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ADGENE LABORATOIRE 1 rue des conquérants -14 220 THURY-HARCOURT www.adgene.fr –info.adgene@adm.com – 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.
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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
RESULTS (ANNEX 2) 17
CONCLUSIONS 17
POSITIVITY THRESHOLD 17
METHODOLOGY 17
RESULTS (ANNEX 1) 17
DETERMINATION OF YIELD AND ROBUSTNESS OF EXTRACTION 17
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METHODOLOGY 17
RESULTS (ANNEX 3) 18
INCLUSIVITY/EXCLUSIVITY 19
METHODOLOGY 19
RESULTS (ANNEX 4) 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
Calibration function
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
Range 1 Range 2 Range 3
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
Range 1 Range 2 Range 3
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
Range 1 Range 2 Range 3
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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Calibration function
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
Xi Level LQ 2LQ 20LQ 200LQ
25 250 2500 25000
X'i theoretical = Log Xi 1.40 2.40 3.40 4.40
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
Xi Level LQ 10LQ 100LQ 1000LQ
25 250 2500 25000
X'i theoretical = Log Xi 1.40 2.40 3.40 4.40
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
2.40 31.62 Intercept value 39.26
3.40 28.35
4.40 24.74 Slope -3.26
Xi Level LQ 10LQ 100LQ 1000LQ
25 250 2500 25000
X'i theoretical = Log Xi 1.40 2.40 3.40 4.40
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
Slope: - 3.26 criteria was validated
Elin: all levels < 0.15 were validated
RotorGene 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 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
1.40 32.96 Intercept value 37.82
2.40 29.49
3.40 25.97 Slope -3.48
4.40 22.55
Xi Level LQ 2LQ 20LQ 200LQ
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
Slope: - 3.37 criteria was validated
Elin: all levels < 0.15 were validated
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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.
AFNOR VALIDATION
Summary report
DI-CHECK L.pneumophila
V0
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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
Summary report
DI-CHECK L.pneumophila
V0
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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
Summary report
DI-CHECK L.pneumophila
V0
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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
Summary report
DI-CHECK L.pneumophila
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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
Summary report
DI-CHECK L.pneumophila
V0
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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
Summary report
DI-CHECK L.pneumophila
V0
26
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
AFNOR VALIDATION
Summary report
DI-CHECK L.pneumophila
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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
Summary report
DI-CHECK L.pneumophila
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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
Doping value Analysis results Yield
Sample GU/well A (Log) Ct GU/well B (Log) Log %
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
Summary report
DI-CHECK L.pneumophila
V0
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Domestic hot water
Level 100 000 GU/L
Doping value Analysis results Yield
Sample GU/well A (Log) Ct GU/well B (Log) Log %
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
Doping value Analysis results Yield
Sample GU/well A (Log) Ct GU/well B (Log) Log %
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
Summary report
DI-CHECK L.pneumophila
V0
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Cooling tower water
Level 100 000 GU/L
Doping value Analysis results Yield
Sample GU/well A (Log) Ct GU/well B (Log) Log %
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
Doping value Analysis results Yield
Sample GU/well A (Log) Ct GU/well B (Log) Log %
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
Summary report
DI-CHECK L.pneumophila
V0
32
CFX 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 1.19E+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
Doping value Analysis results Yield
Sample GU/well A (Log) Ct GU/well B (Log) Log %
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
Summary report
DI-CHECK L.pneumophila
V0
33
Domestic hot water
Level 100 000 GU/L
Doping value Analysis results Yield
Sample GU/well A (Log) Ct GU/well B (Log) Log %
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
Doping value Analysis results Yield
Sample GU/well A (Log) Ct GU/well B (Log) Log %
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
Summary report
DI-CHECK L.pneumophila
V0
34
Cooling tower water
Level 100 000 GU/L
Doping value Analysis results Yield
Sample GU/well A (Log) Ct GU/well B (Log) Log %
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
Doping value Analysis results Yield
Sample GU/well A (Log) Ct GU/well B (Log) Log %
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
Summary report
DI-CHECK L.pneumophila
V0
35
Annex 4: Inclusivity and Exclusivity
Inclusivity strains
Name Origin Ct Green (target) Ct Yellow (IAC)
Legionella pneumophila ser 1 CNRL 29.32 29.04
Legionella pneumophila ser 2 CNRL 28.63 29.19
Legionella pneumophila ser 3 CNRL 27.62 28.52
Legionella pneumophila ser 4 CNRL 29.54 29.04
Legionella pneumophila ser 5 CNRL 28.66 29.10
Legionella pneumophila ser 6 CNRL 27.14 28.41
Legionella pneumophila ser 7 CNRL 29.88 29.42
Legionella pneumophila ser 8 CNRL 27.23 28.46
Legionella pneumophila ser 9 CNRL 27.98 28.51
Legionella pneumophila ser 10 CNRL 30.49 29.07
Legionella pneumophila ser 11 CNRL 29.25 29.37
Legionella pneumophila ser 12 CNRL 28.20 28.99
Legionella pneumophila ser 13 CNRL 31.30 29.49
Legionella pneumophila ser 14 CNRL 29.04 29.08
Legionella pneumophila ser 15 CNRL 28.01 28.67
AFNOR VALIDATION
Summary report
DI-CHECK L.pneumophila
V0
36
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
Summary report
DI-CHECK L.pneumophila
V0
37
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
Summary report
DI-CHECK L.pneumophila
V0
38
Results QuantStudio
Verification carried out using DI-Check analysis software for Legionella pneumophila
v2.2
Verification carried out using the QuantStudio software
AFNOR VALIDATION
Summary report
DI-CHECK L.pneumophila
V0
39
Results CFX
Verification carried out using DI-Check analysis software for Legionella pneumophila
v2.2
Verification carried out using the CFX software
AFNOR VALIDATION
Summary report
DI-CHECK L.pneumophila
V0
40
Annex 6: Inhibition
Verification carried out using RotorGene cycler
AFNOR VALIDATION
Summary report
DI-CHECK L.pneumophila
V0
41
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
17 I8a Unknown 26.93 7 250 Standard 27.14
18 I8b Unknown 27.14 8 250 Standard 26.87
19 I10a Unknown 26.85 9 2500 Standard 26.87
20 I10b Unknown 26.81 10 2500 Standard 26.47
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
I5a: replicate a with 5% Inhibitory, I5b: replicate b with 5% Inhibitory
I8a: replicate a with 2% Inhibitory, I8b: replicate b with 2% Inhibitory
I10a: replicate a with 10% Inhibitory, I10b: replicate b with 10% Inhibitory
I20a: replicate a with 20% Inhibitory, I20b: replicate b with 20% Inhibitory
I50a: replicate a with 50% Inhibitory, I50b: replicate b with 50% Inhibitory
Recommended