Summary report - NF Validationnf-validation.afnor.org/en/wp-content/uploads/sites/2/2014/03/Synt... · Summary report EN ISO 16140 validation study of the BAX® System PCR Assay Salmonella

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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

OXOID

ADRIA Développement 2/60 December 4, 2014

Summary Report – BAX PCR Salmonella (Version 0)

1 INTRODUCTION _______________________________________________ 4

1.1 Dates of the initial validation, extension and renewal studies ___________ 4

1.2 Alternative method ______________________________________________ 4

1.3 Reference method ______________________________________________ 6

2 VALIDATION STUDIES RESULTS _________________________________ 6

2.1 Methods comparison study _______________________________________ 6

2.1.1 Relative accuracy, relative specificity and relative sensitivity _______________ 6

2.1.2 Relative detection level ___________________________________________ 13

2.1.3 Inclusivity / exclusivity ____________________________________________ 15

2.2 Inter-laboratory study ___________________________________________ 16

2.2.1 Study organization _______________________________________________ 16

2.2.2 Control of experimental parameters __________________________________ 17

2.2.3 Results analyses ________________________________________________ 18

2.2.4 Results interpretation _____________________________________________ 19

2.2.5 Interpretation ___________________________________________________ 21

2.3 Practicability __________________________________________________ 23

2.4 Conclusion ____________________________________________________ 25

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

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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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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) –


October 2006 Renewal according to the ISO 16140 standard –


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) –


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


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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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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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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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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Meat products

Responses

Reference method

positive (R+)

Reference method

negative (R-)


(A+)


PA = 37


PD = 0


(A-)


ND = 2


NA = 42


Dairy products

Responses

Reference method

positive (R+)

Reference method

negative (R-)


(A+)


PA = 31


PD = 1


(A-)


ND = 0


NA = 33


Fishery products and vegetables

Responses

Reference method

positive (R+)

Reference method

negative (R-)


(A+)


PA = 28


PD = 0


(A-)


ND = 2


NA = 45


Various products

Responses

Reference method

positive (R+)

Reference method

negative (R-)


(A+)


PA = 32


PD = 0


(A-)


ND = 0


NA = 47

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Feed stuff

Responses

Reference method

positive (R+)

Reference method

negative (R-)


(A+)


PA = 29


PD = 0


(A-)


ND = 1


NA = 35


Environmental samples

Responses

Reference method

positive (R+)

Reference method

negative (R-)


(A+)


PA = 29


PD = 0


(A-)


ND = 1


NA = 30


Specific protocols (enrichment broth: MP 24 h at 42°C)

Responses

Reference method

positive (R+)

Reference method

negative (R-)


(A+)


PA = 21


PD = 5


(A-)


ND = 7 (PPND = 0)


NA = 32 (PPNA = 0)

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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+)


PA = 25


PD = 4

Alternative

method negative (A-)


ND = 4 (PPND = 0)


NA = 31 (PPNA = 0)

24 h

Alternative

method positive (A+)


PA = 28


PD = 5

Alternative

method negative (A-)


ND = 1 (PPND = 0)


NA = 30 (PPNA = 0)

Table 12 – Calculation of relative accuracy (AC),

relative sensitivity (SE) and relative specificity (SP)

Category PA NA ND PD N Relative

accuracy AC (%) [100x(PA+NA])/N]

N+ PA + ND

Relative sensitivity SE (%)

[100xPA]/N+]

N- NA + PD

Relative specificity SP (%)

[100xNA]/N-]

Meat products 37 42 2 0 81 98.5 39 94.9 42 100.0

Dairy products 31 33 0 1 65 98.5 31 100.0 34 97.10


28 45 2 0 75 97.3 30 93.3 45 100.0

Various products

32 47 0 0 79 100.0 32 100.0 47 100.0

Feed stuff 29 35 1 0 65 98.5 30 96.7 35 100.0


29 30 1 0 60 98.3 30 96.7 30 100.0

All products 186 232 6 1 425 98.4 192 96.9 233 99.6

Category PA NA ND PD N

Relative

accuracy AC (%)

[100x(PA+NA])/N]

N+

PA + ND

Relative

sensitivity SE (%)

[100xPA]/N+]

N-

NA + PD

Relative

specificity SP (%)

[100xNA]/N-]

Raw meats 24 h 21 13 7 5 65 81.5 28 75.0 37 86.5

Category PA NA ND PD N

Relative

accuracy AC (%)

[100x(PA+NA])/N]

N+

PA + ND

Relative

sensitivity SE (%)

[100xPA]/N+]

N-

NA + PD

Relative

specificity SP (%)

[100xNA]/N-]

Beef meat

9 h 25 31 4 4 64 87.5 29 86.2 35 88.6

24 h 28 30 1 5 64 90.6 29 96.6 35 85.7

PA = positive agreement (R+/A+) NA =negative agreement (R-/A-) PD =positive deviation (R-/A+) ND = negative deviation (A-/R+)

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2.1.1.4 Calculation of relative accuracy (AC), relative sensitivity (SE) and relative

specificity (SP)

For the alternative method, the percentage values for three criteria according

to the ISO 16140 are the following:

General protocol

Raw meats Beef meats

24 h 9 h 24 h

Relative accuracy 98.4 % 81.5 % 87.5 % 90.6 %

Relative specificity 99.6 % 86.5 % 88.6 % 85.7 %

Relative sensitivity 96.9 % 75.0 % 86.2 % 96.6 %

Sensitivity of both methods, when the positive deviations of the alternative

method are considered, is presented below:

Alternative method

(PA + PD) / PA + PD + ND) (SE)

Reference method

(PA + ND) / PA + PD + ND) (SE)

General protocol 96.9 % 99.5 %

Specific protocol – Raw meats 78.8 % 84.7 %

Specific protocol – Beef meats 9 h 87.9 % 87.9 %

24 h 97.1 % 85.3 %

2.1.1.5 Analysis of discordant results

The analysis of discordant results is presented per protocol in the following

table:

Y = ND + PD m M Conclusion

General protocol Y = 6 + 1 = 7 1 0 The methods are not

different at < 0.05.

Specific protocol – Raw meats Y = 7 + 5 = 12 5 2 The methods are not


Specific protocol – Beef meats 9 h Y = 4 + 4 = 8 4 0 The methods are not


24 h Y = 1 + 5 = 6 1 0 The methods are not


The BAX® System PCR Assay Salmonella spp. shows satisfying

relative accuracy, relative specificity and sensitivity.

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2.1.1.6 Confirmation protocols

For the general protocol, the last enrichment broth (BPW, BPW + novobiocin

or BHI) was streaked onto selective agar plates as detailed in ISO 6579. Any

typical colonies were then confirmed by the tests described in the ISO

method.

For protocols dedicated to raw meats and raw beef meats enriched with

BAX® System MP Broth, the positive samples were confirmed by subculture

of the MP Broth into RVS and then by streaking onto Oxoid BrillianceTM

Salmonella Agar. Any typical colonies were then confirmed by a latex test

and the tests described in the ISO method.

2.1.2 Relative detection level

The relative detection level is the smallest number of culturable micro-organisms that can be

detected in the sample in 50% of occasions by the alternative and reference methods.

2.1.2.1 Matrices

The objective of this study is (i) to determine the target species minimal

quantity that can be detected in food matrices, (ii) to compare both method

results.

Detection limits have been defined by analyzing the different matrix/strain

pairs. Four levels were tested. Six replicates of each combination were

prepared.

The following matrices were tested:

- ground poultry meat inoculated with Salmonella Hadar,

- raw milk inoculated with Salmonella Typhimurium,

- whole egg product inoculated with Salmonella Enteritidis,

- fish fillet inoculated with Salmonella Virchow,

- pâté for dog inoculated with Salmonella Senftenberg,

- process water inoculated with Salmonella Infantis.

- ground beef / Salmonella Infantis.

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2.1.2.2 Contamination protocol

Contaminations and enumerations were realized according to the AFNOR

technical rules (protocol for low level inoculations). The contamination levels

are presented below:

- level 1: 0 UFC/g or /ml

- level 2: level necessary to obtain 0 to 50% positives,

- level 3: level necessary to obtain 50 to 75% positives,

- level 4: level necessary to obtain 100% positives.

The samples were analyzed by both methods, and the background microflora

was enumerated.

2.1.2.3 Results

Detection levels are presented in Table 13.

Table 13 – Relative detection level results

Strain / matrix pairs

Relative detection level (CFU / 25 g or 25 ml) according to Spearman-Kärber test

Reference method Alternative method

Ground poultry / Salmonella Hadar 0.3 [0.2 ; 0.5] 0.4 [0.3 ; 0.6]

Raw milk / Salmonella Typhimurium 0.5 [0.3 ; 1.0] 0.6 [0.3 ; 1.0]

Whole egg product / Salmonella Enteritidis 0.4 [0.2 ; 0.8] 0.4 [0.2 ; 0.8]

Fish fillet / Salmonella Virchow 0.3 [0.2 ; 0.4] 0.3 [0.2 ; 0.4]

Pâté for dog / Salmonella Senftenberg 0.4 [0.3 ; 0.7] 0.4 [0.3 ; 0.7]

Process water / Salmonella Infantis 0.5 [0.3 ; 0.9] 0.54 [0.3 ; 0.9]

Ground beef / Salmonella Infantis - BAX 9 h 0,7 [0,4 ; 1,3]

0,4 [0,2 ; 1,1]

Ground beef / Salmonella Infantis - BAX 24 h 0,3 [0,1 ; 0,8]

2.1.2.4 Conclusion

The detection levels are comprised between:

- 0.2 and 1.3 CFU/25 g or 25 ml for the reference method,

- 0.1 and 1.1 CFU/25 g or 25 ml for the alternative method.

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2.1.3 Inclusivity / exclusivity

Inclusivity is the ability of the alternative method to detect the target analyte from a wide range

of strains.

Exclusivity is the lack of interference from a relevant range of non-target strains of the

alternative method.

The inclusivity and the exclusivity of the method are defined by analysis,

respectively of 50 positive strains and 30 negative strains.

2.1.3.1 Test protocols

Inclusivity

- Study performed in 2002: the strains were grown in BHI broth and the BAX

Salmonella test applied.

- Studies performed in 2003 and 2005: the strains were grown in BHI for

24 h at 35°C; the BAX Salmonella tests were then applied on 1/10 diluted

cultures (2003) or pure culture (2005).

- Study performed in 2008: the strains were grown in BHI and diluted in

order to inoculate, between 10 and 100 cells/225 ml, MP broth and then

incubated for 9 h and 24 h at 42°C. The alternative method protocol was

then applied.

In order to be in agreement with the AFNOR technical rules, 5 additional

strains were tested for the renewal project in 2014: they all gave

positive PCR results.

Exclusivity

The studies were performed in 2002, 2003 and 2005 with the protocols as

described for the inclusivity studies.

2.1.3.2 Results

The raw data (in French) are given Appendix 5.

The results observed for the different studies are summarised Table 14.

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Table 14 – Inclusivity and exclusivity study results

Year of realisation

Inclusivity Exclusivity

Number of

strains tested

Number of

positive results

obtained

Number of

negative results

obtained

Number of

strains tested

Number of

negative results

obtained

2002 55 55 0 47 0

2003 194 194 0 35 0

2005 49 49 0 20 0

2008 9 h 57 55 2 0 /

24 h 57 57 0 0 /

2014 5 5 0 / /

(2)

Salmonella gallinarum Ad 300

Salmonella gallinarum 1

The BAX® System PCR Assay Salmonella spp. is specific and selective.

2.2 Inter-laboratory study

The general protocol was tested in the inter-laboratory study (the study was

performed in 2006).

2.2.1 Study organization

12 laboratories received samples. The matrix used for the study was a

delicatessen (pâté) in order to test the general protocol. The strain used for

inoculation was Salmonella Typhimurium isolated from pork liver.

24 samples were prepared per laboratory. Each sample was divided in

3 levels of contamination, with 8 samples per level.

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2.2.2 Control of experimental parameters

2.2.2.1 Contamination levels obtained after artificial contamination

The contamination levels and the confidence intervals were:

Level Samples

Theoretical target level

(b/25 g)

True level (b/25 g sample)

Low limit / 25 g sample

High limit / 25 g sample

Level 0 (L0) 8-9-15-16-

17-22-23-24 0 0 / /

Low level (L1) 1-2-3-6

7-13-14-18 3 5.1 1.7 11.9

High level (L2) 4-5-10-11

12-19-20-21 30 51 38 67

2.2.2.2 Logistic conditions

Temperature conditions are given below:

Laboratory Temperature measured

at receipt

Temperature measured

by the probe Comment

A 7.1°C 4.9°C

B 9.5°C 3.9°C

C 8.6°C -0.1°C

D 10.0°C 5.9°C

E 7.5°C 6.9°C

F 10.7°C 3.4°C

G 3.3°C 3.4°C

H 7.8°C 0.0°C

I 7.0°C 3.9°C

J 1.3°C 4.0°C

K 4.0°C Not received Delivery at Day 2

L 8.0°C 5.4°C

Among the 12 laboratories, one received the samples at Day 2 (Lab K) and

did not realise the analyses.

Labs B, C, D and F measured temperature at receipt above 8.5°C but the

register curves show clearly that the temperatures were correct.

2.2.2.3 Conclusion

Due to the delivery conditions, the results of 11 laboratories were kept for

interpretations.

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2.2.3 Results analyses

2.2.3.1 Results obtained by collaborators

The following tables give a summary of the collaborators results.

Table 15

Positive results obtained with the reference method

Labs

Contamination Level

L0 L1 L2

Obtained Number of samples



A 0 8 8 8 8 8

B 0 8 8 8 8 8

C 0 8 8 8 8 8

D 0 8 8 8 8 8

E 1 8 8 8 8 8

F 0 8 8 8 8 8

G 0 8 8 8 8 8

H 0 8 8 8 8 8

I 0 8 8 8 8 8

J 1 8 8 8 8 8

L 0 8 8 8 8 8

Total 2 88 88 88 88 88

(a) (b) (c)

Positive results obtained with the alternative method

Labs

Contamination level

L0 L1 L2




A 0 8 8 8 8 8

B 0 8 8 8 8 8

C 0 8 8 8 8 8

D 0 8 8 8 8 8

E 0 8 8 8 8 8

F 0 8 8 8 8 8

G 0 8 8 8 8 8

H 0 8 8 8 8 8

I 0 8 8 8 8 8

J 0 8 8 8 8 8

L 0 8 8 8 8 8

Total 0 88 88 88 88 88

(a) (b) (c)

(a) : False positive

(b) : True positive result observed for Level 1

(c) : True positive result observed for Level 2

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The results of the reference method and of the alternative method were in

agreement with those expected for 10 labs.

Lab B found one control sample positive with the BAX Salmonella test, the

confirmatory tests concluded to the absence of the target in the enrichment

broth. The lysis and PCR tests were repeated on the BHI broth after one

night storage at 2 – 8°C and the test was also negative. A cross

contamination probably occurred during the lysis or the PCR step.

Lab E found a control sample (E16) positive with the reference method, but

only one colony was isolated on selective plate. This Lab suspected a cross

contamination, but the result was considered as positive.

The same result was observed for Lab J on sample J22.

2.2.4 Results interpretation

2.2.4.1 Specificity and sensitivity for each method

For the L0 level and for each method, specificity percentages are calculated

according to:

%1001 x

N

FPSP

with : N- = total number of all L0 assays

FP = number of false positive results

For each contamination level and each method, the sensitivity percentages

are calculated according to:

%100xN

TPSE

with : N+ = total number of all L1 or L2 assays

TP = number of true positive results

Results are reported in Table 16.

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Table 16 – Interpretation

Level Reference method Alternative method

SP/SE % LCL% SP/SE % LCL%

L0 (SP) 97.7 96.0 100.0 98.0

L1 (SE) 100.0 98.0 100.0 98.0

L2 (SE) 100.0 98.0 100.0 98.0

L1+L2 (SE) 100.0 98.0 100.0 98.0

LCL: confidence interval

2.2.4.2 Relative accuracy (AC)

Results for all levels are given below:

Table 17 - Paired results of the alternative and reference methods

Alternative method Reference method

Total + -

+ PA = 176 PD = 0 176

- ND = 2 NA = 86 (PPNA = 1) 88

Total N+ = 178 N- = 86 N =264

Relative accuracy (AC) (in %) is calculated according to:

%100)(x

N

NAPAAC

with : N = number of samples analysed

PA = number of positive agreement NA = number of negative agreement

The relative accuracy is 99.2 %

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2.2.5 Interpretation

2.2.5.1 Comparison of the relative accuracy, specificity and sensitivity values

The values obtained for the two parts of the validation study (comparative

and inter-laboratory studies) are reported in Table 18.

Table 18 - Alternative method values calculated

during the comparative and inter-laboratory studies

Inter-laboratory study Method comparison study

Relative accuracy (AC) 99.2 % 98.4 %

Sensitivity (SE) 100.0 % 99.6 %

Specificity (SP) 100.0 % 96.9 %

The AFNOR Technical Committee requests the sensitivity of the two

methods to be recalculated considering all the confirmed positives (true

positive results):

Alternative method Reference method

(PA + PD) / (PA + PD + ND) = 100.0 % (PA + ND) / (PA + PD + ND) = 99.2%

2.2.5.2 Accordance (DA)

Accordance values for both methods are:


Level Reference method (DA) Alternative method (DA)

L0 96.0 % 100.0 %

L1 100.0 % 100.0 %

L2 100.0 % 100.0 %

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2.2.5.3 Concordance

Both methods concordance values are:


Level Reference method Alternative method

L0 95.5 % 100.0 %

L1 100.0 % 100.0 %

L2 100.0 % 100.0 %

2.2.5.4 Odds Ratio (COR)

The odds ratio value is determined according to:

)100(

100

accordancexeConcordanc

econdorcancxAccordanceCOR

Both method odds ratio values are:


Level Reference method (COR) Alternative method (COR)

L0 1.13 1.00

L1 1.00 1.00

L2 1.00 1.00

OXOID



2.3 Practicability

Practicability is studied according to the criteria defined by the AFNOR

technical rules, by comparing the EN ISO 11290 reference method to the

BAX® System PCR Assay Salmonella spp. method performances.

1. Packaging

2. Reagent volumes

The BAX ® System PCR Assay Salmonella spp kit contains the reagents

required for 96 analyses:

- PCR tubes with tablets composed of strips (8 wells)

- caps

- lysis reagent (2 x 12 mL) and protease (400 µL)

3. Storage conditions The storage temperature is between 2 to 8°C.

Expiration date is shown on the kit package on the different reagent vials.

4. Modalities of use after

first use Lysis reagent + protease, once reconstituted, should be stored 15 days at 2-8°C

5. Required equipment

Among the required equipment,

- 1 air incubator at 37°C ± 1°C

- 3 heating blocks at 37°C and 95°C

- BAX or BAX Q7

6. Ready-to-use reagents or

requiring reconstitution All the reagents are ready-to-use, except the lysis reagent

7. Training For an operator trained in standard techniques of microbiology, 2 or 3 days

training are required.

For an operator trained in PCR techniques, one day training is required.

8. Workflow study

Steps

Average time for a sample

(min)

Average time for 48 samples

(min)

ISO 6579

standard BAX method ISO 6579 BAX method

Sampling, stomaching 7 7 120 120

Subculture in

- RVS and MKTTn

- BHI

3

1

90

45

BAX® PCR test / 1 / 45

Streaking enrichment

broth onto selective

agar plates

10 / 150 /

20 min 9 min 7 min 30 4 min 30

In case of positive samples, time for confirmation has to be added.

The time needed to confirm one colony is estimated to 5 minutes.

The major interest of the method is to screen the negative samples.

OXOID



9. Time to result Negative samples

Step ISO 6579

BAX method

General protocol

(19 – 24 h)

Short protocol

(9 h)

Sampling, enrichment step Day 0 Day 0 Day 0

Subcultures (RVS/MKTTn, BHI) Day 1 Day 1 /

BAX PCR test / Day 1 Day 0

Streaking onto selective agar

plates Day 2 / /

Plates reading Day 3 / /

Negative results obtention Day 3 Day 1 Day 0

Presumptive samples or and positive samples

Step ISO 6579

BAX method

General protocol

(19 – 24 h)

Short protocol

(9 h)

Sampling, enrichment step Day 0 Day 0 Day 0

Subcultures (RVS/MKTTn, BHI) Day 1 Day 1 /

BAX PCR test / Day 1 Day 0

Streaking onto selective agar

plates Day 2 Day 2 Day 0

Plates reading Day 3 Day 3 Day 1

Confirmatory tests Day 5 Day 3 Day 1

Positive results obtention Day 5 Day 3 Day 1

10. Technician qualification Technician in food microbiology

11. 11. Common step with

reference method Pre-enrichment step (16 – 20 h at 37°C in BPW)

12.Traceability All the results are saved in an electronic file format.

13.Maintenance The user’s guide explains some possibly encountered problems

OXOID



2.4 Conclusion

The methods comparative study conclusions are:

The BAX® System PCR Assay Salmonella spp. shows satisfying

relative accuracy, relative specificity and sensitivity.

The detection levels are comprised between:

- 0.2 and 1.3 CFU/25 g or 25 ml for the reference method,

- 0.1 and 1.1 CFU/25 g or 25 ml for the alternative method.

The BAX® System PCR Assay Salmonella spp. is specific and

selective.

The inter-laboratory study conclusions are:

The BAX® System PCR Assay Salmonella spp. and the reference

method show equivalent performances (accordance,

concordance, odds ratio).

OXOID



Appendix 1 – Flow diagrams of the alternative method

General protocol (raw meats and raw poultry meats excluded)

25 g + 225 ml BPW

16 – 20 h at 37°C ± 1°C

Transfer 10 µl into 500 µl BHI

Incubation for 3 h at 37°C ± 1°C

Lysis

PCR

Streaking onto selective agar plates

Tests described in the ISO 6579 standard

Protocol for raw meats and raw poultry meats

25 g + 225 ml BPW

16 – 20 h at 37°C ± 1°C

Lysis

PCR



OXOID



Protocol for dairy products (milk powder excluded)

25 g + 225 ml BPW + 20 mg/l novobiocin

20 - 24 h at 42°C ± 1°C

Lysis

PCR

Confirmation:

0.1 ml enrichment broth + 10 ml/RVS

24 h ± 2h at 41.5°C



Protocol for raw meats

25 g + 225 ml MP broth

24 h at 42°C ± 1°C

Lysis

PCR

Confirmation:

subculture in RVS broth for 24 h ± 3 h at 41.5°C ± 1°C

Streaking onto BrillianceTM Salmonella

Latex tests or tests described in the ISO 6579 standard

OXOID



Protocol for raw beef meats

25 g + 225 ml MP broth (pre-warmed)

9 - 24 h at 42°C ± 1°C

Lysis

PCR

Confirmation:

subculture in RVS broth for 24 h ± 3 h at 41.5°C ± 1°C

Streaking onto BrillianceTM Salmonella

Latex tests or tests described in the ISO 6579 standard

OXOID



Appendix 2 – NF EN ISO 6579: 2002 reference method:

Microbiology of food and animal feeding stuffs –

Horizontal method for the detection of Salmonella spp.

25 g of sample + 225 ml BPW

Incubation

18 h 2 h at 37°C 1°C

0.1 ml BPW 1 ml BPW

10 ml RVS 10 ml MKTTn

Incubation 24 h 3 h at 41.5°C 1°C Incubation 24 h 3 h at 37°C 1°C

Streak on XLD and a second medium

Incubation 24 h 3 h at 37°C 1°C

Streak 1 characteristic colony

onto Nutrient agar

(Take 4 other colonies if

the first one is negative)

Incubation 24 h 3 h at 37°C 1°C

Biochemical and serological confirmation

OXOID



Appendix 3 – Relative accuracy: raw data in French

(Study realized by Institut Pasteur de Lille)

OXOID



Meat products

OXOID



Meat products

OXOID



Meat products

OXOID



Dairy products

OXOID



Dairy products

OXOID




OXOID




OXOID




OXOID



Various products

OXOID



Various products

OXOID



Various products

OXOID



Feed stuff

OXOID



Feed stuff

OXOID




OXOID




OXOID



Appendix 4 – Relative accuracy: raw data (Study realized by ADRIA Développement)

June 2008 Bold typing: artificial contaminations d: doubtful colonies NC : non typical colonies on nutrient agar ni: non isolated colonies PD: positive deviation PA: positive agreement ND: negative deviation NA: negative agreement

Sample N°

Product (French name)

Artificial contaminations

Global result

Reference method ISO 6579 BAX Salmonella MP method

RVS MKTTn

Result

Incubation for 24h a 42°C

Strain Origin Injury applied Injury

evaluation

Inoculation level

(CFU/sample) XLD Hektoen XLD Hektoen PCR

Confirmation Result Agreement

OSCMII Latex Reference

tests

257 Paupiette de veau / / / / / - - - - +ni(Citrobacter

freundii) - - / / / - NA

258 Cuisse de poule / / / / / + + + + + + - - / / - ND

259 Pieds arrière de porc / / / / / - - - - +d

(Providencia) - - / / / - NA

260 Filet de poule / / / / / - +d(Citrobacter

youngae) - - - - - / / / - NA

261 Poule / / / / / + + + + +ni + + + + + + PA 262 VSM volaille / / / / / + + + + + + + + + + + PA

263 Poule / / / / / + - + +ni +(Citrobacter

freundii) + + + + + + PA

264 Jambon frais / / / / / - + +dni +d +d - - - / / - NA 265 Poule / / / / / + + + + +?ni + + + + + + PA 266 Viande de porc hachée / / / / / - - - - - - - / / / - NA 267 Jambon frais / / / / / - - - - - - - / / / - NA 268 Cuisse de poule / / / / / - - - - - - - / / / - NA

269 Morceaux de poule avec peau

/ / / / / + + + + + + + + + + + PA

270 Paupiette e veau / / / / / + + +ni +ni +ni + + + + + + PA 271 Cuisse de poule / / / / / + + + + + + + + + + + PA 272 Cuisse de poule / / / / / + + + + + + + + + + + PA

273 Paupiette de veau / / / / / - +ni(Citrobacter

freundii) - - - - - / / / - NA

274 Paupiette de veau / / / / / + +ni + +ni +ni + - - / / - ND 275 Viande blanche / / / / / - - - +d - - - / / / - NA 276 Lapin au paprika / / / / / - - - - - - - / / / - NA 289 Steak haché frais Salmonella Newport 586 Beef carcass -20°C 2,19 0 + + +ni + + + + + + + + PA 290 Bavette Salmonella Newport 586 Carcasse de bœuf -20°C 2,19 0 + + + + + + + + + + + PA 291 Steak haché frais Salmonella Newport 586 Carcasse de bœuf -20°C 2,19 0 - - - - - - + + + + + PD 292 Tranche à bifteck Salmonella Infantis 128 Ground beef -20°C >2,13 2,6 - - - - - - + + + + + PD 293 Viande hachée Salmonella Infantis 128 Ground beef -20°C >2,13 2,6 - - - - - - + + + + + PD 294 Jarret de mouton avec os Salmonella Typhimurium ST391 Environmental sample -20°C 0,51 1,2 + + + + + + + + + + + PA 295 Côte d'agneau Salmonella Typhimurium ST391 Environmental sample -20°C 0,51 1,2 + + + + + + + + + + + PA 296 Poitrine d'agneau Salmonella Typhimurium ST391 Environmental sample -20°C 0,51 1,2 + + + + + + + + + + + PA 297 Rumsteak en tournedos Salmonella Infantis 128 Ground beef -20°C >2,13 2,6 + + +ni + + + - - / / - ND 298 Tranche à bifteck Salmonella Infantis 128 Ground beef -20°C >2,13 2,6 - - - - - - + + + + + PD 342 Paupiette de veau / / / / / + + + + + + - - / / - ND 343 Paupiette de veau / / / / / + + + + + + - + + + - ND 344 Jambon frais / / / / / - - - - - - - / / / - NA 345 Blanc de poule sans peau / / / / / - - - - - - - / / / - NA 346 Blanc de poule sans peau / / / / / + + + + + + - - / / - ND 347 Blanc de poule sans peau / / / / / - - + - - - - / / / - NA 348 Viande blanche / / / / / - - - - - - - / / / - NA 349 Mêlée de porc / / / / / + + + + + + + + + + + PA

Analyses performed according to the COFRAC accreditation

OXOID



Sample N°



Global result

Reference method ISO 6579 BAX Salmonella MP method

RVS MKTTn

Result

Incubation for 24h a 42°C

Strain Origin Injury applied Injury

evaluation

Inoculation level

(CFU/sample) XLD Hektoen XLD Hektoen PCR

Confirmation Result Agreement


tests


/ / / / / - - - - - - - / / / - NA


/ / / / / + + + + + + - + + + - ND

352 Poule / / / / / + + + + + + + + + + + PA 431 Crépinettes

- - - - - - - / / / - NA

432 Crépinettes

+ + + + + + + + + + + PA 433 Poitrine de porc

- - - - - - - / / / - NA

434 Poitrine de porc

- - - - - - - / / / - NA 435 Poitrine de porc

- - - - +d(NC) - - / / / - NA

436 Langue de porc

- - - - - - - / / / - NA

437 Langue de porc

- - - - +(Citrobacter

koseri) - - / / / - NA

438 Langue de porc

+ + + + + + + + + + + PA 439 Steak haché Cross contamination with carcass wipe - - +(NC) - - - - / / / - NA 440 Steak haché Cross contamination with carcass wipe - - +(NC) - - - - / / / - NA 441 Steak haché Cross contamination with carcass wipe - - +(NC) - - - - / / / - NA 442 Steak haché Cross contamination with carcass wipe - +(NC) - - +(NC) - - / / / - NA 443 Steak haché Cross contamination with carcass wipe - +(NC) +(NC) - - - - / / / - NA 444 Steak haché Cross contamination with carcass wipe - - +(NC) - - - - / / / - NA 445 Steak haché Cross contamination with carcass wipe - - - - - - - / / / - NA

543 Boulettes au bœuf Salmonella Bredeney 396 Beef 2 days at 4°C / 2 days at -20°C

0,73 15-15-15-13-8

(13,2) + + + + + + + + + + + PA

544 Steak haché Salmonella Typhimmurium AOOCO60

Beef 2 days at 4°C / 2 days at -20°C

0,62 1-1-2-3-52

(1,8) + + + + + + + + + + + PA

545 Steak grill oignons Salmonella Bredeney 396 Beef -20°C 0,79 8-2-6-3-2

(4,2) + - + + + + + + + + + PA

546 Steak haché pur bœuf Salmonella Newport 586 Beef -20°C 1,12 3-1-0-7-1

(2,4) - - - - - - + + + + + PD

547 Viande bovine à bourguignon

Salmonella Dublin Ad 529 Beef -20°C 0,63 5-3-2-5-2

(4) + - - + + + + + + + + PA

579 VSM volaille

- - - - - - - / / / - NA 580 VSM volaille

- - - - - - - / / / - NA

581 Peau de poule

- - - - - - - / / / - NA 582 Cuisse de poule

- - - - - - - / / / - NA

OXOID



November 2008

Bold typing: artificial contaminations d: doubtful colonies NC : non typical colonies on nutrient agar ni: non isolated colonies PD: positive deviation PA: positive agreement ND: negative deviation NA: negative agreement

Sample N°



Global result

Reference method ISO 6579 Bax Salmonella MP method

RVS MKTTn

Result

Incubation 9h-42°c Incubation 24h-42°c

Strain Origin Injury

applied Injury

evaluation

Inoculation level

(CFU/ sample)

XLD Hektoen XLD Hektoen PCR

Confirmation

Result Agreement PCR

Confirmation

Result Agreement OSCMII Latex

Reference tests


tests

289 Steak haché frais Salmonella Newport 586 Beef carcass -20°C 2,19 0 + + +ni + + + + + + + + PA + + + + + PA

290 Bavette Salmonella Newport 586 Beef carcass -20°C 2,19 0 + + + + + + - + + + - ND + + + + + PA

291 Steak haché frais Salmonella Newport 586 Beef carcass -20°C 2,19 0 - - - - - - + + + + + PD + + + + + PD

292 Tranche à bifteck Salmonella Infantis 128 Ground beef -20°C >2,13 2,6 - - - - - - + + + + + PD + + + + + PD

293 Viande hachée Salmonella Infantis 128 Ground beef -20°C >2,13 2,6 - - - - - - + + + + + PD + + + + + PD

297 Rumsteak en tournedos Salmonella Infantis 128 Ground beef -20°C >2,13 2,6 + + +ni + + + - - / / - ND - - / / - ND

298 Tranche à bifteck Salmonella Infantis 128 Ground beef -20°C >2,13 2,6 - - - - - - + + + + + PD + + + + + PD

439 Steak haché Cross contamination with carcass wipe - - +(NC) - - - - - / / - NA - - / / - NA



442 Steak haché Cross contamination with carcass wipe - +

(NC) - - +(NC) - - - / / - NA - - / / - NA

443 Steak haché Cross contamination with carcass wipe - +

(NC) +(NC) - - - - - / / - NA - - / / - NA


445 Steak haché Cross contamination with carcass wipe - - - - - - - - / / - NA - - / / - NA

543 Boulettes au bœuf Salmonella Bredeney 396 Beef

2 days at 4°C / 2 days at -20°C

0,73 15-15-15-13-8 (13,2)

+ + + + + + - + + + - ND + + + + + PA

544 Steak haché Salmonella Typhimmurium AOOCO60

Beef

2 days at 4°C / 2 days

at -20°C

0,62 1-1-2-3-52

(1,8) + + + + + + + + + + + PA + + + + + PA

545 Steak grill oignons surgelé

Salmonella Bredeney 396 Beef -20°c 0,79 8-2-6-3-2

(4,2) + - + + + + - + + + - ND + + + + + PA

546 Steak haché pur bœuf Salmonella Newport 586 Beef -20°c 1,12 3-1-0-7-1

(2,4) - - - - - - - + + + - NA + + + + + PD


Salmonella dublin Ad 529 Beef -20°c 0,63 5-3-2-5-2

(4) + - - + + + + + + + + PA + + + + + PA

1281 Steak de bœuf mariné

+ + + + + + + + + + + PA + + + + + PA

1282 Hampe (1)

+ + 1 col

+ 2 col + 2 col + 2 col + + + + + + PA + + + + + PA

1283 Hampe (2)

+ - + 2 col +/- + NC + + + + + + PA + + + + + PA

1284 Carpaccio de bœuf surgelé

Salmonella Panama 195 Ground beef -20°C 0,42 6-6-7-2-5(5,2)

+ + + + + + + + + + + PA + + + + + PA

1285 Viande hachée pur bœuf surgelé


+ + + + + + + + + + + PA + + + + + PA

1286 Tranche de tournedos surgelé


+ + + + + + + + + + + PA + + + + + PA

1287 Macreuse à braiser Salmonella Panama 195 Ground beef -20°C 0,42 6-6-7-2-5(5,2)

+ + + + + + + + + + + PA + + + + + PA

1288 Viande hachée de bœuf surgelée

- - - - - - - / / / - NA - / / / - NA

1289 Rumsteak à griller

- - - - - - - / / / - NA - / / / - NA

Analyses performed according to the COFRAC accreditation

OXOID



Sample N°



Global result


RVS MKTTn

Result



applied Injury

evaluation

Inoculation level

(CFU/ sample)


Confirmation


Confirmation


Reference tests


tests

1290 Viande bovine Aiguillette

- - - +?3 col

Hafnia alvei - - - / / / - NA - / / / - NA

1291 Viande bovine bavette surgelé

- - - - - - - / / / - NA - / / / - NA

1292 Viande bovine bourguignon collier poitrine

Salmonella Panama 195 Ground beef 1 month at 4°C

0,49 1-1-0-0-3(1) + + + + + + + + + + + PA + + + + + PA

1293 Viande bovine rumsteak à griller


0,49 1-1-0-0-3(1) + + + + + + + + + + + PA + + + + + PA

1294 Gîte de noix à bifteak Salmonella Panama 195 Ground beef 1 month at 4°C

0,49 1-1-0-0-3(1) + + + + + + + + + + + PA + + + + + PA

1295 Viande bovine bavette d'aloyau

Salmonella Newport 586 Beef carcass 1 month at 4°C

0,57 1-0-0-1-2(1) + + + + + + + + + + + PA + + + + + PA

1296 Steak hachée frais 5% MG


0,57 1-0-0-1-2(1) + + + + + + + + + + + PA + + + + + PA

1297 Steak hachée frais 15% MG


0,57 1-0-0-1-2(1) + + + + + + + + + + + PA + + + + + PA

1298 Viande bovine tranche à fondue


0,49 1-1-0-0-3(1) + + + + + + + + + + + PA + + + + + PA



0,57 1-0-0-1-2(1) + + + + + + + + + + + PA + + + + + PA


- - - +/-Hafnia

alvei - - - / / / - NA - / / / - NA

1301 Macreuse à braiser surgelé

- - - - - - - / / / - NA - / / / - NA

1302 Viande hachée pur bœuf surgelé

- - - +/-

Providencia stuartii

- - - / / / - NA - / / / - NA

1303 Steak hachée frais pur bœuf 5%MG

- +NC - +

Citrobacter youngae

+ Citrobacter youngae

- - / / / - NA - / / / - NA

1304 Gîte de noix à bifteak

- - - - - - - / / / - NA - / / / - NA


- - - - - - - / / / - NA - / / / - NA

1307 Viande bovine bavette d'aloyau

- - - - - - - / / / - NA - / / / - NA

1308 Viande bovine basse côte à griller

- - - - - - - / / / - NA - / / / - NA


- - - - - - - / / / - NA - / / / - NA

1310 Viande bovine tranche en tournedos

- - - - - - - / / / - NA - / / / - NA

1311 Plat de côte avec os pour pot-au-feu

- - - - - - - / / / - NA - / / / - NA

1312 Gîte de noix à bifteak Salmonella Panama 8 Ground beef -20°C 0,57 6-4-2-4-5(4,2)

+ + + + + + + + + + + PA + + + + + PA

1313 Viande bovine rumsteak à griller


+ + + + + + + + + + + PA + + + + + PA

1314 Viande hachée de bœuf surgelée

Salmonella Typhimurium AOOCO60

Ground beef -20°C 0,90 2-6-6-3-8(5) + + + + + + + + + + + PA + + + + + PA

1315 Boulettes au bœuf surgelé

Salmonella Typhimurium AOOCO60

Ground beef -20°C 0,90 2-6-6-3-8(5) + + + + + + + + + + + PA + + + + + PA


- - - - - - - / / / - NA - / / / - NA


- - - - - - - / / / - NA - / / / - NA

1318 Steak haché 5%MG surgelé

- - - - - - - / / / - NA - / / / - NA

OXOID



Sample N°



Global result


RVS MKTTn

Result



applied Injury

evaluation

Inoculation level

(CFU/ sample)


Confirmation


Confirmation


Reference tests


tests

1319 Steak haché 15%MG surgelé

- - - - - - - / / / - NA - / / / - NA

1320 Haché pur bœuf surgelé

Salmonella Infantis 128 Ground beef -20°C >1,70 7-7-8-8-9(7,8)

+ + + + + + + + + + + PA + + + + + PA

1321 Steak haché surgelé 100% Charolais

- - - - - - - / / / - NA - / / / - NA

1322 Steak haché extra moelleux surgelé


+ - - +/- + + + + + + + PA + + + + + PA

1323 Préparation à 50% de viande hachée de bœuf

- - - - - - - / / / - NA - / / / - NA

1324 Carpaccio de bœuf surgelé


+ - - + + + + + + + + PA + + + + + PA

1325 Plat de côte avec os pour pot-au-feu

- - +/- - +/- - - / / / - NA - / / / - NA


- - - - - - - / / / - NA - / / / - NA

OXOID



Appendix 5 – Inclusivity / exclusivity: raw data (in French)

Inclusivity

OXOID



Inclusivity

OXOID



Inclusivity

OXOID



Inclusivity

OXOID



Inclusivity

OXOID



Inclusivity (ADRIA Développement – 2008)

N° Strain Reference Origin

Inoculation

level

cfu/225ml

PCR BAX BrillianceTM

Salmonella Latex

1. Salmonella Anatum Ad 298 Dairy product 12 + + +

2. Salmonella diarizonae 47:Iv:z53 Ad 478 Seafood 37 + + +

3. Salmonella Bongori Ad 599 Environmental

sample 46 + + pale colonies + (weak)

4. Salmonella Bovismorficans 132 Meat product 22 + + +

5. Salmonella Braenderup 111 Meat product 26 + + +

6. Salmonella Brando 596 Meat product 16 + + +

7. Salmonella Bredeney 396 / 20 + + +

8. Salmonella Cerro Ad 689 Pet food 17 + + +

9. Salmonella Derby 18 Meat product 25 + + +

10. Salmonella Diarizonae Ad 594 Meat product 27 + + +

11. Salmonella Diarizonae Ad 595 Dairy product 13 + + + (weak)

12. Salmonella Dublin Ad 529 Meat product 50 + + +

13. Salmonella Enteritidis 657 Egg product 23 + + +

14. Salmonella Gallinarum Ad 300 Environmental

sample 14

- (9h)/

+(24h)

+ microscopic

colonies +

15. Salmonella Hadar 24871 Meat product 10 + + +

16. Salmonella Heidelberg A00E005 Environmental

sample 39 + + +

17. Salmonella houtenae Ad 596 Dairy product 35 + + microscopic

colonies -

18. Salmonella Indiana 2 Seafood 47 + + +

19. Salmonella indica Ad 600 Environmental

sample 30 + + +

20. Salmonella Infantis F401B Dairy product 28 + + +

21. Salmonella Kottbus 1 Environmental

sample 62 + + +

22. Salmonella Lagos 173 Meat product 38 + + +

23. Salmonella Lille 37 / 9 + + +

24. Salmonella Livingstone F104 Pet food 20 + + +

25. Salmonella London 326 Meat product 32 + + +

26. Salmonella Manhattan 900 Environmental

sample 26 + + +

27. Salmonella Mbandaka 81 Egg product 35 + + + fin

28. Salmonella Montevideo Ad 912 Dairy product 20 + + +

OXOID



N° Strain Reference Origin

Inoculation

level

cfu/225ml

PCR BAX BrillianceTM

Salmonella Latex

29. Salmonella Newbrunswick 436 Meat product 22 + + +

30. Salmonella Newport 586 Meat product 37 + + +

31. Salmonella Panama 195 Meat product 42 + + +

32. Salmonella Paratyphi A ATCC 9150 / 26 + + microscopic

colonies + fin

33. Salmonella Paratyphi B Ad 301 Clinical sample 61 + + +

34. Salmonella Paratyphi C ATCC 13428 / 46 + + +

35. Salmonella Regent 328 Meat product 23 + + +

36. Salmonella Rissen 39 / 50 + + +

37. Salmonella Saintpaul F31 Seafood 20 + + +

38. Salmonella salamae 42:r:- 105 / 8 + + +

39. Salmonella salamae

1,13,23:gmt:enx Ad 450 Dairy product 29 + + + (weak)

40. Salmonella salamae

42:b:enxz15 Ad 593 Vegetables 3

-

(9h)/+(24h) + + (weak)

41. Salmonella Senftenberg Ad 355 Seafood 35 + + +

42. Salmonella Sternhauze Ad 500 / 24 + + +

43. Salmonella Tennesse A00E006 Environmental

sample 15 + + +

44. Salmonella Thompson AER301 Meat product 27 + + +

45. Salmonella Typhi Ad 302 Clinical sample 100 + + +

46. Salmonella Typhimurium A00C060 Meat product 74 + + +

47. Salmonella Veneziana 233 / 57 + + +

48. Salmonella Virchow F276 Vegetables 25 + + +

49. Salmonella Wayne Ad 502 / 10 + + microscopic

colonies

+/-

(weak)

50. Salmonella Worthington 3506 Ready to eat meal 46 + + +

OXOID



Inclusivity (ADRIA Développement – 2014)

Strain Reference Origin cfu/225ml

BAX Real Time Salmonella method MP broth pre-warmed for 9 h at 41.5°C

PCR Confirmatory

tests Latex

Result Ct Brilliance

Salmonella

1 Salmonella arizonae SIIIa 51:z4,z23:-

CIP 5523 Turkey 2 + 43.5 + +

(weak)

2 Salmonella diarizonae SIIIb 38:IV:z53

Ad451 Raw milk cheese

8 + 35.4 + +

(weak)

3 Salmonella Typhimurium SI 1,4,[5],12:-:- (variant immobile)

Ad 1233 Tiramisu 17 + 32.7 + +

4 Salmonella Typhimurium SI 1,4,[5],12:i:- (variant monophasique)

Ad 1334 Ready-to-eat meal (meat)

9 + 34.5 + +

5 Salmonella Typhimurium SI 1,4,[5],12:-:1,2 (variant monophasique)

Ad 1335 Enviromental sample

7 + 42.8 + +

OXOID



Exclusivity

OXOID



Exclusivity

Documents

Summary report - NF Validationnf-validation.afnor.org/en/wp-content/uploads/sites/2/2014/03/Synt... · Summary report EN ISO 16140 validation study of the BAX® System PCR Assay Salmonella