCTION 11

METHODOLOGY 11

CFX RESULTS 11

CFX CONCLUSION 12

QS3 RESULTS 13

QS3 CONCLUSION 14

ROTORGENE RESULTS 14

ROTORGENE CONCLUSION 15

GENERAL CONCLUSION 16

LIMIT OF DETECTION (LODPCR) 16

METHODOLOGY 16

RESULTS (ANNEX 1) 16

CONCLUSION 16

LIMIT OF QUANTIFICATION (LOQPCR) 16

METHODOLOGY 16


CONCLUSIONS 17

POSITIVITY THRESHOLD 17

METHODOLOGY 17


DETERMINATION OF YIELD AND ROBUSTNESS OF EXTRACTION 17

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METHODOLOGY 17


INCLUSIVITY/EXCLUSIVITY 19

METHODOLOGY 19


VERIFICATION OF CALCULATIONS AND INTERPRETATIONS ESTABLISHED BY THE SOFTWARE 19

INHIBITION 20

PRACTICABILITY 20

CONDITIONING MODE AND VOLUME OF REAGENTS 20

CONDITION OF STORAGE OF THE ELEMENTS AND EXPIRY OF THE PRODUCTS 20

TERMS OF USE AFTER FIRST USE 21

REAGENTS READY TO USE OR TO RECONSTITUTE 21

TRAINING DURATION OF THE UNINITIATED OPERATOR TO THE METHOD 21

TYPE OF QUALIFICATION FOR THE OPERATOR 21

TRACEABILITY OF THE ANALYSIS RESULTS 21

MAINTENANCE BY THE LABORATORY 21

STABILITY OF REAGENTS AND RANGES 22

UNG 22

UV REAGENT PROTECTION 22

INTERLABORATORIES STUDY 23

METHODOLOGY 23

RESULTS 24

CONCLUSIONS 25

ANNEX 1: LOD 26

ANNEX 2: LOQ 28

ANNEX 3: YIELD AND ROBUSTNESS 29

ANNEX 4: INCLUSIVITY AND EXCLUSIVITY 35

ANNEX 5: VERIFICATION OF CALCULATIONS AND INTERPRETATION MADE BY THE

SOFTWARE 37

ANNEX 6: INHIBITION 40

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Introduction

This report presents the results of the validation of a method for the detection and

quantification of Legionella pneumophila in all types of water.

Reference Methods

The tests were carried out according to:

PCR Validation protocol (September 2015) «Validation protocol for the

commercial methods of detection and enumeration of Legionella and Legionella

pneumophila by concentration and gene amplification by reaction of

Polymerase chain reaction (PCR) v 3»

NFT 90 471 (June 2015) «Water quality–Detection and quantification of

Legionella and/or Legionella pneumophila by concentration and genomic

amplification by real time polymerase chain reaction (qPCR)»

ISO/TS 12869 (November 2012) «Water quality-Detection and quantification of

Legionella spp. and/or Legionella pneumophila by concentration and genic

amplification by quantitative polymerase chain reaction (qPCR)»

Presentation of the method

DI-Check Legionella pneumophila is a molecular method for the detection and

quantification of Legionella pneumophila using real-time PCR in any types of water.

This system consists of three main steps:

1st step: Water sample filtration

The water sample is filtered using 0.4 µm membrane filters positioned on a filtration

apparatus.

2nd step: DNA extraction and purification

The DNA extraction and purification are made starting from the filter using the

DNApure Water Isolation kit.

3rd step: Real-Time PCR

The purified DNA is amplified by Real-Time PCR using the DI-Check Legionella

pneumophila kit.

The analysis of results is carried out using the program of PCR instrument and the

interpretation is done using the DI-Check Analysis software for L. pneumophila v 2.2.

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Principle of the alternative method

The DNApure Water Isolation kit contains all the reagents necessary for the extraction

of microbial DNA from Gram negative bacteria present in the water samples.

Briefly, the sample is subjected to thermal lysis in presence of a resin (Chelex®) which

limits the destruction of the DNA by inactivating nucleases and chelating heavy metals

that may damage DNA.

Subsequently the lysate sample is loaded in a Centrifugal Filter Unit to obtain efficient

DNA concentration and purification.

The purified DNA is amplified by Real-Time PCR using the DI-Check Legionella

pneumophila kit. This product is a probe-based detection system that consists of a

duplex reaction with fluorescently dual labeled probes. Forward and reverse primers

specifically hybridize to the Legionella DNA. The probe included in the reaction mixture

is labeled with a 5`-dye and a 3`-quencher. During PCR amplification, the probe is

cleaved and the reporter dye and quencher are separated. The resulting increase in

fluorescence can be detected using a software that determines the Ct (cycle from

which fluorescence is higher than background signal) that allows to detect presence of

Legionella pneumophila target sequences, therefore the presence of the bacteria in

water sample.

The quantification of the Legionella pneumophila in the sample can be carried out

based on a standard curve covering a concentration range between 25000 and 25 GU

copies per PCR reaction. The standard curve is prepared from purified Legionella

Standard DNA WDCM 00107.

Moreover, the reaction mixture used for amplification contains a synthetic Internal

Control DNA sequence which is co-amplified using the same primers but a different

dual labeled probe. The Internal Control system provides information regarding the

presence of inhibitors in tested samples and the overall success of the PCR.

The threshold value depends on the cycler used and is fixed for both targets (L.

pneumophila and internal control system).

The analysis of results is done with the program of PCR instrument and the

interpretation is done automatically using the DI-Check Analysis software for L.

pneumophila.

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Objective of the study

The aim of the study is to establish the performance of the DI-Check Legionella

pneumophila.

The validation was performed on 3 thermalcyclers listed in Table 1:

Table 1: Thermalcyclers

Threshold value

PCR cycler L. pneumophila

FAM

Internal Amplification Control

VIC/HEX

CFX96 Touch™ Real-Time PCR

Detection System; CFX96

Touch™ Deepwell detection

system

200 100

QuantStudio 3-5 96 well 0.3 0.05

RotorGene Q 3-5plex 0.085 0.18

Validation of the method using RotorGene focuses on the following parameters:

LOD and positivity threshold for Legionella pneumophila detection

LOQ of Legionella pneumophila

Calibration function

Inclusivity and exclusivity

Connection of the standard curve and the reference material to the primary

standard

Yield and robustness

Software verification

Practicability

Validation of the method using QS3 and CFX focuses on the following parameters:

LOD and positivity threshold for Legionella pneumophila detection

LOQ of Legionella pneumophila


Connection of the standard curve and the reference material to the primary

standard (only using CFX)

Yield and robustness (only using CFX)

Software verification

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Summary of results

Connection of the standard curve and the reference material to

the primary standard

Methodology

Three independent ranges of four levels were prepared by serial dilutions starting from

the primary standard (Centre National de Référence de Legionelles, CNRL) and from

the calibrated working solution (Diatheva), respectively. Dilutions covered the linear

quantification range.

Three different lots of reference material provided with the DI-Check Legionella

pneumophila kit were used for the connection of reference material to primary

standard.

RotorGene Results

The results obtained from the primary standard are shown in the Table 2.

Table 2 : Primary standard results

CNRL range

Q (GU/well) Log (Q) Ct

Range 1 Range 2 Range 3 Average

25 1.4 32.15 32.93 32.71 32.60

250 2.4 29.61 29.55 30.83 30.00

2500 3.4 26.21 26.31 26.33 26.28

25000 4.4 22.79 22.99 22.77 22.85

Slope -3.295

Intercept value 37.48

R2 0.998

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The results obtained from the working calibration solution are shown in the Table 3.

Table 3: Working calibration results

Q (GU/well) Log (Q) Ct Ct

average

Log (Q)

Recalculated

value

Error of

Calibration

by Level

Conclusion

Range 1 Range 2 Range 3

25 1.4 32.67 32.84 32.87 32.79 1.43 -0.03 validated

250 2.4 29.56 29.41 29.37 29.45 2.44 -0.04 validated

2500 3.4 26.07 26.07 26.19 26.11 3.45 -0.05 validated

25000 4.4 22.95 23.02 23.03 23.00 4.39 0.01 validated

Slope -3.265 -3.280 -3,270 % slope (-3,32) 98 99 98 Criteria 75% and 125% validated validated validated Average calibration Error -0.03 validated

Check slope equivalency: Absolute value (Calibration Error Log (250000)-Log(25)) 0.02 validated

Recalculated Log (Q) values were obtained from the data in the CNRL range.

For each range, the slope was between 75% and 125%.

For each level, the difference between the logarithm of the calculated value and that

of the expected value was less than 0.2. The absolute value of the difference between

the high and low points of the range (calibration error) was less than 0.2 Log.

The result obtained from the reference material solution is shown in the Table 4.

Table 4: Reference material solution result

Q

(GU/well) Log (Q)

Ct

Ct average

Log (Q)

Recalculated

value

Error of

calibration Conclusion


1200 3.08 27.81 27.71 27.59 27.70 2.97 0.11 validated

The result obtained with the reference material was validated (Error of calibration

<0.15).

Results obtained were consistent with those expected.

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CFX Results

The results obtained from the primary standard are shown in the Table 5.

Table 5 : Primary standard results

CNRL range

Q (GU/well) Log (Q) Ct

Range 1 Range 2 Range 3 Average

25 1.4 34.79 34.67 34.65 34.70

250 2.4 31.57 31.23 31.31 31.37

2500 3.4 28.01 27.87 27.86 27.91

25000 4.4 24.32 24.47 24.39 24.39

Slope -3.439

Intercept value 39.56

R2 1.000

The results obtained from the working calibration solution are shown in the Table 6.

Table 6 : Working calibration solution results

Q (GU/well) Log (Q) Ct Ct

average

Log (Q)

Recalculated

value

Error of

Calibration

by Level

Conclusion


25 1,4 35.69 34.49 34.89 35.02 1.41 -0.01 validated

250 2,4 31.44 31.65 31.58 31.56 2.38 0.02 validated

2500 3,4 28.14 28.17 28.14 28.15 3.39 0.01 validated

25000 4,4 25.31 24.61 24.73 24.88 4.41 -0.01 validated

Slope -3.444 -3.312 -3.392 % slope (-3.32) 104 100 102 Criteria 75% and 125% validated validated validated Average calibration Error 0.00 validated

Check slope equivalency: Absolute value (Calibration Error Log (250000)-Log(25)) 0.02 validated

Recalculated Log (Q) values were obtained from the data in the CNRL range.

For each range, the slope was between 75% and 125%.

For each level, the difference between the logarithm of the calculated value and that

of the expected value was less than 0.2. The absolute value of the difference between

the high and low points of the range (calibration error) was less than 0.2 Log.

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The result obtained from the reference material solution is shown in the Table 7.

Table 7: Reference material result

Q

(GU/well) Log (Q)

Ct

Ct average

Log (Q)

Recalculated

value

Error of

calibration Conclusion


1200 3.08 29.28 29.47 28.94 29.23 3 0.08 validated

The result obtained with the reference material was validated (Error of calibration

<0.15).

The results obtained were consistent with those expected.

General conclusion:

Connection of the Diatheva working solution and reference material to the CNRL

primary standard was conform.

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The calibration function allows to define the effectiveness of the PCR and to verify the

performance of the linear regression through the accuracy of linearity.

Methodology

Study of calibration function was carried out testing 4 levels of concentration (25, 250,

2500, and 25000 GU of Legionella pneumophila per reaction) prepared from DNA

standards provided in the DI-Check Legionella pneumophila kit.

Five measurements were made using the DI-Check Legionella pneumophila kit for

each level of concentration in reproducibility conditions.

Results were analyzed to calculate the slope of the calibration line and the accuracy

of linearity.

The acceptance criteria were:

The slope had to be between – 4.115 and – 2.839.

Linearity accuracy had to be less than 0.15 Log for all levels in the range.

CFX Results

sum

Xi Level LQ 2LQ 20LQ 200LQ

25 250 2500 25000

X'i theoretical = Log Xi 1.40 2.40 3.40 4.40

rep1 35.36 32.03 28.58 25.08

rep2 35.72 31.72 28.46 24.51

y'i,j rep3 35.07 31.99 28.61 25.22

rep4 35.2 32.26 28.54 25.19

rep5 34.39 31.32 28.11 24.64

Ti= 176 159 142 125 602

Mi = 35.15 31.86 28.46 24.93

X'i Ti 246 382 484 548 1659

∑𝒀𝒊, 𝒋

𝒌

𝒋=𝟏

𝑻𝒊

𝒌

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LOG (Q) Ct average

1.40 35.15 Intercept value 39.97

2.40 31.86

3.40 28.46 Slope -3.41

4.40 24.93


25 250 2500 25000


rep1 1.35 2.33 3.34 4.37

rep2 1.25 2.42 3.38 4.54

x'i,j rep3 1.44 2.34 3.34 4.33

rep4 1.40 2.26 3.36 4.34

rep5 1.64 2.54 3.48 4.50

average X'i 1.42 2.38 3.38 4.42

through 0.02 -0.02 -0.02 0.02

Standard

deviation 0.1435 0.1055 0.0598 0.0968

Elin 0.145 0.107 0.063 0.098

Ulin 0.460 0.341 0.199 0.313

CFX conclusion

Slope: - 3.41 criteria was validated

Elin: all levels < 0.15 were validated

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QS3 Results

sum


25 250 2500 25000


rep1 34.39 31.40 28.60 24.77

rep2 34.97 31.70 28.150 24.82

y'i,j rep3 34.91 32.01 28.70 25.05

rep4 34.25 31.59 28.27 24.47

rep5 34.098 31.406 28.009 24.591

Ti= 173 158 142 124 596

Mi = 34.5 31.6 28.3 24.7

X'i Ti 241 379 482 544 1646

LOG (Q) Ct average

1.40 34.52


3.40 28.35

4.40 24.74 Slope -3.26


25 250 2500 25000


rep1 1.493 2.410 3.268 4.441

rep2 1.315 2.318 3.406 4.426

x'i,j rep3 1.335 2.223 3.236 4.358

rep4 1.536 2.353 3.369 4.535

rep5 1.583 2.408 3.449 4.497

average X'i 1.452 2.342 3.346 4.451

through 0.05 -0.06 -0.05 0.05

∑𝒀𝒊, 𝒋

𝒌

𝒋=𝟏

𝑻𝒊

𝒌

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Standard

deviation 0.1208 0.0771 0.0907 0.0681

Elin 0.133 0.095 0.105 0.086

Ulin 0.422 0.302 0.333 0.275

QS3 conclusion



RotorGene Results

sum


25 250 2500 25000


rep1 32.29 29.27 25.46 22.16

rep2 32.88 29.66 26.14 22.8

y'i,j rep3 33.02 29.44 25.97 22.44

rep4 33.6 29.87 26.51 22.78

rep5 33.01 29.23 25.78 22.55

Ti= 165 147 130 113 555

Mi = 32.96 29.49 25.97 22.55

X'i Ti 230 354 441 496 1521

∑𝒀𝒊, 𝒋

𝒌

𝒋=𝟏

𝑻𝒊

𝒌

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LOG (Q) Ct average


2.40 29.49

3.40 25.97 Slope -3.48

4.40 22.55


25 250 2500 25000

X'i theoritical = Log Xi 1.40 2.40 3.40 4.40

rep1 1.59 2.46 3.55 4.50

rep2 1.42 2.35 3.36 4.32

x'i,j rep3 1.38 2.41 3.41 4.42

rep4 1.21 2.29 3.25 4.33

rep5 1.38 2.47 3.46 4.39

average X'i 1.40 2.39 3.41 4.39

through 0.00 0.00 0.01 -0.01

Standard

deviation 0.1342 0.0777 0.1129 0.0760

Elin 0.134 0.078 0.113 0.076

Ulin 0.427 0.247 0.361 0.242

RotorGene conclusion



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General conclusion

Linear regression satisfied exigence of exactitude lower than 0.15 Log for each level

of standard curve, for the three thermocyclers tested. Linearity was verified on the

whole range of calibrated DNA solution DI-Check Legionella pneumophila standards

provided by the DI-Check Legionella pneumophila kit.

Limit of detection (LODPCR)

The LODPCR is estimated from the smallest number of genome units generating a

positive result (an amplification) at the 90% confidence level. The LODPCR announced

by the supplier to be verified is 5 GU per well.

Methodology

Evaluation of limit of detection was carried out testing 30 independent dilutions

prepared using a DNA solution extracted from Legionella pneumophila WDCM 00107

strain connected to the primary standard (reference: ALEGPNE205) in concentration

of 5 GU per PCR reaction.

Results (Annex 1)

CFX: 93.3 % of presence

QS3: 96.7 % of presence

RotorGene: 93.3 % of presence

Conclusion

Limit of detection was validated for 5 GU per PCR reaction. All Ct values were lower

than intercept.

Qualitative detection was conforming.

Limit of quantification (LOQPCR)

The limit of quantification is the first level of the calibration range.

The verification of the limit of quantification is to ensure that the accuracy at the limit

of quantification (ELQ) is ≤0.15 Log critical value.

Methodology

Evaluation of limit of quantification was carried out testing 30 independent dilutions

prepared using a DNA solution extracted from Legionella pneumophila connected to

the primary standard in concentration of 25 GU per PCR reaction. Each dilution was

amplified under conditions of intermediate reproducibility.

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Results (Annex 2)

The accuracies at the level of the LQ (ELQ) were:

- CFX = 0.14, validated

- QS = 0.15, validated

- RotorGene = 0.14, validated

Conclusions

Limit of quantification was validated for 25 GU per PCR reaction for the three thermal

cyclers.

Positivity Threshold

Methodology

The positivity threshold was verified during the LOD validation. Ct values obtained

were verified to be lower than the Ct value (Intercept value) defined by the

manufacturer as follows:

Validation criteria: positivity threshold < Intercept value

Ct defined (Intercept value):

CFX = 42

QS3 = 43

RotorGene = 38

Results (Annex 1)

All Ct values for characterization of limit of detection of the three thermal cyclers were

lower than the respective Intercept value. 42, 43 and 38 were the positivity thresholds

of CFX, QS3 and RotorGene, respectively.

Determination of yield and robustness of extraction

Methodology

The performance of the extraction kit "DNApure Water Isolation kit" (Ref MBK0080)

was evaluated.

The 3 types of matrices, used for the test, were the following:

1. MW Control Mineral Water (Evian)

2. DHW domestic hot water

3. CTW Cooling tower water

The yield was evaluated on 10 independent samples on the 3 matrices which were

artificially contaminated with two levels of Legionella pneumophila (100 000 and 5000

GU/L) under intermediate reproducibility conditions (5 different days). 0.1 L and 1 L

water samples were artificially contaminated using bacterial suspensions for level 1

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and level 2, respectively. The concentration was determined after an extraction step

by direct DNA lysis on 3 aliquots.

Detection and amplification were performed using 2 thermal cyclers, CFX and

RotorGene.

Results (Annex 3)

Table 8: RotorGene results

Day 1 Day 2 Day 3 Day 4 Day 5

Log % Log % Log % Log % Log %

MW LV1 -0.36 43 -0.22 60 -0.16 70 -0.24 57 -0.16 70

LV2 -0.16 69 -0.07 86 -0.15 71 -0.06 88 0.00 100

DHW LV1 -0.36 43 -0.17 68 -0.16 69 -0.28 52 -0.38 42

LV2 -0.01 80 -0.19 65 -0.26 55 0.03 106 0.01 103

CTW LV1 -0.28 53 -0.15 70 -0.14 73 -0.25 57 -0.28 52

LV2 -0.05 90 -0.13 74 -0.03 93 -0.22 60 -0.02 95

MW: Mineral Water, DHW: Domestic Hot Water, CTW: Cooling Tower Water

LV1: Level 1 (100 000 GU/L), LV2: Level 2 (5 000 GU/L)

Log: Log efficiency of extraction method

%: efficiency of the extraction method expressed as percentage

Table 9 : CFX results

Day 1 Day 2 Day 3 Day 4 Day 5

Log % Log % Log % Log % Log %

MW LV1 -0.43 37 -0.28 52 -0.25 56 -0.25 57 -0.13 74

LV2 -0.13 74 0.16 144 -0.13 74 -0.18 67 -0.12 76

DHW LV1 -0.30 50 -0.24 57 -0.25 56 -0.21 62 -0.18 66

LV2 -0.07 84 -0.02 95 -0.16 70 -0.14 72 -0.19 64

CTW LV1 -0.30 50 -0.28 52 -0.27 54 -0.17 67 -0.19 64

LV2 -0.06 87 0.00 100 -0.14 72 -0.07 85 -0.14 72

MW: Mineral Water, DHW: Domestic Hot Water, CTW: Cooling Tower Water

LV1: Level 1 (100 000 GU/L), LV2: Level 2 (5 000 GU/L)

Log: Log efficiency of extraction method

%: efficiency of the extraction method expressed as percentage

The average yields obtained were greater than 25%.

All the calculated yields had values conforming to criteria -0.6 Log and +0.3 Log

equivalent to efficiency comprise between 25% and 199%.

Results were therefore in line with those expected.

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Inclusivity/Exclusivity

Methodology

Inclusivity tests were performed on DNA extracts prepared from 15 serogroups of L.

pneumophila to obtain 100 GU per well. Concentrations were estimated by O.D.600 nm

of bacterial suspension.

The exclusivity tests were performed on DNA extracts prepared from 20 strains of

Legionella spp. and 16 bacterial strains other than Legionella to obtain at least 10000

GU per well. Concentrations were estimated by O.D.600 nm of bacterial suspension.

Results (Annex 4)

DNAs of the 15 Legionella pneumophila strains tested were all detected.

Among the 36 tested DNA extracts not belonging to the specie Legionella

pneumophila, no cross reaction (no amplification) was detected.

The selectivity of the method was satisfactory.

Verification of calculations and interpretations established by the

software

From the data export of runs, the software used "DI-Check Analysis Sofware for L.

pneumophila 2.2" allowed to exploit the quantitative (and the qualitative) results

obtained.

The software accounted for the criteria and indicates their validation of the Intercept,

Slope, R2, NTC control, LOQ (Log validated), inhibition indicator and Positive control

(Reference Material).

The data review showed the following results (Annex 5) and Table 10:

Table 10

Software

RotorGene

Q Series v

2.1.0

DI-Check

Analysis

Software for

L.

pneumophila

2.2

QuantStudio

Design &

Analysis

Software

v1.4.3

DI-Check

Analysis

Software for

L.

pneumophila

2.2

Biorad

CFX

Manager

3.1

DI-Check

Analysis

Software for

L.

pneumophila

2.2

R2 0.999 0.999 1 1 0.998 1

Slope -3.293 -3.293 -3.332 -3.332 -3.445 -3.445

Intercept 35.903 35.90 38.858 38.86 39.565 39.56

As shown in Table 10, results obtained with all suppliers software and with "DI-Check

Analysis Sofware for L. pneumophila 2.2» were similar.

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The validation was therefore in line with the results:

-Intercept: Validated

-Slope: Validated

-R2: Validated

-NTC Indicator: Validated

-LOQ: Validated

-Positive Control (Reference Material): Validated

Inhibition

A solution concentrated 700 GU/5ul was tested with an inhibitor sample by successive

dilution. Concentrations were set at 2-5-8-10-20-50% of inhibitory samples.

The results obtained (Annex 6) allowed detection of inhibition from 20% of the addition

of inhibitory sample. Indeed, there was no amplification (FAM and HEX) for these

concentrations.

For other concentrations (2 to 10% of inhibitory samples), no underestimation was

detected (Ct of the Internal Amplification Control = CtNTC +/-0.5, see Annex 6).

Practicability

Conditioning mode and volume of reagents

The reagents are presented in the following packs.

• DNApure Water Isolation kit extraction reagents and consumables are:

2 bottles x 100 ml «Lysis Buffer»

1 bottle x 3.5 ml "Elution Buffer"

1 sachet x 100 units "DNApure Column"

2 bags x 100 units "1.5 ml Tubes"

2 bags x 50 units "Cryotube vials"

• Amplification reagents "DI-Check Legionella pneumophila kit" are:

2 tubes x 1200 μl "L. pneumophila PCR Mix"

1 tube x 10 μl "Standard DNA"

5 tubes x 1500 μl "Dilution Buffer"

1 tube x 100 μl "Negative PCR Control"

2 tubes x 50 μl "Reference Material"

Condition of storage of the elements and expiry of the products

The storage temperature and expiry date are shown on page 1 of the kit instructions.

The "DNApure Water Isolation" kit must be stored at (5 ± 3) °C, with the exception of

"DNApure Columns" that must be stored at room temperature.

The kit "DI-Check Legionella pneumophila" must be stored at -20 ° C

The expiry date is indicated on the kit packs, as well as on each kit component.

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Terms of use after first use

The reagents are used until exhaustion in accordance with the expiry date.

Specific equipment and premises needed

The necessary equipment and consumables are indicated on page 1 of the "DNApure

Water Isolation" kit and page 2 of the "DI-Check Legionella pneumophila" kit.

The necessary safety measures are indicated on page 2.

Reagents ready to use or to reconstitute

All reagents are ready to use.

Training duration of the uninitiated operator to the method

The initial training of the technician is 2 days.

Real time handling

Table 11

Step Timing

Filtration depend the type of water from 5 to 30 minutes

DNA Extraction 70 minutes

PCR 1 hour and 55 minutes (in average between the 3 cyclers)

Results analyses 10 minutes

Time to obtain results

• Minimum time: The minimum time for obtaining results is approximately 3h 30 min

• Realization of the PCR after extraction: the analysis can be interrupted after the

extraction. The extract is thus stored at -20 °C ± 3 °C if the PCR analysis is not carried

out within 6 hours after extraction. This makes it possible to optimize the organization

of the analyzes.

Type of qualification for the operator

Technician.

Traceability of the analysis results

The results are kept in the form of computer files and / or paper. Steps other than PCR

are plotted in documents provided by the laboratory.

Maintenance by the laboratory

No maintenance is performed by the laboratory.

Minimum Volume to pipette

The minimum volume to be pipetted is 1.3 μl.

AFNOR VALIDATION

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Stability of reagents and ranges

The expiry date and stability are indicated on the kit boxes. The storage conditions are

described on the kits.

UNG

Contamination prevention tips are listed on page 1 and 2 of the "DNApure Water

Isolation" kit leaflet and page 2 of the "DI-Check Legionella pneumophila" kit. In fact,

prevention involves wearing gloves, decontaminating filtration accessories and

complying with Good Laboratory Practices.

Negative PCR Control guarantees the absence of DNA contamination during the

analysis.

UV reagent protection

The reagents are stored in their original packaging (protect from light).

AFNOR VALIDATION

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Interlaboratories Study

Methodology

The interlaboratory study was realized by thirteen (13) collaborating laboratories in

October 2019.

Every PCR machine were present.

Phase 1 (For only amplification step): DNA Extracts of L. anisa (WDCM 00106)

and L. pneumophila S1 (WDCM 00107)

Phase 2 (For complete analysis): Samples free of Legionella DNA artificially

contaminated with bacterial suspension of Legionella pneumophila (WDCM

00107), Legionella spp. (WDCM 00106) and non-Legionella (WDCM 00026

Pseudomonas aeruginosa)

Phase 3 (For whole analysis in real situation): Naturally contaminated domestic

hot water

Results of one laboratory were not taken into account because of delay in

transportation of samples to test.

For the analysis of results:

12 laboratories were retained for phase 1.

10 laboratories were retained for phase 2 level 1 and 11 laboratories were retained for

phase 2 level 2 due to technical problems.

8 laboratories were retained for phase 3. One laboratory had technical problems and

other laboratories obtained quantification results under the LOQ.

The three thermalcyclers had been used in all the three phases, including the phase 3

for which 8 laboratories were retained.

AFNOR VALIDATION

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Results

Table 12

Type of samples DNA extracts Bacterial suspensions Natural

water Level 1 Level 2 Level 1 Level 2

Theoretical

levels

L. pneumophila S1

WDCM 00107

1000

GU/well

10000

GU/well

400000

GU/L

40000

GU/L Naturally

contaminated

hot tap water

Pseudomonas

aeruginosa

WDCM 00026

/ / 4x105

CFU/L

4x104

CFU/L

Number of

laboratories

Participant 13 13 13 13 13

Retained 12 12 10 11 8

Homogeneity

Test

Number of analyses 20 20 9 9 9

Average (Log) 3.009 4.006 5557 4.564 4.094

Results ILS Average (Log) 2.937 3.900 5.297 4.233 3.975

Sr (Log) 0.029 0.039 0.083 0.104 0.096

SR (Log) 0.093 0.087 0.139 0.168 0.105

RSTDr % 0.99 1.00 1.56 2.45 2.42

RTSDR % 3.15 2.22 2.63 3.96 2.64

Conclusion

In terms of repeatability (Sr), the maximum standard deviations observed were 0.039

for the PCR step (DNA extracts), 0.104 for the entire method (preparation of DNA &

PCR) with bacterial suspensions and 0.096 for naturally contaminated samples.

The DI-CHECK Legionella method was repeatable.

The maximum reproducibility (SR) standard deviations express the inter-laboratory

variability: those obtained for the PCR analysis of calibrated DNA solutions were 0.093

while those corresponding to all of the analysis steps (preparation of DNA & PCR) were

0.168 and 0.105 for naturally contaminated samples.

The DI-CHECK Legionella method was reproducible.

AFNOR VALIDATION

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Conclusions

The results of the study showed that the performance of the kit for the detection of

Legionella pneumophila complies with the requirements of NF T 90-471, ISO/TS 12869

and PCR Validation protocol v3.

The method can be used to test any type of water, without restriction of use.

The "DI-Check Legionella pneumophila" is a method validated for detection and for

quantification of Legionella pneumophila.

AFNOR VALIDATION

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Annex 1: LOD

LOD CFX LOD QS3 LOD RotorGene

Ct Ct IAC Ct Ct IAC Ct Ct IAC

1 36.70 32.63 38.41 31.09 35.73 27.77

2 36.89 32.89 39.38 30.88 N/A 27.72

3 38.49 32.42 36.77 30.83 36.66 27.61

4 38.40 32.86 36.82 30.90 36.3 27.64

5 N/A 32.78 36.10 31.14 36.33 27.87

6 38.29 32.37 37.78 30.85 35.08 27.81

7 37.60 32.44 36.47 30.95 35.71 28.05

8 37.65 32.68 36.63 30.98 34.86 27.86

9 37.01 32.36 35.82 30.98 36.74 27.77

10 36.59 32.14 36.68 31.21 35.11 27.77

11 37.38 32.18 36.19 30.88 34.85 28.03

12 36.98 32.30 36.34 30.75 36.68 28

13 38.47 32.52 37.14 30.99 37.9 27.59

14 37.01 32.27 36.40 30.98 37.01 27.87

15 38.53 32.09 42.74 30.89 34.43 27.53

16 37.83 32.69 36.77 30.71 35.61 27.68

17 38.20 32.28 35.75 30.92 35.88 27.57

18 36.75 32.27 37.89 30.99 35.26 27.94

19 38.21 32.88 35.91 30.81 34.91 27.58

20 41.41 32.34 37.22 30.75 36.05 27.81

21 36.92 32.28 37.75 31.08 35.42 27.63

22 37.74 32.50 38.44 30.97 35.05 27.77

23 35.58 32.51 36.84 30.93 34.83 27.51

24 37.26 33.09 38.33 30.82 34.84 27.61

25 36.10 32.69 37.43 30.99 36.76 27.72

26 38.32 32.47 36.30 30.98 N/A 27.63

27 36.28 32.17 37.42 30.98 36.67 27.63

28 38.61 32.07 37.98 31.02 34.98 27.78

29 N/A 32.44 35.69 30.96 34.42 27.97

30 37.12 32.77 N/A 30.73 35.31 27.96

TOTAL 28 29 28

% 93.3 96.7 93.3

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Annex 2: LOQ

CFX QS3 RotorGene

Ct GU/well Log Ct GU/well Log Ct GU/well Log

35.59 19 1.28 34.39 31 1.49 33.31 20 1.30

35.36 23 1.35 34.97 21 1.31 32.14 43 1.63

35.53 20 1.30 35.41 15 1.18 32.65 31 1.49

35.72 18 1.25 34.90 22 1.34 33.65 16 1.20

34.73 35 1.54 35.10 19 1.27 33.02 24 1.38

35.12 27 1.42 34.99 20 1.31 33.07 23 1.36

34.57 39 1.58 35.43 15 1.17 33.31 20 1.30

34.62 37 1.57 35.38 16 1.19 32.4 36 1.56

34.86 32 1.50 34.36 32 1.50 33.01 24 1.38

34.39 44 1.64 35.48 14 1.16 32.29 39 1.59

35.23 25 1.39 35.37 16 1.19 32.32 38 1.58

35.15 26 1.41 34.28 34 1.53 33.45 18 1.26

34.71 35 1.54 34.64 26 1.42 33.66 16 1.20

34.36 44 1.65 35.17 18 1.25 32.95 25 1.40

35.55 20 1.30 35.40 15 1.18 33.6 16 1.21

34.29 47 1.67 34.95 21 1.32 32.23 40 1.61

34.22 49 1.69 34.55 28 1.44 32.96 25 1.40

35.6 19 1.28 34.10 38 1.58 33.02 24 1.38

34.77 34 1.52 34.48 29 1.47 32.88 26 1.42

35.24 25 1.39 34.29 34 1.52 33.31 20 1.30

34.42 43 1.63 35.29 17 1.22 32.28 39 1.59

35.16 26 1.41 35.32 16 1.21 32.46 35 1.54

35.20 25 1.40 34.25 35 1.54 33.19 21 1.33

35.07 27 1.44 34.39 31 1.49 32.59 32 1.50

35.59 19 1.28 34.59 27 1.43 33.58 17 1.22

35.17 22 1.41 35.27 17 1.22 33.44 18 1.26

35.40 22 1.34 34.44 30 1.48 31.88 51 1.71

35.40 19 1.34 34.23 35 1.54 32.79 27 1.65

35.58 27 1.29 34.44 30 1.48 32.59 31 1.57

35.11 22 1.43 34.71 25 1.39 32.73 29 1.46

a -3.41 -3.26 -3.48

b 39.97 39.26 37.82

Average 1.44 1.361 1.416

Bias 0.041 -0.039 0.018

Standard deviation 0.132 0.140 0.143

ELQ 0.14 0.15 0.14

AFNOR VALIDATION

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Annex 3: Yield and robustness RotorGene results

Mineral water

Level 100 000 GU/L

Doping value Analysis results Yield

Sample GU/well A (Log) Ct GU/well B (Log) Log %

EM1-1 1.24E+02 9.72 28.64 4.65E+02 4.47 -0.24 57

EM1-2 1.24E+02 9.72 29.64 2.42E+02 4.19 -0.53 30

EM1-3 1.19E+02 9.70 28.99 4.21E+02 4.43 -0.27 54

EM1-4 1.19E+02 9.70 28.7 5.20E+02 4.52 -0.18 67

EM1-5 9.37E+01 9.59 28.86 5.07E+02 4.51 -0.08 83

EM1-6 9.37E+01 9.59 29.34 3.50E+02 4.35 -0.24 57

EM1-7 1.50E+02 9.80 28.76 5.89E+02 4.58 -0.22 60

EM1-8 1.50E+02 9.80 28.91 5.32E+02 4.53 -0.26 54

EM1-9 1.29E+02 9.73 28.84 5.33E+02 4.53 -0.20 63

EM1-10 1.29E+02 9.73 28.56 6.47E+02 4.62 -0.12 77

Mineral water

Level 5 000 GU/L



EM2-1 1.56E+02 9.82 31.42 8.07E+01 3.71 -0.10 79

EM2-2 1.56E+02 9.82 31.84 6.12E+01 3.59 -0.22 60

EM2-3 1.74E+02 9.86 30.73 1.27E+02 3.91 0.05 112

EM2-4 1.74E+02 9.86 31.68 6.80E+01 3.64 -0.22 60

EM2-5 1.50E+02 9.80 31.56 7.36E+01 3.67 -0.13 75

EM2-6 1.50E+02 9.80 31.74 6.53E+01 3.62 -0.18 66

EM2-7 6.07E+01 9.4 33.21 3.50E+01 3.35 -0.05 89

EM2-8 6.07E+01 9.4 33.25 3.41E+01 3.34 -0.06 86

EM2-9 6.04E+01 9.4 32.80 4.59E+01 3.47 0.06 116

EM2-10 6.04E+01 9.4 33.30 3.30E+01 3.33 -0.08 84

AFNOR VALIDATION

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Domestic hot water

Level 100 000 GU/L



DHW1-1 1.24E+02 9.72 29.025 3.61E+02 4.36 -0.35 44

DHW1-2 1.24E+02 9.72 28.63 4.68E+02 4.48 -0.24 57

DHW1-3 1.19E+02 9.70 28.775 4.92E+02 4.50 -0.20 63

DHW1-4 1.19E+02 9.70 28.59 5.62E+02 4.56 -0.14 72

DHW1-5 9.37E+01 9.59 29.13 4.13E+02 4.42 -0.17 67

DHW1-6 9.37E+01 9.59 29.075 4.30E+02 4.44 -0.15 70

DHW1-7 1.50E+02 9.80 28.935 5.23E+02 4.52 -0.27 54

DHW1-8 1.50E+02 9.80 29.025 4.91E+02 4.50 -0.30 50

DHW1-9 1.29E+02 9.73 29.32 3.81E+02 4.39 -0.35 45

DHW1-10 1.29E+02 9.73 29.52 3.30E+02 4.32 -0.41 39

Domestic hot water

Level 5 000 GU/L



DHW2-1 1.56E+02 9.82 31.39 8.23E+01 3.72 -0.01 80

DHW2-2 1.56E+02 9.82 31.39 8.23E+01 3.72 -0.01 80

DHW2-3 1.74E+02 9.86 31.66 6.89E+01 3.64 -0.22 61

DHW2-4 1.74E+02 9.86 31.46 7.86E+01 3.70 -0.16 69

DHW2-5 1.50E+02 9.80 32.00 5.50E+01 3.55 -0.25 56

DHW2-6 1.50E+02 9.80 32.04 5.36E+01 3.54 -0.26 55

DHW2-7 6.07E+01 9.40 32.98 4.08E+01 3.42 0.01 103

DHW-8 6.07E+01 9.40 32.89 4.32E+01 3.44 0.04 109

DHW2-9 6.04E+01 9.40 32.93 4.22E+01 3.43 0.03 107

DHW2-10 6.04E+01 9.40 33.04 3.91E+01 3.40 0.00 99

AFNOR VALIDATION

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Cooling tower water

Level 100 000 GU/L



CTW1-1 1.24E+02 9.72 28.955 3.78E+02 4.38 -0.33 46

CTW1-2 1.24E+02 9.72 28.575 4.85E+02 4.49 -0.23 60

CTW1-3 1.19E+02 9.70 28.45 6.22E+02 4.60 -0.10 80

CTW1-4 1.19E+02 9.70 28.835 4.72E+02 4.48 -0.22 61

CTW1-5 9.37E+01 9.59 28.845 5.13E+02 4.52 -0.08 84

CTW1-6 9.37E+01 9.59 29.22 3.85E+02 4.39 -0.20 63

CTW1-7 1.50E+02 9.80 28.94 5.21E+02 4.52 -0.27 53

CTW1-8 1.50E+02 9.80 28.77 5.85E+02 4.57 -0.22 60

CTW1-9 1.29E+02 9.73 29.08 4.49E+02 4.46 -0.27 53

CTW1-10 1.29E+02 9.73 29.13 4.34E+02 4.44 -0.29 51

Cooling tower water

Level 5 000 GU/L



CTW2-1 1.56E+02 9.82 30.87 1.16E+02 3.87 0.05 113

CTW2-2 1.56E+02 9.82 31.67 6.84E+01 3.64 -0.18 67

CTW2-3 1.74E+02 9.86 31.02 1.05E+02 3.83 -0.03 93

CTW2-4 1.74E+02 9.86 31.78 6.36E+01 3.61 -0.25 56

CTW2-5 1.50E+02 9.80 30.94 1.11E+02 3.85 0.05 113

CTW2-6 1.50E+02 9.80 31.59 7.21E+01 3.66 -0.13 73

CTW2-7 6.07E+01 9.4 33.76 2.45E+01 3.20 -0.21 62

CTW2-8 6.07E+01 9.4 33.84 2.32E+01 3.17 -0.23 59

CTW2-9 6.04E+01 9.4 33.20 3.54E+01 3.35 -0.05 89

CTW2-10 6.04E+01 9.4 33.01 3.99E+01 3.41 0.01 101

AFNOR VALIDATION

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CFX results

Mineral water

Level 100 000 GU/L



EM1-1 1.19E+02 9.70 30.28 4.01E+02 4.41 -0.29 52

EM1-2 1.19E+02 9.70 31.31 2.00E+02 4.11 -0.59 26

EM1-3 1.59E+02 9.82 30.75 5.17E+02 4.52 -0.30 50

EM1-4 1.59E+02 9.82 30.63 5.63E+02 4.55 -0.26 54

EM1-5 1.43E+02 9.77 30.71 5.31E+02 4.53 -0.24 57

EM1-6 1.43E+02 9.77 30.80 5.00E+02 4.51 -0.27 54

EM1-7 9.89E+01 9.61 31.26 3.59E+02 4.36 -0.25 56

EM1-8 9.89E+01 9.61 31.22 3.68E+02 4.37 -0.24 57

EM1-9 1.00E+02 9.62 31.10 4.24E+02 4.43 -0.19 64

EM1-10 1.00E+02 9.62 30.75 5.47E+02 4.54 -0.08 83

Mineral water

Level 5 000 GU/L



EM2-1 1.25E+02 9.72 33.52 7.09E+01 3.66 -0.06 87

EM2-2 1.25E+02 9.72 34.03 5.02E+01 3.51 -0.21 61

EM2-3 1.21E+02 9.71 32.97 1.03E+02 3.82 0.11 129

EM2-4 1.21E+02 9.71 32.67 1.26E+02 3.91 0.20 158

EM2-5 1.46E+02 9.79 33.39 7.74E+01 3.69 -0.09 81

EM2-6 1.46E+02 9.79 33.65 6.49E+01 3.62 -0.17 68

EM2-7 1.33E+02 9.75 33.80 6.71E+01 3.63 -0.11 77

EM2-8 1.33E+02 9.75 34.26 4.92E+01 3.5 -0.25 57

EM2-9 1.62E+02 9.83 33.47 8.36E+01 3.73 -0.11 78

EM2-10 1.62E+02 9.83 33.58 7.77E+01 3.69 -0.14 73

AFNOR VALIDATION

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Domestic hot water

Level 100 000 GU/L



DHW1-1 1.19E+02 9.70 30.38 3.74E+02 4.38 -0.32 48

DHW1-2 1.19E+02 9.70 30.26 4.06E+02 4.41 -0.28 52

DHW1-3 1.59E+02 9.82 30.62 5.67E+02 4.56 -0.26 55

DHW1-4 1.59E+02 9.82 30.50 6.15E+02 4.59 -0.23 59

DHW1-5 1.43E+02 9.77 30.77 5.12E+02 4.51 -0.26 55

DHW1-6 1.43E+02 9.77 30.75 5.17E+02 4.52 -0.25 56

DHW1-7 9.89E+01 9.61 31.13 3.94E+02 4.40 -0.21 61

DHW1-8 9.89E+01 9.61 31.10 4.02E+02 4.41 -0.20 62

DHW1-9 1.00E+02 9.62 31.09 4.28E+02 4.44 -0.19 65

DHW1-10 1.00E+02 9.62 31.06 4.36E+02 4.45 -0.18 66

Domestic hot water

Level 5 000 GU/L



DHW2-1 1.25E+02 9.72 33.39 7.74E+01 3.69 -0.02 95

DHW2-2 1.25E+02 9.72 33.75 6.07E+01 3.59 -0.13 74

DHW2-3 1.21E+02 9.71 33.14 9.16E+01 3.77 0.06 115

DHW2-4 1.21E+02 9.71 33.80 5.87E+01 3.57 -0.13 74

DHW2-5 1.46E+02 9.79 34.02 5.06E+01 3.51 -0.28 53

DHW2-6 1.46E+02 9.79 33.29 8.28E+01 3.72 -0.06 87

DHW2-7 1.33E+02 9.75 33.55 7.90E+01 3.70 -0.04 91

DHW-2-8 1.33E+02 9.75 34.04 5.70E+01 3.56 -0.18 65

DHW2-9 1.62E+02 9.83 33.55 7.90E+01 3.70 -0.13 74

DHW2-10 1.62E+02 9.83 34.04 5.70E+01 3.56 -0.27 54

AFNOR VALIDATION

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Cooling tower water

Level 100 000 GU/L



CTW-1 1.19E+02 9.70 30.51 3.43E+02 4.34 -0.35 44

CTW-2 1.19E+02 9.70 30.17 4.31E+02 4.44 -0.25 56

CTW-3 1.59E+02 9.82 30.60 5.74E+02 4.56 -0.26 56

CTW-4 1.59E+02 9.82 30.78 5.06E+02 4.51 -0.31 49

CTW-5 1.43E+02 9.77 30.86 4.80E+02 4.49 -0.28 52

CTW-6 1.43E+02 9.77 30.77 5.12E+02 4.51 -0.26 55

CTW-7 9.89E+01 9.61 31.03 4.23E+02 4.43 -0.18 66

CTW-8 9.89E+01 9.61 30.99 4.37E+02 4.44 -0.17 68

CTW-9 1.00E+02 9.62 31.12 4.19E+02 4.43 -0.20 64

CTW-10 1.00E+02 9.62 31.11 4.22E+02 4.43 -0.19 64

Cooling tower water

Level 5 000 GU/L



CTW2-1 1.25E+02 9.72 33.07 9.60E+01 3.79 0.07 117

CTW2-2 1.25E+02 9.72 34.13 4.69E+01 3.48 -0.24 57

CTW2-3 1.21E+02 9.71 33.45 7.43E+01 3.68 -0.03 93

CTW2-4 1.21E+02 9.71 33.26 8.45E+01 3.73 0.03 106

CTW2-5 1.46E+02 9.79 33.33 8.06E+01 3.71 -0.07 84

CTW2-6 1.46E+02 9.79 33.84 5.71E+01 3.56 -0.22 60

CTW2-7 1.33E+02 9.75 33.65 7.39E+01 3.67 -0.07 85

CTW2-8 1.33E+02 9.75 33.65 7.41E+01 3.67 -0.07 85

CTW2-9 1.62E+02 9.83 33.72 7.07E+01 3.65 -0.18 66

CTW2-10 1.62E+02 9.83 33.50 8.16E+01 3.72 -0.11 77

Legend

A: Reference value for the concentration of the mother suspension, expressed as

decimal logarithm of the number of genome units per milliliter

B: Value measured from the spiked sample, expressed as decimal logarithm of the

number of genome units per sample

For the Yield calculation the formula reported in the ISO 12869 and NF148 was used

considering also the following parameters

D: 4 for the level 1; 5 for the level 2

Vpe: 100µl

AFNOR VALIDATION

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Annex 4: Inclusivity and Exclusivity

Inclusivity strains

Name Origin Ct Green (target) Ct Yellow (IAC)

Legionella pneumophila ser 1 CNRL 29.32 29.04















AFNOR VALIDATION

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Exclusivity strains

Name Origin Ct Green (target) Ct Yellow (IAC)

Legionella anisa ATCC 35292 N/A 30.00

Legionella erythra CNRL N/A 29.82

Legionella maceachernii CNRL N/A 29.33

Legionella dumofii CNRL N/A 29.3

Legionella parisensis CNRL N/A 29.24

Legionella micdadei CNRL N/A 26.25

Legionella feeleii CNRL N/A 29.14

Legionella sainthelensi CNRL N/A 29.48

Legionella jordanis ATCC33623 N/A 27.76

Legionella gormanii CNRL N/A 27.27

Legionella bozemanii CNRL N/A 29.25

Legionella oakridgensis CNRL N/A 29.25

Legionella birminghamsis CNRL N/A 29.34

Legionella cincinnatiensis CNRL N/A 29.52

Legionella cherii CNRL N/A 27.33

Legionella longbeachae CNRL N/A 29.03

Legionella tuconensis CNRL N/A 29.37

Legionella hackeliae CNRL N/A 29.11

Legionella larsingensis CNRL N/A 29.75

Legionella wadsworthii CNRL N/A 30.08

Enterobacter aerogenes ATCC 15038 N/A 29.14

Proteus vulgaris ATCC 19181 N/A 31.12

Pseudomonas putida ATCC 12633 N/A 27.28

Serratia marcescens ATCC 13880 N/A 27.43

Alcaligenes faecalis ATCC8750T N/A 29.14

Aeromonas hydrophila ATCC 7766 N/A 31.27

Klebsiella oxytoca WDCM 00096 N/A 29.02

Stenotrophomonas maltophila ATCC 13637 N/A 27.35

Flavobacterium ATCC 15997 N/A 29.11

Burkholderia cepacia ATCC 17616 N/A 28.80

Escherichia coli WDCM 00012 N/A 28.82

Pseudomonas aeroginosa ATCC 10145 N/A 27.96

Pseudomonas fluorescens ATCC 13525 N/A 29.29

Clostridium perfringens WDCM 00007 N/A 26.4

Bacillus subtillis WDCM 00070 N/A 29.16

Listeria monocytogenes WDCM 00020 N/A 29.15

AFNOR VALIDATION

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Annex 5: Verification of calculations and interpretation

made by the software

Results RotorGene

Verification carried out using DI-Check analysis software for Legionella pneumophila

v2.2

Verification carried out using the RotorGene software

AFNOR VALIDATION

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Results QuantStudio


v2.2

Verification carried out using the QuantStudio software

AFNOR VALIDATION

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Results CFX


v2.2

Verification carried out using the CFX software

AFNOR VALIDATION

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Annex 6: Inhibition

Verification carried out using RotorGene cycler

AFNOR VALIDATION

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Quantitative analysis of Cycling A.Yellow (Page

1)

No. Name Type Ct No. Name Type Ct

1 NTC NTC 27.38 1 NTC NTC 27.38

2 NTC NTC 27.39 2 NTC NTC 27.39

13 I2a Unknown 27.24 3 PTC Positive Control 26.94

14 I2b Unknown 27.18 4 PTC Positive Control 27.33

15 I5a Unknown 27.18 5 25 Standard 27.56

16 I5b Unknown 26.99 6 25 Standard 27.58





21 I20a Unknown 11 25000 Standard 25.28

22 I20b Unknown 12 25000 Standard 25.33

23 I50a Unknown 24 I50b Unknown

I2a: replicate a with 2% Inhibitory, I2b: replicate b with 2% Inhibitory






Documents

Legionella pneumophila for the detection and ... · Legionella spp. and/or Legionella pneumophila by concentration and genic amplification by quantitative polymerase chain reaction