NF Validation Validation of alternative‘s analysis method ... · Version 1.1 14 september 2016 Cancels and replaces the previous ... reactional volume of heating block, user interface

AdGène laboratoire 1 rue des conquérants – 14220 Thury-Harcourt – www.adgene.fr

Email : [email protected] / tel : 02 31 15 62 80 / fax : 02 31 15 62 85

BIO-RAD 3 boulevard Raymond Poincaré 92430 MARNES LA COQUETTE

NF Validation

Validation of alternative‘s analysis method for Legionella spp

detection and quantification

Renewal study for iQ-Check® Legionella spp. for the detection and enumeration of

Legionella in sanitary hot water and cooling towers

N° agreement BRD 07/15 – 12/07

Summary Report

This study include 15 pages whose 0 annexe. Reproduction of this report is only authorized in its integral form.

Version 1.1

14 september 2016 Cancels and replaces the previous version The old version must be destroyed.

http://www.adgene.fr/

mailto:[email protected]

2 AdGène laboratoire iQ-Check® Legionella spp. summary report V1.1

Summary

Reminder of the method of analysis ....................................................................................................... 3

Date of first attribution of NF Validation trade mark .......................................................................... 3

Method principle ................................................................................................................................. 3

Protocol references: ............................................................................................................................ 4

Application domain ............................................................................................................................. 4

Restrictions .......................................................................................................................................... 4

Methods of reference ......................................................................................................................... 4

Validation history .................................................................................................................................... 5

Summary of results.................................................................................................................................. 7

Linking of calibration solution and reference material to primary standard ...................................... 7

Calibration function ............................................................................................................................. 8

Validation of limit of detection ........................................................................................................... 8

Validation of limit of quantification .................................................................................................. 10

Study of efficiency and robustness of extraction .............................................................................. 11

Selectivity: inclusivity and exclusivity ................................................................................................ 12

Inclusivity ....................................................................................................................................... 12

Exclusivity ...................................................................................................................................... 12

Practicability ...................................................................................................................................... 12

Ease of use: .................................................................................................................................... 12

Fast results report: ........................................................................................................................ 12

Results security: ............................................................................................................................. 12

Inter-laboratories study .................................................................................................................... 13

Bibliography study ................................................................................................................................. 14

Overview of customer reclamation ....................................................................................................... 14

Conclusions ............................................................................................................................................ 15


Reminder of the method of analysis

Date of first attribution of NF Validation trade mark iQ Check® Legionella spp. and iQ Check® Legionella pneumophila kits was validated to allow

utilization of trademark NF VALIDATION in 2007. Authorization was renewed in 2011, follows

by two extensions in 2012 (see chapter Validation History).

Method principle iQ Check® Legionella spp kit is intended to detect or to quantify bacteria genius Legionella in

water sample, due to Polymerase Chain Reaction (PCR). PCR allows amplification and

detection of specific sequences with specific primers and fluorescent probe.

Principle is based on three steps (see Annex 1):

- Sample filtration

- DNA extraction with Aquadien kit (and protocol W2 for clogging samples).

- Legionella spp target sequences amplification.

DNA extraction with Aquadien kit is based on alkaline lysis wih thermal choc. It is followed by

an ultrafiltration purification step. A DNA fraction is amplified by real-time PCR.

Primers hybridize to target sequence during PCR reaction. Taq polymerase use primers and

triphosphates desoxynucleotides (dNTPs) to stretch DNA and to create copies of Legionella

spp target DNA.

Specific probe hybridize to amplicons during PCR. This probe is labelled with a fluorophore

which emit fluorescence only after hybridization. Fluorescence intensity increases

proportionally with increasing of PCR products.

Fluorescence is directly measured by optical machinery of the thermocycler during

hybridization step. Thermocycler software cast in real-time the measured fluorescence function

of number of amplification cycles. Software determines a Ct (cycle from which fluorescence is

higher than background signal). Reading Ct permits to detect presence of Legionella spp target

sequences. Detection of target sequences indicates presence of the bacteria in analysed water

sample.

Quantification is possible by using calibrated DNA solutions calibré iQ Check® Legionella

Quantification Standards. These standards are connected to primary standard of Centre

National de Référence des Légionelles.

PCR inhibition phenomenon is detected by utilisation of a synthetic DNA (internal control –

IPC) included in amplification solution with each sample. IPC is amplified during same time

than target sequences, with same primers but with a different probe and a different fluorphore.


iQ Check® Legionella spp. kits are validated with following materials:

Software Opticon Monitor 3.4

CFX manager Software Industrial Diagnostic Edition v2.2

CFX manager Software Industrial Diagnostic Edition V2.2

Chassis PTC-200 C1000 C1000 touch Optical Head / heating block Chromo4 CFX96 CFX96 Deep

well touch

Protocol references: Aquadien™ (Réf. 3578121) : 10/2013 – Code : 881116

iQ-Check® Legionella spp (Réf. 3578102) : 10/2013 – Code : 881117

Application domain Samples of all types of water.

Restrictions Certification of the kit deals with it utilization with thermocyclers Bio-Rad Chromo™4 and

CFX96 Deep well Touch.

Methods of reference 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) : Water quality -- Detection and quantification of Legionella

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

polymerase chain reaction (qPCR)

PCR Validation protocol:

Protocole de validation pour les méthodes commerciales de détection et de dénombrement

de Legionella et Legionella pneumophila par concentration et amplification génique par

réaction de polymérisation en chaîne (PCR) V3.0


Validation history

Certified method Certificate Date

approval Object Commentaries

Expert laboratory

PCR validation protocol

iQ-check Legionella spp.

BRD 07/15-12/07

18/12/2007 Validation initial IPL SED

Nord Rev. 0 (2006)

10/06/2011 Renewal 1

Mix PCR evolution 2 extractions method

(protocol W2) Update according to protocol version 1

IPL SED Nord

Rev. 1 (2011)

04/04/2012 Extension 1 New thermocycler (Deep Well touch)

Eurofins IPL Nord

Rev. 1 (2011)

27/05/2013 Extension 2 Validation protocol

V.2 NA

Rev. 2 (2013)

05/11/2013 Modification Software V2.1 NA Rev. 2 (2013)

09/03/2015 Modification Software V2.2 NA Rev. 2 (2013)

18/12/2015 Renewal 2

AdGène with

extension on

qualitative test

Rev 3 (2015)

2007:

Method was validated initially in 2007.

2011:

2010/211 study for renewal of validation considered the modifications of validated kit and

of validation protocol (renewal n°1considering norm NT T90-471 published in April 2010).

A third party study has focused on two first phases of validation protocol aiming to verify

supplier announced performances for new formulation of iQ-Check® L. spp kit:

Phase 1: Study of limit of detection and limit of quantification of PCR step, calibrating

function, link to primary standard, efficiency and robustnessof extraction with Aquadien™

kit. New thermocycler CFX 96 was implemented.

Phase 2: Study of inclusivity and of exclusivity, of practicability and of reagents quality.

Interlaboratory study realised in 2007 was not made again

New modification from initial validation was :

- iQ-Check® L. spp kit: New origin of Taq polymerase and chemical evolution of IPC

probe (TEXAS RED fluorophore was replaced by HEX fluorophore)

- Aquadien Kit: two modalities of utilization according to sample filterability (protocol

W2 for clogging samples added to classical protocol) and horizontal double- tangential microfiltration for DNA purification step. Membranes and materials

composition do not change.

New thermocycler can be used: CFX96 with CFX Manager Software Industrial

Diagnostic Edition version V1.1.


2012:

Validation extension was pronounced in 2012 after evolution of characteristics of

thermocycler CFX96 which becomes CFX96 Deep Well Touch. Modifications concern

reactional volume of heating block, user interface (keyboard and screen), and software

CFX Manager which pass in version V1.2

AFNOR Certification Technical office qualified theses evolutions as minority and without

impact on kit performance. No new assays were performed.

2013:

Late May 2013: Validation of iQ-Check® L. spp method was extended to norm ISO/TS

12869. No study complement was necessary: Assays performed according to norm NF

T90-471 answers to requirements of ISO/TS 12869 and follow migration to revision 2 of

validation protocol.

November 2013: Evolution of software CFX manager IDE v2.1. No study complement

was necessary.

2015:

March 2015: Evolution of software CFX manager IDE v2.2. No study complement was

necessary.

October 2015: Renewal of iQ-Check® L. spp method with extension on detection

(qualitative research) of Legionella spp without supplementary test. AFNOR Certification

Technical office qualified this evolution without impact on kit performance. No new

assays were performed.


Summary of results Next results were obtained with revision V1.0 of validation protocol for commercial method of

detection and quantification of Legionella and Legionella pneumophila by concentration and

gene amplification by Polymerase Chain Reaction (PCR). Results are conforms with version

V2.0 because it contains no major modification causing complementary study.

Linking of calibration solution and reference material to primary standard

Methodology

Linking of working calibration solution to primary standard is made to cover the quantification

domain with 3 ranges of calibrated DNA iQ-Check® Legionella spp. which contain 4 levels of

concentrations of Genome Unity of Legionella pneumophila serogroup (QS1, QS2, QS3, QS4)

and 3 independent ranges of primary standard aiming at the 4 levels of concentrations of range

of calibrated DNA iQ-Check® Legionella Quantification Standards.

Linking of reference material to primary standard is evaluated analysing results of 2 deposits

of reference material given with iQ-Check® Legionella spp. kit.

Results

Analysed parameters for evaluation of linking of calibration solution and of reference material

to primary standard on thermocycler CFX96 and chromo 4 are submitted in next table:

Regression curve Correlation Efficiency (%)

Reference range (CFX96) C(t) average = -3.198.log(x) + 39.076 0.998 105.5

Reference range (Chromo 4) C(t) average = -2.891.log(x) + 38.674 0.995 121.75

Calibration solution Calibration error

QS1 QS2 QS3 QS4

Per level (CFX96) 0.07 0.20 0.14 0.07

Per level (Chromo 4) 0.03 0.30 0.23 0.16

Average (CFX96) 0.12

Average (Chromo 4) 0.18*

Slopes equivalence (CFX96) 0.00

Slopes equivalence (Chromo 4) 0.13

Reference material Calibration error

CFX96 0.19

Chromo 4 0.19

* Calibration error of calibration solution is 0.18log with thermocycler Chromo4. However, equivalence of slopes from reference range and calibration solution range is verified.

Calibration error of calibration solution is lower than 0.15log. Slopes from reference range and calibration solution range are equivalent. Conclusion


Calibration solution and reference material of iQ-Check® Legionella spp kit satisfy conditions

of linking to primary standard with thermocycler CFX96.

Calibration solution globally satisfies conditions of linking to primary standard with

thermocycler Chromo 4. Reference material of iQ-Check® Legionella spp kit satisfies

conditions of linking to primary standard with thermocycler Chromo 4.

Calibration function

Methodology

Study of calibration function is made deposit 5 different reference ranges of calibrated DNA

solution iQ-Check® Legionella Quantification Standards (comprising 4 levels of concentration

of Genome Unity of Legionella pneumophila), given with iQ-Check® Legionella spp. kit.

5 measures are made with iQ-Check® Legionella spp kit for each level of concentration in

reproducibility conditions.

Results

Equation of regression curve and efficiency of PCR reaction are defined in these conditions.

Results are obtained on CFX96.

Regression curve -3.197.log(x) + 41.347

Efficiency 105.5%

Performances of linear regression:

QS1 QS2 QS3 QS4

Bias 0.06 -0.10 0.00 0.04

Standard deviation 0.12 0.06 0.08 0.05

Exactitude of linearity 0.13 0.12 0.08 0.07

uncertainty of linearity 0.42 0.37 0.27 0.22

Conclusion

Linear regression satisfies exigence of exactitude lower than 0.15log for each level of reference

range. Linearity is verified on the whole domain cover by the range of calibrated DNA solution

iQ-Check® Legionella Quantification Standards given with iQ-Check® Legionella spp. kit.

Validation of limit of detection

Methodology


Evaluation of limit of detection is made from 30 independent dilutions of Legionella

pneumophila DNA in concentration of 5UG per PCR reaction. Duplicate amplifications are

made in repeatability conditions. Results are obtained on CFX96.

Results

Conclusion

The 30 duplicate are positives. Limit of detection is validated for 5 UG per PCR reaction.

All Ct in previous table are lower than intercept. Qualitative detection is conforming.


Validation of limit of quantification

Methodology

Evaluation of limit of quantification is made from 30 independent dilutions of Legionella

pneumophila DNA in concentration of 15UG per PCR reaction. Duplicate amplifications are

made in repeatability conditions. Results are obtained on CFX96.

Results

Results Theoretical values or validation criteria

Average x’ (Log UG/reaction) 1.309 1.279 Standard deviation (Log UG/ reaction)

0.097

Bias 0.030 LQ Exactitude 0.101 0.15 LQ Uncertainty 0.207

Conclusion

Value of exactitude of limit of quantification is estimated at 0.101. This value is lower than 0.15.

Limit of quantification is validated for 15 UG per PCR reaction for iQ-Check®Legionella spp.

kit.

Positivity threshold

User manual foresees a Ct of 43 hereafter whose samples are considered as lower than the

limit of detection

All values for characterisation of limit of detection have Ct lower than 43. This value

corresponds to the positivity threshold lower than limit of detection.


Study of efficiency and robustness of extraction

Methodology

Studies of extraction efficiency were realized with extraction kit Aquadien™ (for clean waters)

and Aquadien W2 (for clogging waters). Efficiency was evaluated on 10 independent samples,

which were artificially contaminated with two levels of concentrations of Legionella

pneumophila ATCC 33152 (1000 and 100 000 UG / PCR reaction). Samples were 3 different

matrixes: sterile water, domestic hot water and water from air cooling-tower.

Samples were artificially contaminated by primary bacterial suspension. It concentration was

determined by 3 quantifications after an extraction step of DNA by direct lysis on 3 aliquots.

Results are obtained on CFX96.

Results

Extraction efficiency

Aquadien Protocol Aquadien W2 Protocol

Log Average Log Average

Domestic hot water 1000 UG/L 100 000 UG/L

-0.29 -0.39

-0.34 -0.16 -0.45

- 0.30

Water from air cooling-tower

1000 UG/L 100 000 UG/L

-0.09 -0.31

-0.20 -0.45 -0.47

- 0.46

Mineral water 1000 UG/L 100 000 UG/L

-0.25 -0.42

-0.33 -0.55 -0.46

- 0.50

Average efficiency (log) -0.29 -0.41 Variance (log) 0.04 0.03 Global extended uncertainty (log) 0.71 0.89

Conclusion

Study of efficiency and robustness of extraction method allows evaluating average efficiency

of:

- Aquadien method: -0.29log

- Aquadien W2 method: -0.41log

Efficiencies with two extraction methods are conforming to criteria -0.6 log / +0.3 log

(equivalent to efficiency comprise between 25% and 199%).


Selectivity: inclusivity and exclusivity DNA was extracted from pure bacterial suspension for each strain.

Inclusivity

Inclusivity assays were realized on DNA extracts with concentration about 100 UG/ PCR

reaction. Concentrations were estimated by O.D.600nm of bacterial suspension.

DNA of 35 strains of tested Legionella (15 Legionella pneumophila et 20 Legionella spp) were

amplified.

Exclusivity

Exclusivity assays were realized on DNA extracts with concentration about 10 000 UG/ PCR

reaction. Concentrations were estimated by O.D.600nm of bacterial suspension.

DNA of 16 strains of tested were not amplified, except 5 of them which show weak

amplification.

Conclusion

iQ-Check® Legionella spp kit selectivity is satisfying.

Practicability

Ease of use: reagents are all supplied with kits and are ready-to-use. Serial analyses from 1

to 30 samples, for quantification, are easy to make. A technician, who knows microbiology and

molecular biology techniques and the specific thermocycler and it software, can be trained in

1 day.

Fast results report: duration of different phases is compatible with a short results report

(5hours).

Results security: It guarantees by utilization of inhibition internal control (in same reaction well

than sample) and by a software of results analysis. Use of software ensures traceability of

complete information.


Inter-laboratories study

Methodology

Inter-laboratories study was realized in 2007 with 14 collaborating laboratories. Results of one

laboratory were not taken into account because of technical problem which invalidated

standardization. 13 laboratories were retained for statistical exploitation.

Goal of this study is to evaluate fidelity (repeatability and reproducibility) of iQ-Check®

Legionella spp. method:

- For only amplification step (2 DNA solutions of L. anisa et L. pneumophila sg1 at 2

different levels of concentration).

- For complete analysis (concentration, lysis, extraction, purification and gene

amplification) on characterized bacterial suspensions of L. pneumophila and

Escherichia coli (CIP 54.8) at 2 different levels of concentration).

- For whole analysis in real situation (hot domestic water naturally contaminated by

L. pneumophila and Legionella spp).

- For a water guarantees without any DNA of Legionella.

Results

Sample types Calibrated DNA

solutions

Contaminated hot

domestic water Natural water

Contamination

levels

(UG/L)

L. pneumophila

ATCC 33152

2000

UG/µl

20000

UG/µl

4000

UG/200

ml

40000

UG/200

ml

Hot domestic

water naturally

contaminated

L. anisa 500

UG/µl

5000

UG/µl

1000

UG/200

ml

10000

UG/200

ml

E. coli

5000

UG/200

ml

50000

UG/200

ml

Number of

laboratories

participating 14 14 14 14 14

retain 13 13 13 13 13

Homogeneity

assay

Analysis number 20 20 9 9 9

Average (Log) 2.91 3.97 3.42 4.41 3.76

Results

Average (Log) 3.02 4.11 3.52 4.47 3.69

r (Log) 0.18 0.15 0.28 0.34 0.46

R (Log) 0.43 0.32 0.72 0.66 0.8

Sr (Log) 0.06 0.06 0.10 0.12 0.16

SR (Log) 0.14 0.10 0.24 0.20 0.23


Conclusion

Repeatability values in r (log) are about 0.15 for DNA solutions (only PCR step) and about 0.7

for bacterial suspensions (global method). This is acceptable. Signification of these results is

that we can wait for factor 2 measurement of deviation in a same laboratory. Repeatability is

not a major source of error.

Reproducibility values in R (log) are about 0.4 for DNA solutions (only PCR step) and about

0.7 for bacterial suspensions (global method). Compared to repeatability, this order of

magnitude is equivalent to values that we can obtain for environmental microbiology analyses.

Signification of these results is that we can wait for factor 5 of measurement deviation between

2 different laboratories. Reproducibility does not participate in a unreasonable way to result

dispersion.

Bibliography study

- No new publication found since the last method publication

- No external validation realized by other certification organism.

Overview of customer reclamation

No customer reclamation.


Conclusions

Performances of iQ-Check® Legionella spp. method are conforming to requirement of norms NF T90-471 and ISO/TS 12869, and of AFNOR validation protocol: “Protocole de validation pour les méthodes commerciales de détection et de dénombrement de Legionella et Legionella pneumophila par concentration et amplification génique par réaction de polymérisation en chaîne (PCR) V3.0”. Modifications between versions 2.0 and 3.0 of AFNOR validation protocol concern positivity threshold (qualitative detection) and do not need complementary study. iQ-Check® Legionella spp kit is a kit validated for detection and for Detection and Quantification of Legionella and/or Legionella pneumophila by concentration and gene amplification by real-time Polymerase Chain Reaction (qPCR).

IPL santé, environnement durables Nord – 1 rue du Professeur Calmette – 59046 LILLE cedex

iQ-Check™ Quanti Legionella spp. - Ext 2011 - v01

ANNEXE C

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ANNEXE D

FONCTION DE CALIBRAGE

Fonction de calibrage

Niveau (UG/puits) x i 19 390 3900 39000 19 390 3900 39000x' i =Log(x i ) 1,28 2,59 3,59 4,59 1,28 2,59 3,59 4,59

gamme gamme 1 37,08 33,33 29,60 26,49yij 36,49 33,27 29,84 26,72

gamme 2 37,02 33,18 29,44 26,36k=5 répétitions 36,77 33,33 29,71 26,24

gamme 3 37,78 33,30 29,69 26,5436,69 33,31 29,99 26,46

gamme 4 37,32 33,81 30,14 26,6537,16 33,32 30,32 26,84

gamme 5 37,40 33,57 30,01 26,5236,90 33,41 30,05 26,50

Moyenne mi 37,06 33,38 29,88 26,53 37,06 33,38 29,88 26,53

Estimation de la droite de régression

Pente a = -3,197Ordonnée à l'origine b = 41,347

Estimation de l'efficacité

Efficacité e = 105,5%

Vérification des performances de la régression linéaire

Niveau x i 19 390 3900 39000 19 390 3900 39000x' i =Log(x i ) 1,28 2,59 3,59 4,59 1,28 2,59 3,59 4,59

gamme gamme 1 1,33 2,51 3,67 4,65yij 1,52 2,53 3,60 4,57

gamme 2 1,35 2,55 3,72 4,69k=5 répétitions 1,43 2,51 3,64 4,73

gamme 3 1,12 2,52 3,65 4,631,46 2,51 3,55 4,66

gamme 4 1,26 2,36 3,51 4,601,31 2,51 3,45 4,54

gamme 5 1,23 2,43 3,55 4,641,39 2,48 3,53 4,64

Moyenne mi 1,34 2,49 3,59 4,63 1,34 2,49 3,59 4,63

Biais 0,06 -0,10 0,00 0,04

Ecart type S = 0,12 0,06 0,08 0,05

Exactitude de linéarité ELIN = 0,13 0,12 0,08 0,07

Incertitude de linéarité ULIN = 0,42 0,37 0,27 0,22

1,31 2,46 3,54 4,64

1,28 2,43 3,48 4,57

1,29 2,52 3,60 4,64

1,39 2,53 3,68 4,71

1,43 2,52 3,64 4,61

37,15 33,49 30,03 26,51

37,24 33,57 30,23 26,75

37,24 33,31 29,84 26,50

36,90 33,26 29,58 26,30

36,79 33,30 29,72 26,61

Droite de calibrage

20

22

24

26

28

30

32

34

36

38

40

0,00 1,00 2,00 3,00 4,00 5,00x'i

C(t)

gamme 1

gamme 2

gamme 3

gamme 4

gamme 5





ANNEXE E

LIMITE DE DETECTION

Limite de détection à 5UG

Echantillons à la concentration 5UG

Well Sample C(t) I.C. C(t) SQe1 37,88 33,12 6,445

A02 e1 37,73 33,12 7,232B02 e2 39,38 36,36 1,982C02 e2 38,41 34,8 4,257D02 e3 37,65 34,56 7,690E02 e3 38,19 34,14 5,046F02 e4 37,92 34,09 6,237G02 e4 38,12 34,01 5,328H02 e5 38,25 32,97 4,826A03 e5 37,77 33,71 7,028B03 e6 38,99 35,71 2,685C03 e6 38,34 34,12 4,496D03 e7 38,1 34,39 5,432E03 e7 38,02 34,24 5,775F03 e8 39,21 34,53 2,268G03 e8 37,86 34,21 6,526H03 e9 37,39 32,78 9,424A04 e9 37,82 33,79 6,734B04 e10 41,57 39,12 0,356C04 e10 37,97 34,42 5,989D04 e11 38,9 34,49 2,887E04 e11 38,13 34,04 5,271F04 e12 38,93 34,35 2,816G04 e12 37,85 34,08 6,557H04 e13 37,15 32,87 11,400A05 e13 38,08 34,53 5,489B05 e14 38,19 34,68 5,041C05 e14 38,56 34,31 3,786D05 e15 37,91 34,44 6,301E05 e15 38,43 34,93 4,193F05 e16 38,04 34,35 5,667G05 e16 37,42 34,07 9,195 Contrôle Gamme StandardH05 e17 37,32 32,73 9,950A06 e17 38,47 35,11 4,058 Well Content C(t) I.C. C(t) SQB06 e18 40,81 37,79 0,645 A01 QS1 36,02 33,04 19,00C06 e18 38,08 34,36 5,515 B01 QS1 36,06 33,22 19,00D06 e19 37,78 34,27 6,964 C01 QS2 34,01 35,6 390,00E06 e19 37,9 34,15 6,307 D01 QS2 42,26 N/A 390,00F06 e20 38,16 34,63 5,147 E01 QS3 29,99 34,11 3900,00G06 e20 39,2 34,48 2,280 F01 QS3 29,72 33,4 3900,00H06 e21 37,28 33,31 10,260 G01 QS4 26,4 33,75 39000,00A07 e21 41,82 35,38 0,293 H01 QS4 26,37 33,98 39000,00B07 e22 39,31 35,69 2,096C07 e22 37,61 33,71 7,956D07 e23 37,95 34,28 6,091 Contrôle négatifE07 e23 38,17 34,39 5,128F07 e24 38,14 34,38 5,261 Content C(t) I.C. C(t) SQG07 e24 37,92 34,65 6,214 G09 Neg Ctrl N/A 34,62 N/AH07 e25 37,33 33,41 9,875 H09 Neg Ctrl N/A 34,39 N/AA08 e25 37,47 33,94 8,870B08 e26 37,84 34,55 6,629C08 e26 37,97 34,48 5,997D08 e27 38,04 34,37 5,654E08 e27 37,53 34,6 8,487F08 e28 38,98 34,64 2,712G08 e28 38,15 34,36 5,218H08 e29 37,74 33,16 7,195A09 e29 41,52 39 0,372B09 e30 38,16 34,32 5,146C09 e30 38,22 34,72 4,908D09





ANNEXE F

LIMITE DE QUANTIFICATION

Limite de quantification LQ à 15UG

Gamme de calibrage QS

UG/puits Moy Log (UG/puits) C(t)

QS1 1,278753601 36,87

1,278753601 37,23

QS2 2,591064607 33,65

2,591064607 33,71

QS3 3,591064607 29,73 Pente -3,241

3,591064607 29,87 Ordonnée origine 41,514

QS4 4,591064607 26,41 Corrélation (r2) 0,992

4,591064607 26,51 Efficacité (%) 103,474

LQPCR à 15UG : 30 mesures en réplicat

Réplicat Moyenne UG/puits Moy UG/puits x' (Log) Moyenne x'LQ-1 37,55 16,7 1,223

37,87 13,4 1,124LQ-2 37,57 16,5 1,217

37,27 20,4 1,309LQ-3 37,17 22,0 1,340

37,08 23,3 1,368LQ-4 37,21 21,4 1,328

37,49 17,5 1,241LQ-5 37,53 17,0 1,229

37,63 15,8 1,198LQ-6 37,73 14,7 1,167

37,21 21,3 1,328LQ-7 36,83 27,9 1,445

37,00 24,8 1,393LQ-8 37,07 23,5 1,371

37,21 21,3 1,328LQ-9 37,73 14,8 1,167

37,68 15,2 1,183LQ-10 37,82 13,8 1,140

38,15 11,0 1,038LQ-11 37,25 20,7 1,315

37,09 23,1 1,365LQ-12 36,54 34,4 1,534

37,06 23,7 1,374LQ-13 37,35 19,3 1,285

37,26 20,5 1,312LQ-14 37,56 16,7 1,220

37,57 16,4 1,217LQ-15 37,50 17,3 1,238

37,04 24,0 1,380LQ-16 36,48 35,7 1,553

37,37 19,0 1,278LQ-17 37,13 22,5 1,352

37,43 18,3 1,260LQ-18 37,39 18,7 1,272

37,65 15,6 1,192LQ-19 36,69 30,8 1,488

37,00 24,7 1,393LQ-20 36,57 33,5 1,525

37,13 22,5 1,352LQ-21 37,70 15,0 1,177

37,18 21,8 1,337LQ-22 37,75 14,5 1,161

37,68 15,2 1,183LQ-23 37,29 20,2 1,303

37,25 20,7 1,315LQ-24 37,00 24,7 1,393

36,94 25,8 1,411LQ-25 37,29 20,1 1,303

37,62 15,9 1,201LQ-26 37,55 16,7 1,223

36,63 32,1 1,507LQ-27 36,54 34,3 1,534

37,02 24,4 1,386LQ-28 36,83 28,0 1,445

36,99 24,9 1,396LQ-29 37,58 16,4 1,214

37,15 22,3 1,346LQ-30 37,12 22,8 1,356

37,34 19,5 1,288

1,309

0,097

0,030

0,101

0,207

37,37

37,23

37,46

37,09

36,78

36,91

37,44

37,72

37,27

36,97

37,28

37,52

36,85

36,85

37,31

37,57

37,27

36,93

37,7

37,99

37,17

36,8

37,58

37,47

36,91

37,14

37,71

37,42

37,13

37,35

C(t) UG/puits

Biais

Incertitude ULQ

Ecart-type s

Moyenne x'

Exactitude de LQ ELQ

1,50E+01

1,84E+01

2,26E+01

2,63E+01

1,94E+01

1,64E+01

2,19E+01

1,50E+01

1,24E+01

1,80E+01

1,99E+01

2,80E+01

2,77E+01

1,72E+01

2,04E+01

2,73E+01

2,07E+01

1,66E+01

1,49E+01

2,44E+01

2,04E+01

2,52E+01

2,11E+01

2,94E+01

2,64E+01

1,285

1,214

1,248

1,93E+01

1,80E+01

1,84E+01

2,91E+01

1,173

1,263

1,354

1,419

1,349

1,175

1,089

2,24E+01

39000

3900

390

19

1,340

1,454

1,298

1,218

1,309

1,416

1,306

1,232

1,440

1,439

1,257

1,172

1,309

1,402

1,252

1,365

1,460

1,420

1,280

1,322





ANNEXE G

RENDEMENT ET ROBUSTESSE

Ren

dem

ent e

t rob

uste

sse

Rob

uste

sse

Eau

Cha

ude

San

itaire

Prot

ocol

e A

quad

ien

Prot

ocol

e A

quad

ien

W2

Niv

eau

N1

100

000

UG

/LU

G/p

uits

A (l

og)

C(t)

UG

/pui

tsM

oyen

ne U

G/p

uits

B (l

og)

log

%U

G/p

uits

A (l

og)

C(t)

UG

/pui

tsM

oyen

ne U

G/p

uits

B (l

og)

log

%E

C1N

16,

55E

+02

5,02

29,4

31,

31E

+03

EC

2N1W

2,32

E+0

46,

5725

,79

3,27

E+0

429

,67

1,10

E+0

325

,69

3,53

E+0

4E

C2N

16,

55E

+02

5,02

29,7

1,08

E+0

3E

C2N

1W2,

32E

+04

6,57

25,8

83,

03E

+04

29,7

31,

06E

+03

25,9

72,

83E

+04

EC

3N1

6,28

E+0

25,

0029

,41

1,19

E+0

3E

C3N

1W4,

31E

+02

4,84

30,3

6,48

E+0

229

,35

1,25

E+0

330

,32

6,40

E+0

2E

C4N

16,

28E

+02

5,00

29,3

21,

27E

+03

EC

4N1W

4,31

E+0

24,

8430

,16

7,22

E+0

229

,31

1,29

E+0

330

,22

6,89

E+0

2E

C5N

14,

31E

+02

4,84

29,2

41,

46E

+03

E5N

1W-1

003,

27E

+04

6,72

30,5

37,

23E

+02

29,5

1,20

E+0

330

,57

7,02

E+0

2E

C6N

14,

31E

+02

4,84

29,3

11,

39E

+03

E6N

1W-1

003,

27E

+04

6,72

30,4

8,00

E+0

229

,38

1,31

E+0

330

,36

8,29

E+0

2E

7N1-

100

3,27

E+0

46,

7231

5,01

E+0

2E

7N1W

-100

3,27

E+0

46,

7230

,38

8,17

E+0

230

,59

6,91

E+0

230

,31

8,58

E+0

2E

8N1-

100

3,27

E+0

46,

7230

,88

5,52

E+0

2E

C8N

1W7,

10E

+03

6,06

26,8

79,

45E

+03

30,6

26,

78E

+02

26,7

41,

04E

+04

E9N

1-10

03,

27E

+04

6,72

30,8

5,89

E+0

2E

9N1W

7,00

E+0

47,

0523

,82

9,00

E+0

430

,76,

32E

+02

23,8

98,

51E

+04

EC

10N

17,

10E

+03

6,06

25,8

2,11

E+0

4E

10N

1W7,

00E

+04

7,05

23,5

71,

08E

+05

25,6

62,

34E

+04

23,6

31,

03E

+05

Ren

dem

ent m

oyen

pou

r le

nive

au 1

00 0

00 U

G/L

-0,3

941

%R

ende

men

t moy

en p

our l

e ni

veau

100

000

UG

/L-0

,45

36%

Niv

eau

N2

1 00

0 U

G/L

UG

/pui

tsA

(log

)C

(t)U

G/p

uits

Moy

enne

UG

/pui

tsB

(log

)lo

g%

UG

/pui

tsA

(log

)C

(t)U

G/p

uits

Moy

enne

UG

/pui

tsB

(log

)lo

g%

EC

1N2

2,32

E+0

46,

5730

,25

9,11

E+0

2E

C1N

2W6,

55E

+02

5,02

34,5

3,21

E+0

130

,23

9,23

E+0

235

,31,

78E

+01

EC

2N2

2,32

E+0

46,

5730

,13

1,00

E+0

3E

C2N

2W6,

55E

+02

5,02

34,7

42,

68E

+01

30,1

21,

01E

+03

35,0

12,

20E

+01

EC

3N2

4,04

E+0

46,

8130

,24

8,24

E+0

2E

C3N

2W6,

28E

+02

5,00

34,9

72,

10E

+01

30,0

59,

53E

+02

36,8

5,51

E+0

0E

C4N

24,

04E

+04

6,81

30,0

89,

29E

+02

EC

4N2W

6,28

E+0

25,

0034

,74

2,46

E+0

129

,96

1,02

E+0

335

,97

1,01

E+0

1E

C5N

24,

04E

+04

6,81

30,1

29,

00E

+02

EC

5N2W

2,32

E+0

46,

5731

,16

4,39

E+0

229

,98

1,01

E+0

331

,05

4,79

E+0

2E

C6N

23,

27E

+04

6,72

30,2

98,

76E

+02

EC

6N2W

2,32

E+0

46,

5730

,97

5,09

E+0

230

,21

9,28

E+0

231

,21

4,19

E+0

2E

C7N

23,

27E

+04

6,72

30,2

49,

07E

+02

EC

7N2W

4,31

E+0

24,

8434

,78

2,09

E+0

130

,11,

01E

+03

35,5

21,

18E

+01

EC

8N2

7,10

E+0

36,

0631

,82

2,28

E+0

2E

C8N

2W4,

31E

+02

4,84

34,8

2,07

E+0

132

,06

1,91

E+0

235

,57

1,15

E+0

1E

C9N

27,

21E

+04

7,06

30,0

51,

07E

+03

EC

9N2W

7,10

E+0

36,

0632

,14

1,80

E+0

230

,09

1,04

E+0

331

,91

2,14

E+0

2E

C10

N2

7,21

E+0

47,

0630

,38

8,41

E+0

2E

10N

2W7,

00E

+04

7,05

30,0

58,

90E

+02

30,1

59,

95E

+02

29,8

71,

02E

+03

Ren

dem

ent m

oyen

pou

r le

nive

au 1

000

UG

/L-0

,29

51%

Ren

dem

ent m

oyen

pou

r le

nive

au 1

000

UG

/L-0

,16

69%

Ren

dem

ent m

oyen

Eau

cha

ude

sani

taire

Aqu

adie

n-0

,34

46%

Ren

dem

ent m

oyen

Eau

cha

ude

sani

taire

Aqu

adie

n W

2-0

,30

50%

1,07

E+0

34,

53-0

,49

33%

4,59

-0,4

437

%-0

,48

3,40

E+0

46,

0933

%1,

20E

+03

1,22

E+0

34,

59-0

,41

39%

1,28

E+0

34,

61

3,21

E+0

15,

060,

0411

0%

-0,3

941

%

2,43

E+0

14,

94

88%

-0,0

883

%

2,93

E+0

46,

02-0

,55

28%

9,17

E+0

26,

47-0

,10

79%

1,00

E+0

36,

51-0

,06

87%

4,88

4,58

E+0

26,

22-0

,35

45%

4,62

E+0

26,

22-0

,35

45%

1,32

E+0

34,

63-0

,21

61%

-0,2

063

%

6,44

E+0

24,

37-0

,47

34%

7,05

E+0

24,

40-0

,43

37%

2,09

E+0

14,

880,

0410

9%

2,07

E+0

14,

870,

0310

8%

-0,1

275

%2,

10E

+01

-0,0

52,

46E

+01

34%

8,86

E+0

26,

45-0

,28

53%

4,95

9,74

E+0

26,

49-0

,23

58%

9,54

E+0

26,

48-0

,24

57%

5,89

E+0

26,

27-0

,52

30%

1,35

E+0

34,

648,

14E

+02

6,47

-0,3

347

%

6,12

E+0

26,

29-0

,50

31%

6,10

E+0

26,

29-0

,50

31%

7,12

E+0

26,

41-0

,38

41%

8,37

E+0

26,

48-0

,31

48%

9,02

E+0

26,

46-0

,33

46%

9,58

E+0

26,

49-0

,31

49%

1,06

E+0

5

8,75

E+0

46,

50

6,58

0475

6

-0,5

5

-0,4

7

28%

2,22

E+0

45,

85-0

,20

63%

9,92

E+0

35,

55-0

,50

31%

2,09

E+0

25,

82-0

,23

59%

62%

1,05

E+0

36,

53-0

,53

29%

9,54

E+0

26,

48-0

,56

27%

1,96

E+0

25,

85-0

,21

9,15

E+0

26,

47-0

,60

25%

Ech

antil

lon

Val

eur d

u do

page

Rés

ulta

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Ren

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Ech

antil

lon

Val

eur d

u do

page

Rés

ulta

t ana

lyse

Ren

dem

ent

Rés

ulta

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Ren

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ent

Ech

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Rés

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Val

eur d

u do

page

Val

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u do

page

Ren

dem

ent

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lon



Ren

dem

ent e

t rob

uste

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Rob

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sse

Tour

Aér

oréf

rigér

ante

Prot

ocol

e A

quad

ien

Prot

ocol

e A

quad

ien

W2

Niv

eau

N1

100

000

UG

/LU

G/p

uits

A (l

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C(t)

UG

/pui

tsM

oyen

ne U

G/p

uits

B (l

og)

log

%U

G/p

uits

A (l

og)

C(t)

UG

/pui

tsM

oyen

ne U

G/p

uits

B (l

og)

log

%T1

N1

2,32

E+0

46,

5725

,53

4,01

E+0

4T1

N1W

6,55

E+0

25,

0229

,71

1,08

E+0

325

,35

4,64

E+0

429

,72

1,07

E+0

3T2

N1

2,32

E+0

46,

5725

,39

4,52

E+0

4T2

N1W

2,32

E+0

46,

5726

,11

2,52

E+0

425

,26

5,00

E+0

426

,12,

55E

+04

T3N

14,

04E

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ANNEXE H

SELECTIVITE

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s ci

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gion

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Documents

NF Validation Validation of alternative‘s analysis method ... · Version 1.1 14 september 2016 Cancels and replaces the previous ... reactional volume of heating block, user interface