ACCREDITATION N°1-0144 PORTEE DISPONIBLE SUR WWW.COFRAC.FR ADRIA DEVELOPPEMENT Creac’h Gwen - F. 29196 QUIMPER Cedex - Tél. (33) 02.98.10.18.18 - Fax (33) 02.98.10.18.08 E-mail : [email protected] - Site web : http://www.adria.tm.fr ASSOCIATION LOI DE 1901 - N° SIRET 306 964 271 00036 - N° EXISTENCE 532900006329 - N°TVA FR4530696427100036 Oxoid Ltd, part of Thermo Fisher Scientific Wade Road, Basingstoke, Hampshire, RG24 8PW, UK NF VALIDATION Validation of alternative analytical methods Application in food microbiology Summary report EN ISO 16140 validation study of the BAX® System PCR Assay Salmonella spp. Qualitative method This document includes 60 pages, with 5 appendixes. Only copies including the totality of this document are authorised. Competences of the laboratory are certified by COFRAC accreditation for the analyses marked with symbol . Version 0 December 4, 2014
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Appendix 1 – Flow diagrams of the alternative method ______________________________________ 26
Appendix 2 – NF EN ISO 6579: 2002 reference method: Microbiology of food and animal feeding stuffs –
Horizontal method for the detection of Salmonella spp. ______________________________________ 29
Appendix 3 – Relative accuracy: raw data in French (Study realized by Institut Pasteur de Lille) _____ 30
Appendix 4 – Relative accuracy: raw data (Study realized by ADRIA Développement) _____________ 46
Appendix 5 – Inclusivity / exclusivity: raw data (in French) ___________________________________ 51
OXOID
ADRIA Développement 3/60 December 4, 2014
Summary Report – BAX PCR Salmonella (Version 0)
Before comment
Quality assurance documents related to this study can be consulted upon
request from OXOID, part of Thermo Fisher Scientific.
The technical protocol and the result interpretation were realized according to
the EN ISO 16140 and the AFNOR technical rules.
o Company: Oxoid Ltd,
part of Thermo Fisher Scientific
Wade Road, Basingstoke,
Hampshire, RG24 8PW, UK
o Expert Laboratory: ADRIA Développement
ZA Creac’h Gwen
F-29196 QUIMPER Cedex
o Studied method: BAX® System PCR Assay Salmonella spp.
o Validation standard: EN ISO 16140 (October 2003): Food microbiology
– Protocol for the validation of alternative methods
o Standard method: EN ISO 6579 (2002): Horizontal method for the
detection of Salmonella spp.
o Scope: Food and feed products, environmental
samples (excluding samples from primary
production)
o Certification organism: AFNOR Certification
Analysis performed according to the COFRAC accreditation
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ADRIA Développement 4/60 December 4, 2014
Summary Report – BAX PCR Salmonella (Version 0)
1 INTRODUCTION
1.1 Dates of the initial validation, extension and renewal studies
BAX® System PCR Assay Salmonella spp performances were assessed
on November 28, 2002 (certificate number QUA 18/03 – 11/02). Extension
studies and renewal studies were performed:
March 2004 Extension for two specific protocols dedicated to raw
meat and dairy products (milk products excluded) –
Study realised by IPL
May 2006 Extension for a second automate (BAX Q7) –
Study realised by IPL
October 2006 Renewal according to the ISO 16140 standard –
Study realised by IPL
June 2008 Extension for a new protocol dedicated to raw meat
(seasoned or not, using MP broth) –
Study realised by ADRIA Développement
November 2008 Extension for a new protocol for raw meat samples with
a short incubation time (9 h at 42°C) –
Study realised by ADRIA Développement
May 2009 Extension for a new version of the software
September 2010 Renewal study and extension for a new version of the kit
March 2011 Extension to version 2.8 of the BAX software
March 2012 Extension to version 2.9 of the BAX software
November 2014 Renewal according to the ISO 16140 standard
Study realised by ADRIA Développement
1.2 Alternative method
Principle
The BAX® system is based on the gene amplification of a Salmonella spp.
specific nucleic sequence by PCR technology. The reagents necessary for
the PCR reaction and for the internal control are included in the same PCR
tube.
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Summary Report – BAX PCR Salmonella (Version 0)
The BAX® Q7 system is a system composed by a thermocycler and an
optical module detecting the fluorescence. The software program analyses
the level of fluorescence and provides results, i.e. positive or negative.
Protocols (See Appendix 1)
The protocols are described below:
- Protocols:
All products, excluding raw meat
and raw poultry
Pre-enrichment: 16 h - 20 h at 37°C in BPW (d 1/10)
Subculture: 3 h – 4 h in BHI (10 µl BPW/500 µl BHI)
Raw poultry meat (non seasoned) Pre-enrichment: 16 h - 20 h at 37°C in BPW (d 1/10)
Dairy products
(except milk powders)
Pre-enrichment: 20 h - 24 h at 42°C in BPW
supplemented with novobiocin (20 mg/l) (d 1/10)
Raw meat Pre-enrichment: 24 h at 42°C in MP broth
Raw beef meat Pre-enrichment: 9 h – 24 h at 42°C in MP broth
- DNA extraction lysis:
* addition of 5 l enrichment broth to 200 l lysis reagent
* 30 minutes at 55°C
* 10 minutes at 95°C
- Amplification:
* transfer 50 l of the lysate in a PCR tube
* run the PCR in the automate
- Detection
The fluorescence is measured directly by the BAX® system, which
provides positive or negative results.
- confirmation of positive results
1) according to the tests described in the reference method after
streaking the last enrichment broth onto selective agar plates,
or
2) by applying a subculture in RVS broth (24 h at 41.5°C) prior to
streaking onto BrillianceTM Salmonella for raw meats. Typical
colonies are then confirmed by latex test.
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Summary Report – BAX PCR Salmonella (Version 0)
1.3 Reference method
The reference method is the EN ISO 6579 (2002): Horizontal method for the
detection of Salmonella spp (See Appendix 2).
2 VALIDATION STUDIES RESULTS
2.1 Methods comparison study
2.1.1 Relative accuracy, relative specificity and relative sensitivity
Accuracy is the closeness of agreement between a test result and the accepted reference value. Relative specificity is defined as the degree to which a method is affected (or not) by the other components present in a multi-component sample; that is, it is the ability of the method to measure exactly a given analyte, or its amount, within the sample without interference from non-target components such as matrix effect or background noise. Relative sensitivity is defined as the ability of the alternative method to detect two different amounts of analyte measured by the reference method within a given matrix over the whole measurement range; that is, it is the minimal quantity variation (increase of the analyte concentration x) which gives a significant variation of the measured signal (response y).
2.1.1.1 Number and nature of samples
In 2002, 2004 and 2006, the studies were realised by IPL (See raw data in
Appendix 3). In 2008 (June and November), they were performed by ADRIA
Développement (See raw data Appendix 4).
The repartition per tested category and per study is provided Table 1.
Analysis performed according to the COFRAC accreditation
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Summary Report – BAX PCR Salmonella (Version 0)
Table 1 – Repartition per tested category and per study
2002-
20042006 2008
(June)
2008(Nov.)
2002-
20042006 2008
(June)
2008(Nov.)
Raw meat 27 0 0 0 15 0 0 0 42
Poultry 5 0 0 0 13 0 0 0 18
Delicatessen 7 0 0 0 14 0 0 0 21
Total 39 0 0 0 42 0 0 0 81
Poultry 0 0 11 0 0 0 11 0 22
Beef 0 0 12 0 0 0 7 0 19
Others 0 0 10 0 0 0 14 0 24
Total 0 0 33 0 0 0 32 0 65
Fresh 0 0 0 24 0 0 0 25 49
Frozen 0 0 0 8 0 0 0 5 13
Seasoned 0 0 0 2 0 0 0 0 2
Total 0 0 0 34 0 0 0 30 64
Raw milk cheeses 0 9 0 0 16 3 0 0 28
Pasteurised milk
cheeses and ice creams0 13 0 0 6 0 0 0 19
Milks and milk powders 2 8 0 0 8 0 0 18
Total 2 30 0 0 30 3 0 0 65
Fish 0 8 0 0 10 3 0 0 21
Raw vegetables and
spices0 12 0 0 11 9 0 0 32
Ready-to-eat vegetables 0 10 0 0 10 2 0 0 22
Total 0 30 0 0 31 14 0 0 75
Egg products 3 9 0 0 11 2 0 0 25
Pastry , chocolate 5 5 0 0 21 6 0 0 37
Ready-to-cook or ready-
to-heat meals0 10 0 0 1 6 0 0 17
Total 8 24 0 0 33 14 0 0 79
"Tourteaux" 0 8 0 0 4 11 0 0 23
Flours, pellets 3 2 0 0 9 4 0 0 18
Meat 1 13 0 0 1 6 0 21
Total 4 23 0 0 14 21 0 0 62
Process water 0 10 0 0 0 5 0 0 15
Swabs, wipes 0 12 0 0 0 14 0 0 26
Dusts, wastes 0 8 0 0 0 11 0 0 19
Total 0 30 0 0 0 30 0 0 60
53 137 33 34 150 82 32 30 551TOTAL
Various
products
BPW,
16 h - 20 h
Feed stuffBPW,
16 h - 20 h
Environmental
samples
BPW,
16 h - 20 h
Raw beef
meats
MP broth,
9 h- 24 h
Dairy
products
BPW,
16 - 20 h
+ novobiacin
Fishery
products and
vegetables
BPW,
16 h - 20 h
Total
Meat productsBPW,
16 - 20 h
Raw meat
products
MP broth,
24 h
Category Protocol Types
Positive samples Negative samples
2.1.1.2 Artificial contamination of samples
Artificial contaminations were done by using injured bacterial suspensions.
The injured treatment and the efficiency were determined according to EN
ISO 16140 and AFNOR technical rules.
The percentage of naturally and artificially contaminated samples per study is
presented below (Table 2).
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Table 2 - Percentage of naturally and artificially
contaminated samples per study
Year of study
Number of
contaminated
samples
Number of positive
results observed
among the
contaminated samples
Positive
results
observed
Percentage of
artificially
contaminated
samples
Percentage of
naturally
contaminated
samples
2002 – 2004 –
2006 110 95 190 50.0 % 50.0 %
2008 (June) 22 12 33 36.0 % 64.0 %
2008 (November) 38 31 34 91.2 % 8.8 %
All validation
studies 170 138 257 53.7 % 46.3 %
2.1.1.3 Test results
The results are reported below.
Table 3 – Paired results of the reference and alternative methods
All products – General protocol
(enrichment broth: BPW or BPW + novobiocin at 37°C)
Responses
Reference method
positive (R+)
Reference method
negative (R-)
Alternative method positive
(A+)
Positive agreement (A+/R+)
PA = 186
Positive deviation (R-/A+)
PD = 1
Alternative method negative
(A-)
Negative deviation (A-/R+)
ND = 6 (PPND = 0)
Negative agreement (A-/R-)
NA = 232 (PPNA = 1)
PA: positive agreement
PD: positive deviation
NA: negative agreement
ND: negation deviation
PPNA: positive presumptive negative agreement
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Summary Report – BAX PCR Salmonella (Version 0)
Table 4 – Paired results of the reference and alternative methods
Meat products
Responses
Reference method
positive (R+)
Reference method
negative (R-)
Alternative method positive
(A+)
Positive agreement (A+/R+)
PA = 37
Positive deviation (R-/A+)
PD = 0
Alternative method negative
(A-)
Negative deviation (A-/R+)
ND = 2
Negative agreement (A-/R-)
NA = 42
Table 5 – Paired results of the reference and alternative methods
Dairy products
Responses
Reference method
positive (R+)
Reference method
negative (R-)
Alternative method positive
(A+)
Positive agreement (A+/R+)
PA = 31
Positive deviation (R-/A+)
PD = 1
Alternative method negative
(A-)
Negative deviation (A-/R+)
ND = 0
Negative agreement (A-/R-)
NA = 33
Table 6 – Paired results of the reference and alternative methods
Fishery products and vegetables
Responses
Reference method
positive (R+)
Reference method
negative (R-)
Alternative method positive
(A+)
Positive agreement (A+/R+)
PA = 28
Positive deviation (R-/A+)
PD = 0
Alternative method negative
(A-)
Negative deviation (A-/R+)
ND = 2
Negative agreement (A-/R-)
NA = 45
Table 7 – Paired results of the reference and alternative methods
Various products
Responses
Reference method
positive (R+)
Reference method
negative (R-)
Alternative method positive
(A+)
Positive agreement (A+/R+)
PA = 32
Positive deviation (R-/A+)
PD = 0
Alternative method negative
(A-)
Negative deviation (A-/R+)
ND = 0
Negative agreement (A-/R-)
NA = 47
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Summary Report – BAX PCR Salmonella (Version 0)
Table 8 – Paired results of the reference and alternative methods
Feed stuff
Responses
Reference method
positive (R+)
Reference method
negative (R-)
Alternative method positive
(A+)
Positive agreement (A+/R+)
PA = 29
Positive deviation (R-/A+)
PD = 0
Alternative method negative
(A-)
Negative deviation (A-/R+)
ND = 1
Negative agreement (A-/R-)
NA = 35
Table 9 – Paired results of the reference and alternative methods
Environmental samples
Responses
Reference method
positive (R+)
Reference method
negative (R-)
Alternative method positive
(A+)
Positive agreement (A+/R+)
PA = 29
Positive deviation (R-/A+)
PD = 0
Alternative method negative
(A-)
Negative deviation (A-/R+)
ND = 1
Negative agreement (A-/R-)
NA = 30
Table 10 – Paired results of the reference and alternative methods
Specific protocols (enrichment broth: MP 24 h at 42°C)
Responses
Reference method
positive (R+)
Reference method
negative (R-)
Alternative method positive
(A+)
Positive agreement (A+/R+)
PA = 21
Positive deviation (R-/A+)
PD = 5
Alternative method negative
(A-)
Negative deviation (A-/R+)
ND = 7 (PPND = 0)
Negative agreement (A-/R-)
NA = 32 (PPNA = 0)
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Summary Report – BAX PCR Salmonella (Version 0)
Table 11 – Paired results of the reference and alternative methods
Raw beef meats (enrichment broth: MP 9 – 24 h at 42°C)
Incubation
time
Responses
Reference method
positive (R+)
Reference method
negative (R-)
9 h
Alternative
method positive (A+)
Positive agreement (A+/R+)
PA = 25
Positive deviation (R-/A+)
PD = 4
Alternative
method negative (A-)
Negative deviation (A-/R+)
ND = 4 (PPND = 0)
Negative agreement (A-/R-)
NA = 31 (PPNA = 0)
24 h
Alternative
method positive (A+)
Positive agreement (A+/R+)
PA = 28
Positive deviation (R-/A+)
PD = 5
Alternative
method negative (A-)
Negative deviation (A-/R+)
ND = 1 (PPND = 0)
Negative agreement (A-/R-)
NA = 30 (PPNA = 0)
Table 12 – Calculation of relative accuracy (AC),
relative sensitivity (SE) and relative specificity (SP)