Extension for detection and quantification of Legionella spp. · GeneDisc® Legionella spp. and Legionella pneumophila are detection and quantification system for Legionella spp

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

Extension for detection and quantification of

Legionella spp.

Summary report

February 2019

This document presents the extension study of a method of Legionella spp. detection by

molecular biology with GeneDisc by Pall GeneDisc Technologies.

Attestation n° GEN 25/03-12/07

Pall Genedisc technologie 1 rue du Courtil 35170 BRUZ FRANCE

This report includes 25 pages, including 2 appendices. Reproduction of this report is only permitted in

its full form

AdGène Laboratoire Summary report GeneDisc ® legionella spp

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Summary

INTRODUCTION 4

HISTORY 4

PRESENTATION OF THE DETECTION SYSTEM 5

PROTOCOL/ METHOD PRINCIPLE 6

OBJECTIVE OF THE STUDY / EVOLUTIONS OF THE ALTERNAT IVE METHOD 7

REFERENCE METHODS 9

STUDY 9

FITTING THE CALIBRATION AND THE REFERENCE MATERIAL TO THE PR IMARY STANDARD 9 STUDY OF THE CALIBRATION FUNCTION OF THE QUANTITATIVE PCR STEP 10 METHODOLOGY 10 RESULTS 11 CONCLUSION 12 LIMIT OF DETECTION 13 METHODOLOGY 13 RESULTS 13 CONCLUSION 13 LIMIT OF QUANTIFICATION 13 METHODOLOGY 14 RESULTS 14 CONCLUSIONS 14 POSITIVITY THRESHOLD 15 DETERMINATION OF YIELD AND ROBUSTNESS 15 EFFICIENCY OBTAINED WITH THE SIMPLIFIED PROTOCOL FOR SANITA RY WATER AND "CLEAN

WATER" 16 INCLUSIVITY/EXCLUSIVITY 17 PRATICABILITY 18

INTER-LABORATORY STUDY 21

CONCLUSION 22


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GENERAL CONCLUSIONS 22

APPENDIX 1: GENEDISC PLATE DUO (GLEGDUO206006) 23

APPENDIX 2: LIMIT OF QUANTIFICATION 24


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Introduction This document presents the results obtained for the extension validation of the GeneDisc Legionella spp. Method GeneDisc by Pall GeneDisc Technologies. The validation studies were realized according to the « Protocole de validation pour les kits de détection et de dénombrement de Legionella et Legionella pneumophila par concentration et amplification génique par réaction en chaîne de polymérisation (PCR) », Revision 2 published by AFNOR Certification on the september 23th, 2015.

This document presents the results obtained by CEA ENDETEC during the initial validation of the GeneDisc Legionella pneumophila method in 2007, results obtained for the validation of the application extensions (2009, 2011, 2012, 2015) and results obtained by AdGene for the extension study.

History The GeneDisc GeneDisc® Legionella spp. (Reference des GeneDisc Plates: GLEGSPP106006 & GLEGDUO106006) and Legionella pneumophila (Reference des GeneDisc Plates: GLEGPNE106006 & GLEGPNE112006, GLEGDUO106006) has been certified NF Validation by AFNOR Certification (certificate n° GEN 25/04 – 12/07). The first certificate was obtained in December 2007. The following modifications have been implemented since 2007:

Year Type of certification Comments 2007 Initial certification 2008 Extensions

- Modification of the silica column format (miniaturization)

- Modification of the GeneDisc design in duplicate vs. triplicate previously

Expert laboratory: CEA-ENDETEC test for performance criteria:

- LD, LQ - Linearity - Optimal recovery

2012 Extensions - Compliance with

norme NF T90-471 - Extraction with

GeneDisc DNA Extractor

- Amplification by GeneDisc Cycler v3


- connection to the primary DNA standard Linearity

- Optimal recovery


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2012 Renewal and modifications - Use of the Extraction

Pack Environment 3 for the DNA preparation from sanitary water samples with a simplified protocol

- New supplier of BSA for master mix


- Optimal recovery - Robustness - Praticability

2015 Renewal and modifications - New version of

validation protocole - GDUP (GeneDisc

Ultra-Purifier) - GeneDisc Plate

Legionella ID for qualitative detection of Legionella spp. and Legionella pneumophila

No additional tests. Data Work on data for the threshold of positivity and inhibition control.

Presentation of the detection system

GeneDisc® Legionella spp. and Legionella pneumophila are detection and quantification system for Legionella spp. and Legionella pneumophila by real time PCR in all kind of water. The GeneDisc Legionella spp. and pneumophila method includes two steps:

A first step for the DNA preparation from a water sample. � Extraction Pack Environment 1 (reference: PENVI1096) for the DNA

preparation in all kind of type of water. � Extraction Pack Environment 3 (reference: PENVI3100) for the DNA

preparation in sanitary water. � GeneDisc DNA Extractor (GDDE, reference: EGDDE01) or GeneDisc Ultra-

Lyser (GDUL, reference: EGDUL1A) and GeneDisc Ultra-Purifier (GDUP, reference: EGDUP1A)

A second step of Legionella DNA quantification by real time PCR with GeneDisc Cycler V3 (GDC V3, reference: EGDCV3A) and GeneDisc Packs:

� GeneDisc Legionella spp. 6 sectors (reference: GLEGSPP206006) � GeneDisc Legionella pneumophila 6 & 12 sectors (reference:

GLEGPNE206006 & GLEGPNE212006) � GeneDisc Legionella DUO 6 sectors (reference: GLEGDUO206006) � GeneDisc Legionella ID 6 & 12 sectors (reference: GLEGOID206006 &

GLEGOID212006)


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Protocol/ method principle

The Extraction Packs Environment 1 and 3 contain all the consumables and reagents required for the water sample filtration, the cellular lysis and the DNA purification.

The DNA extraction is based on a mechanical and thermal lysis of the cells in presence of detergent, followed by an optional DNA purification by adsorption on mini silica columns.

All steps of the DNA preparation protocol are managed by the GeneDisc® DNA Extractor (GDDE,

reference: EGDDE01) ou GeneDisc Ultra-Lyser (GDUL, reference: EGDUL1A) et GeneDisc Ultra-Purifier (GDUP, reference: EGDUP1A).

Then, Legionella DNA is quantified by real-time PCR with the Legionella GeneDisc Packs.

The PCR assay for Legionella is based on the genic amplification by real-time PCR of a specific nucleic sequence for the Legionella pneumophila species. The use of TaqMan probes labelled by a fluorophore (FAM) enables the detection. During elongation of the amplicon, the probe is cleaved and the fluorophore, physically separated from the Quencher, produces a fluorescent signal. This fluorescence is directly measured by the optical module of the GeneDisc Cycler.

For each analysis, the DNA / Master Mix mix, loaded in the central tank of a GeneDisc Plate sector, is sent by vacuum in the peripheral PCR wells pre-loaded with reagents (primers, probes, DNA).

Regarding the quantitative PCR assay, for each GeneDisc batch, a calibration GeneDisc, Plate corresponding to the PCR amplification of calibrated L. pneumophila WDCM 00107 genomic DNA (ALEGPNE105), is analyzed. The GeneDisc Cycler software automatically calculates the Ct, the parameters of the standard curve and the quadratic corresponding to the following curve: fluorescence amplitude of the internal inhibition controls = f(Ct Legionella pneumophila). These polynomial expresses


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sensitivity of the internal inhibition control to the competition, in presence of Legionella pneumophila genomic DNA (out of inhibition).

The obtained parameters for the calibration GeneDisc Plate and its associated Master Mix are saved and applied to all GeneDisc Plates from the same batch, using a given GeneDisc Cycler, in compliance with definition of the PCR series according to the NF T90-471 and ISO TS/12869 standards.

For each sample analyzed, under the same experimental conditions than the calibration GeneDisc Plate, the inhibition percentage of the internal inhibition control is determined by the software in order to let the user know the dilution factor to apply if required (d5 or d10). If the PCR reaction is not inhibited, the software automatically calculates the Ct value and converts it in Legionella GU number/L as a function of the volume of the protocol applied and the filtered water.

Objective of the study / evolutions of the alternative

method The study is due to a modification of the PCR mix and of the algorithm for the detection and quantification systems of Legionella spp. and Legionella pneumophila, as well as a modification of specific primers and probe of Legionella pneumophila. Primers and probes are used for the detection and quantification of Legionella spp. remain unchanged as well as the entire analysis protocol. The only changes therefore only concern the amplification part of systems on detection and detection of Legionella spp. and Legionella pneumophila. The validation of GeneDisc Plate Legionella pneumophila (GLEGPNE206006 and GLEGPNE212006) and Legionella DUO (GLEGDUO206006) only concerned the following elements:

- LD for the detection of Legionella spp. and Legionella pneumophila - LQ and linearity for Legionella spp. and Legionella pneumophila quantification. - Inclusivity, exclusivity Legionella pneumophila detection

The standard used for the realization of the calibration range remains unchanged, which is why the connection step will not be performed. Indeed, the modification of the primers and probes of a PCR system has no impact on the connection. A second modification concerns the GeneDisc plate IDs which allow a qualitative detection of Legionella spp., Legionella pneumophila and Legionella pneumophila serogroup 1. Besides the modification of the primers and the probe of Legionella pneumophila, the reaction mix, and the algorithm, the Legionella pneumophila


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serogroup 1 target has been added. On the other hand, this GeneDisc originally marketed in 6 sectors is now marketed in 6 and 12 sectors (GLEGOID206006 and GLEGOID212006). This modification leads to the need for a duplex PCR well: L. pneumophila (FAM) and L. pneumophila serogroup 1 (ROX).

Table 1 : Composition of reactional chambers of each sector, for GenenDISC plate Legionella ID (GLEGOID20600 and GLEOID212006)

The validation of the qualitative method concerns the following elements:

� LD and positivity threshold for the detection of Legionella spp., Legionella pneumophila and Legionella pneumophila serogroup 1.

� Linearity for the determination of the intercept for Legionella spp., Legionella

pneumophila and Legionella pneumophila serogroup 1.

� Inclusivity, exclusivity for the detection of Legionella pneumophila and Legionella pneumophila serogroup 1.

� LQ for Legionella spp and Legionella pneumophila


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Reference methods

NF T90-471 (juin 2015) : Qualité de l’eau - Détection et quantification des Legionella et/ou Legionella pneumophila par concentration et amplification génique par réaction de polymérisation en chaîne en temps réel (RT-PCR). ISO/TS 12869 (November 2012): Detection and quantification of Legionella spp. and/or Legionella pneumophila by concentration and genic amplification by quantitative polymerase chain reaction (qPCR) Validation PCR protocol: Validation protocol for commercial methods of detection and quantification of Legionella and Legionella pneumophila by concentration and gene amplification by polymerase chain reaction (PCR) V3.0

Study

Fitting the calibration and the reference material to the primary

standard

Data from previous study (2007 and 2011) Endetec la boratory

With the primary standard, preparation of 3 independent ranges of five level, prepared by “cascade dilution” in the solution used to analyze the PCR blank (diluent supplied by the CNR). The dilutions cover the linear domain of quantification. The same operation was carried out from the calibrating solution with the diluent supplied by Pall GeneDisc® Technologies. The 3 ranges of primary standard and calibrating solution were analyzed in the same PCR series.

The results obtained from the primary standard are shown in the table below

Q (UG/well) Log (Q) Ct obtained Ct average Range 1 Range2 Range3

25 1,40 31,334 32,323 31,6155 31,76 250 2,40 29,3485 28,6005 28,586 28,85 2 500 3,40 25,246 25,422 25,4885 25,39 25 000 4,40 21,8845 21,6405 21,5335 21,69 250 000 5,40 18,248 18,5385 18,2705 18,35


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The results obtained from the calibrating solution are shown in the table below

Q

(UG/well)

Log

(Q)

Ct obtained Average

Ct

Log (Q)

calculating

value

Calibration

error by

level

Conclusion

Range

1

Range

2

Range

3

25 1,40 31,55 31,61 31,62 31,59 1,52 -0,12 Accordance

250 2,40 28,61 28,55 28,39 28,52 2,42 -0,03 Accordance

2 500 3,40 25,47 25,08 24,54 25,03 3,45 -0,05 Accordance

25 000 4,40 21,73 21,47 21,41 21,53 4,48 -0,08 Accordance

250 000 5,40 18,23 18,17 18,35 18,25 5,44 -0,05 Accordance

Average calibration error -0,06 Accordance

Verification equivalence of slopes: Val. Absolute ( calibration error

log (250000) -log (25))

0,07 Accordance

For each level, the difference between the logarithm of the calculated value and that of the expected value is less than 0.15. The absolute value of the deviation difference between the high point and the low point of the range (calibration error) is less than 0.15 log. The results obtained are therefore consistent with those expected.

Study of the calibration function of the quantitative PCR step

Data from this study AdGène laboratory (2018).

The calibration function allows to define the PCR efficiency and to check the performances of the linear regression through the accuracy of linearity. Methodology

Test are followed under intermediate reproducibility conditions. Five range of 5 levels: 25, 250, 2 500, 25 000 et 250 000 genome unit of Legionella pneumophila by reaction tubes are prepared from DNA of a strain Legionella pneumophila WDCM 00107 (reference: ALEGPNE205). The first point of the range is equal to the limit of quantification, ie 25 genome units of Legionella pneumophila per reaction tube. The results are analyzed in order to calculate the slope of the calibration line and the accuracy of linearity.


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Results

GeneDisc Plate Legionella DUO (GLEGDUO206006)

Sum

level Xi LQ 10LQ 100LQ 1 000LQ 10 000LQ

25 250 2 500 25 000 250 000

X'i theorical =

log Xi

1,40 2,40 3,40 4,40 5,40

y'i,j

rep1 33,1 29,8 26,7 23,6 20,15

rep2 33,4 29,95 26,85 23,8 20,2

rep3 33,25 30 26,95 23,8 19,85

rep4 32,75 29,3 26,15 22,95 19,5

rep5 33,8 30,25 27 23,7 20,25

Ti=

166,3 149,3 133,65 117,85 99,95 500,75

Mi = 33,26 29,86 26,73 23,57 19,99

X'i Ti 232,477423 358,012443 454,134682 518,29723 539,524104 1869,96846

x'i,j

rep1 1,44 2,45 3,39 4,34 5,39

rep2 1,35 2,40 3,35 4,28 5,37

rep3 1,40 2,39 3,32 4,28 5,48

rep4 1,55 2,60 3,56 4,53 5,59

rep5 1,23 2,31 3,30 4,31 5,36

Average X'i 1,39428482 2,42992295 3,38331923 4,3458535 5,43631954

Distortion 0,00 0,03 -0,01 -0,05 0,04

Standard

deviation

0,11767556 0,10736861 0,10474413 0,10855029 0,09596091

Elin 0,11773232 0,11203092 0,10575963 0,12040004 0,10335127

Ulin 0,37462424 0,35648238 0,33652715 0,38311292 0,32886375

��, ��

��

��


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LOG (Q) Average Ct

1,40 33,26 2,40 29,86 Origine 37,84 3,40 26,73 4,40 23,57 Slope -3,28 5,40 19,99

Conclusion

Criteria of validation : Slope must be between –4,115 and –2,839 Elin must be minor than 0,15 log for each level Slope: - 3,28 conform Elin < 0,15 : every level have Elin < 0,15 conform GeneDisc Plate Legionella DUO (GLEGDUO212006)

Criteria result Conformity slope - - 4,115 et -

2,839 -3,28 �

Elin < 0,15 log every Elin <

0,15 �


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Limit of detection


Limit of detection of the PCR is estimated from the smallest number of genome units generating a positive result (amplification) at the confidence threshold of 90 %. LDPCR announced by the supplier to be verified is 5 genome units per well. Methodology

30 independent solutions of ADN is done from a DNA solution extracted from a strain Legionella pneumophila WDCM 00107 connected to the primary standard (reference: ALEGPNE205) are realized performed by successive dilutions to reach the limit of detection announced by the supplier.

Results

GeneDisc Plate DUO (GLEGDUO206006) Appendix 1: 100 % of presence

Conclusion

The detection limit announced by the supplier is compliant. Criteria of Validation: at least 90% of the solutions must be positive. All the tests give a usable Ct. Limit of detection announced by the supplier is conform .

Limit of quantification Data from this study AdGène laboratory (2018). The quantification limit is the first level of the range. LQPCR announced by the supplier is 25 units genomes by well. The verification of the limit of quantification is to ensure that the accuracy at the limit of quantification (denoted ELQ) is less than the critical value of 0.15 log.


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Methodology

30 independent dilutions at LQ value are prepared from a solution of Legionella pneumophila DNA connected to the primary standard (reference: ALEGPNE205). Every dilutions is amplified under intermediate reproducibility.

Results

Appendix 2

Conclusions

Criteria of validation: if E LQ ≤ 0,15, the targeted limit of quantification is veri fied and the LQPCR complies with the specifications of t he ISO guide.

Accuracy of LQ (ELQ) is 0,146: criteria conform


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Positivity threshold


The verification of the positivity threshold will be done during the validation limit of the detection limit step. The Ct values obtained during the validation of the LD are verified to be lower than the value of Ct defined by the manufacturer: 38 for the target Legionella spp. Validation criteria : positivity threshold <Ct defined GeneDisc Plate Legionella pneumophila The values found during the study of LD all have a Ct lower than the value of intercept 37.8 and the value defined by the manufacturer (38).

Determination of yield and robustness Data from previous study (2007 and 2011) Endetec la boratory The performance of « Extraction Pack Environment 1 » (PENVI1096) has been evaluated. Detection and amplification were performed with the "GeneDisc Legionella pneumophila 12 sectors" (Ref GLEGPNE112006).

Table 2 : Uncertainties related to direct lysis


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Table 3 : Pall GeneDisc Technologies Method Performance Study

The average yields obtained are greater than 25%. No inhibition was observed during these tests. All the calculated yields have values between 25% and 199% and the associated biases are between -0.6 and 0.3, according to the requirements of NF T90-471. The results are therefore in line with those expect ed.

Efficiency obtained with the simplified protocol for sanitary water

and "clean water" The performance of “Extraction Pack Environment 3 » (PENVI3100) was evaluated. The evaluation includes the use of the centrifugation unit (Nanosep 30K) or not. Detection and amplification were performed with the "GeneDisc Legionella DUO 06 sectors" (Ref GLEGDUO106006).

Table 4 : Results of direct lyses on the suspensions


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The yields by concentration levels and by water types are detailed in the table below.

The average yields obtained for Legionella pneumophila are greater than 25%. No inhibition was observed during these tests. All calculated returns have values between 25% and 199% (which corresponds to bias values between -0.6 and +0.3 log). The results are therefore in accordance with the re quirements of NF T90-471.

Inclusivity/Exclusivity

Data from previous study (2007 and 2011) Endetec la boratory Inclusivity tests were performed on DNA extracts to obtain about 100 GU per well. A total of 21 strains of Legionella spp. and 15 serogroups of L. pneumophila were tested. The exclusivity tests were performed on DNA extracts to obtain at least 10,000 UG per well. A total of 17 bacterial strains other than Legionella were tested. In conclusion, the DNAs of the 36 Legionella strains tested were all detected and among the 17 DNA extracts tested, belonging to genera other than Legionella, no cross reaction (lack of amplification) was detected. The selectivity of the GeneDisc® Legionella spp . is satisfactory.


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Praticability Data from previous study (2007 and 2011) Endetec la boratory

Criterion to be checked

Communication on the criterion

Method used to check the criterion

1

Mode of packaging of items for the method

Packaging and instructions

Extraction Packs: PENVI1096: 96 preparations PENVI3100: 100 preparations GeneDisc Plates Packs: 6 Plates = 30 samples for GeneDisc 6 sectors & 66 samples for GeneDisc 12 sectors Dehydrated DNA: 5 tubes calibrated at 250,000 GU

2 Volume of reagents Instructions Volume to be used is indicated in the

instructions

3 Conditions of storage of items (+ expiry of unopened items)

Packaging All reagents are stored at room temperature except the GeneDisc Plates (5°C ± 3°C) and Standard DNA (-20 °C ± 3°C)

4

Usability after first use. (in particular the existence of limiting dates)

Packaging and instructions

Reagents are used until end of stock in the limit of the expiration date

5 Specific equipment or premises needed

Instructions The list of material and consumable is indicated in the chapter 6 of the Instructions

6

Reagents ready to use or to be made up (in this case, existence of instructions for use)

Packaging or instructions

All reagents are ready-to-use except the “Binding Buffer” and the “Washing Buffer 2” of the PENVI1096

7

Time needed to train operator unfamiliar with the method

Report 2 days for a technician

8

Real handling time / Flexibility of the technique in relation to the number of samples to be analyzed, their bacterial load, etc.

Report See below

9 Time to obtain results

Report and certificate

See below


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10 Type of qualification of the operator

Report Stated by the expert laboratory (the expert laboratory may make use of data from collaborating laboratories)

11 If available, traceability of analysis results

Instructions Verification by the expert laboratory

12 Maintenance by the laboratory

Report See below

13 Minimum pipetting volume

Report 20 µL

14 Stability of reagents and ranges

Packaging Expiration date and storage condition are indicated on the packaging for all reagents. Aliquoting is not required.

15 UNG treatment (prevention of contamination)

Report No UNG treatment are GeneDisc Plates are closed PCR devices

16 Protection of reagents from UV

Report The reagents are kept in their original packaging (opaque to light)

17 External quantitative check of PCR

Report This control is included in sectors 6 and 12 of the GeneDisc Plates 6 and 12 sectors, respectively

18

Check for absence of inhibitor

Report This control is included in each sector of the GeneDisc Plates. It corresponds to a synthetic oligonucleotide calibrated and amplified with the same primers as the Legionella spp. target

� Criteria 8 & 9:

o Handling time:

STEPS TIME FOR PROCESSING 8 SAMPLES

Filtration Between 5 and 30 min, depending of the type of water

DNA Extraction

PENVI1096

1h15

PENVI3100

30 min

PCR 15 min for preparation / PCR run: 55 min Analysis of the results 10 min


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o Flexibility

The analysis could be stopped after the filtration step and/or the DNA extraction. The filter is transferred in the lysis tube and this one is stored at - 20°C ± 3°C. After the DNA extraction, the DNA extract is stored at - 20°C ± 3°C if PCR analysis is not realized within 6 h after DNA extraction. That allows optimizing the analytical workflow.

o Time to obtain results:

2h15 for 5 samples (GeneDisc DUO Legionella pneumophila – spp.),

2h15 for 11 samples with the GeneDisc Packs 12 sectors (Pack Legionella pneumophila)

3h15 for 10 samples with the GeneDisc Packs 6 sectors (Pack Legionella pneumophila).

The result can be delivered at J0.

In case of detection of PCR inhibitors, the time-to-result is delayed for 1h30.

� Criterion 12

No maintenance is performed by the laboratory.

An annual maintenance is realized by Pall GeneDisc Technologies: thermal and optical metrology and biological validation.


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INTER-LABORATORY STUDY Data from previous study (2007 and 2011) Endetec la boratory The inter-laboratory study was conducted in 2007 with 16 participating laboratories. Two laboratories have not been able to give the expected results. The purpose of this study is to assess the precision (repeatability and reproducibility) of the GeneDisc Legionella method concerning:

- the genetic amplification step alone;

- the overall analysis (concentration, lysis, extraction, purification and genetic amplification) on characterized bacterial suspensions;

- the whole analysis in real situation (naturally contaminated hot sanitary water samples).

The results are reported in the following table.

Type of samples

Calibrated DNA solution

Spiked Tap water

Natural sample

rSpiking levels (GU/L)

L. pneumophila WDCM 00107

7,600 94,000 660 7,000 Naturally contaminated hot tap water L. parisiensis CIP

103847 8,800 85,000 1,800 16,000

E. coli 110 1,400 Number of laboratories

participant 16 16 16 16 16 retained 14 14 15 14 11

Homogeneity Test

Number of analyses

20 20 7 11 10

Average (Log) 5.470 6.449 4.490 5.792 5.173

Results

Average (Log) 5.253 6.241 4.667 5.666 5.013 Bias (Log) 0.217 0.208 0.183 0.126 0.160 Sr (Log) 0.051 0.035 0.113 0.084 0.098 SR (Log) 0.130 0.104 0.445 0.440 0.425 Er (√bias2 + Sr2) 0.223 0.211 0.215 0.151 0.188 ER (√bias2 + SR2) 0.253 0.233 0.481 0.458 0.454 ETotal (√bias 2 + Sr2 + SR2 )

0.258 0.236 0.494 0.465 0.465


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In terms of repeatability (r), the standard deviation observed is 0.06 for the PCR step (calibrated DNA solutions), 0.12 for the entire method (preparation of DNA & PCR) with artificially contaminated water samples and 0.16 for natural samples. The GeneDisc Legionella spp. method is repeatable.

The reproducibility (R) standard deviations express the samples degree of complexity: those obtained for the PCR analysis of calibrated DNA solutions are 0.13 whilst those corresponding to all of the analysis steps (preparation of DNA & PCR) are between 0.40 and 0.50.

Conclusion These data are compliant with the performances noti fied by the supplier.

General conclusions The results of the preliminary study as well as the inter-laboratory study show that the performances of the GeneDisc® method for the detection of Legionella pneumophila comply with the requirements of NF T 90-471. The validation of the GeneDisc method was renewed on December 17, 2015 under the certificate number GEN 25 / 04-12 / 07 by AFNOR Validation. The GeneDisc Legionella pneumophila method is applicable to any type of water, without restriction of use. The identification by GeneDisc Legionella ID is consistent, it allows detection and differentiation of Legionella spp, Legionella pneumophila and Legionella pneumophila serogroup 1.


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Appendix 1: GeneDisc Plate DUO (GLEGDUO206006)

Limite of detection

Ct LD (duplicat)

Average Ct LD

35,4 35,6 35,8 35 35,25

35,5 34,9 35,25 35,6 35,7 35,8 35,9 36 36,15

36,3 35 35,15

35,3 35,2 35,35 35,5 35 34,8

34,6 35,1 34,95 34,8 34,7 34,95 35,2 35,6 36,1 36,6 35,7 35,4 35,1 36,5 35,5 34,5 35,7 35,6 35,5 35,8 35,7 35,6 34,9 34,8 34,7 34,6 34,65 34,7

34,5 34,6 34,7 35,5 35,9 36,3 35 34,6

34,2 35,2 35,6 36

35,6 36,05 36,5 36,7 36,25 35,8 36,4 36,4

0 35,9 35,05 34,2 34,3 34,7 35,1 35,2 35,35 35,5 35,4 35,5 35,6 35,6 35,45 35,3 35,5 35,15 34,8

Total 30/30

100%


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Appendix 2: Limit of quantification

CT LQ (duplicat)

CT LQ (average) log

33,10 33,1 1,44

33,10

32,20 32,3 1,69 32,40

33,70 33,4 1,35 33,10

32,90 32,8 1,53 32,70

33,30 33,25 1,40 33,20

32,60 32,75 1,55 32,90

33,70 33,8 1,23 33,90

32,70 32,6 1,60 32,50

34,00 34,05 1,15 34,10

33,20 33,55 1,31 33,90

33,80 33,65 1,28 33,50

34,10 33,8 1,23 33,50

33,80 33,8 1,23 33,80

32,70 33,2 1,41 33,70

33,00 32,85 1,52 32,70

33,50 33,35 1,37 33,20

33,10 33,3 1,38 33,50

32,60 32,25 1,70 31,90

33,4 33,1 1,44


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32,8

33,4 33,5 1,32 33,6

33,1 33,25 1,40 33,4

32,5 32,85 1,52 33,2

33,8 33,8 1,23 33,8

33,00 33,2 1,41 33,40

33,30 33,25 1,40 33,20

33,20 33,1 1,44 33,00

33,00 33,1 1,44 33,20

33,10 33 1,47 32,90

32,5 32,55 1,61 32,6

32,3 32,35 1,67 32,4

moyenne X'i 1,42

Biais 0,03

écart-type 0,1444113

Elin 0,1467861 Ulin 0,46707338

Documents

Extension for detection and quantification of Legionella spp. · GeneDisc® Legionella spp. and Legionella pneumophila are detection and quantification system for Legionella spp