NF VALIDATION Validation study according to the EN ISO standard. Summary report. EN ISO Validation study of the Pathatrix Auto

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1 ACCREDITATION N PORTEE DISPONIBLE SUR LIFE TECHNOLOGIES Route de l Orme des Merisiers Immeuble Le Discovery Parc Technologique SAINT AUBIN NF VALIDATION Validation study according to the EN ISO standard Summary report EN ISO Validation study of the Pathatrix Auto spp. for individual samples and up to 10-pooling linked to selective agar plates in raw beef meats, ready-toreheat and ready-to-eat meat products (including poultry), heat treated milk and dairy products Qualitative method This report includes 75 pages, with 11 appendixes. Only copies including the totality of this report are authorized. Competences of the laboratory are certified by COFRAC accreditation for the analyses marked with symbol. Version 0 March 25, 2014 ADRIA DEVELOPPEMENT Creac h Gwen - F QUIMPER Cedex - Tél. (33) Fax (33) adria.developpement@adria.tm.fr - Site web : ASSOCIATION LOI DE N SIRET N EXISTENCE N TVA FR

2 1 AIM OF THE STUDY 5 2 METHODS PROTOCOLS Reference method protocol Alternative method protocol 5 3 METHOD COMPARISON STUDY Relative accuracy, relative specificity and relative sensitivity Number and nature of samples Artificial contamination of samples Confirmation protocols Test s Calculation of relative accuracy (AC), relative sensitivity (SE) and relative specificity (SP) Analysis of discordants Confirmations Selective enrichment broth storage at 2-8 C for 32 h Relative detection level Matrices Contamination protocol Results Conclusion Inclusivity / exclusivity Test protocols Results Conclusion Practicability 20 ADRIA Développement 2/75 March 25, 2014 (Pathatrix Plate Summary Report Version 0)

3 4 INTERLABORATORY STUDY ORGANISATION AND RESULTS Study organisation Experimental parameters control Contamination level before inoculation, levels obtained after the artificial contaminations of the samples Logistic conditions Conclusion Results analysis Aerobic mesophilic flora enumeration Expert lab s Collaborator lab s Results interpretation Specificity and sensitivity for each method Relative accuracy (AC) Discordant s Interpretation Comparison of the relative accuracy, specificity and sensitivity values Accordance (DA) Concordance Odds Ratio (COR) 32 5 CONCLUSION 33 Appendix 1 Reference method: NF EN ISO 6579: 2002: Microbiology of food and animal feeding stuffs Horizontal method for the detection of spp. 35 Appendix 2 Method alternative protocols 36 Appendix 3 Relative accuracy: raw data 37 Appendix 4 Relative detection levels: raw data 45 Appendix 5 Inclusivity and exclusivity: raw data 48 Appendix 6 Results obtained by the Expert Laboratory 53 Appendix 7 Results obtained by each Collaborator 54 Appendix 8 Specificity and sensitivity 68 Appendix 9 Paired s of the alternative and reference methods for each level 69 Appendix 10 Accordance 70 Appendix 11 Concordance 73 ADRIA Développement 3/75 March 25, 2014 (Pathatrix Plate Summary Report Version 0)

4 Before comment Quality Assurance documents related to this study can be consulted upon request by LIFE TECHNOLOGIES / APPLIED BIOSYSTEMS. The technical protocol and the interpretation were realised according to the EN ISO and the AFNOR technical rules. Company: LIFE TECHNOLOGIES Route de l Orme des Merisiers Immeuble Le Discovery Parc Technologique SAINT AUBIN (France) Expert Laboratory: ADRIA Développement ZA Creac h Gwen QUIMPER Cedex - France Studied method: Pathatrix Auto spp. for individual samples and up to 10-pooling linked to selective agar plates Validation standard : NF EN ISO (October 2003) : Food microbiology Protocol for the validation of alternative methods Reference method : NF EN ISO 6579 (2002): Horizontal method for the detection of spp. Scope: Raw beef meats, ready-to-reheat and ready-toeat meat products (including poultry) Heat treated milk and dairy products Certification body: AFNOR Certification Analysis performed according to the COFRAC accreditation ADRIA Développement 4/75 March 25, 2014 (Pathatrix Plate Summary Report Version 0)

5 1 AIM OF THE STUDY The Pathatrix Auto spp. for individual samples and up to 10-pooling linked to selective agar plates was validated in November 2013 for raw beef meats, ready-to-reheat and ready-to-eat meat products (including poultry), heat treated milk and dairy products, according to the EN ISO protocol. The Pathatrix Auto spp. method allows for sample pooling (up to 10 samples). Thus, the developed validation protocol is based on the analyses on pooled samples. The pooling can be done between samples from the same type. During the validation study, the following criteria were evaluated: Method comparison study: - the practicability, - the inclusivity and the exclusivity, - the relative detection limit, - the relative accuracy, the relative sensitivity and the relative specificity. Inter-laboratory study. 2 METHODS PROTOCOLS 2.1 Reference method protocol The reference method is the ISO 6579: Horizontal method for the detection of spp (See Appendix 1). 2.2 Alternative method protocol The Pathatrix Auto spp. 10-pooling protocol is an automated, large volume, Re-circulating Immuno-Magnetic Separation (RIMS) sample processing device for rapid detection of specific pathogens from pre-enriched pooled food samples. Analysis performed according to the COFRAC accreditation ADRIA Développement 5/75 March 25, 2014 (Pathatrix Plate Summary Report Version 0)

6 After a RIMS step, detection is done by plating strains on and TM Agar (Oxoid). The confirmations are done with a latex test (Oxoid). During the validation, characteristic colonies are confirmed with the ISO 6579 confirmation tests. Pathatrix protocols can be performed on individual samples, and on pooled samples (up to 10 samples per pool). A specific protocol design is proposed in this ISO validation study. Based on the background microflora level of the tested samples, different protocols are proposed: - Protocol 1: for raw beef meats, ready-to-reheat and ready-to-eat meat products (including poultry) * 1/10 dilution in pre-warmed (37 C ± 1 C) BPW, * Incubation for 20 h ± 2 h at 37 C ±1 C - Protocol 2: for heat treated milk and dairy products * 1/10 dilution in pre-warmed BPW + Brilliant Green (0.002 %) * Incubation for 20 h ± 2 h at 37 C ±1 C For potentially acidic and alkaline samples, this additional step must be applied: - dilute the sample according to the enrichment protocol, - leave to stand for 60 ± 5 min at room temperature, - mix by stomaching for 1 min at normal speed and determine the ph. If necessary, adjust the ph to 6.8 ± 0.2 The ph of the BPW suspensions before incubation for potentially acidic samples are provided in Appendix 3 in raw data tables. During the study, it was never necessary to adjust it before incubation. The protocols are described in Appendix 2. ADRIA Développement 6/75 March 25, 2014 (Pathatrix Plate Summary Report Version 0)

7 3 METHOD COMPARISON STUDY 3.1 Relative accuracy, relative specificity and relative sensitivity The relative accuracy is the closeness of agreement between a test and the accepted reference value. The 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. The 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) Number and nature of samples A total of 153 samples were analyzed. The distribution per tested category, type and pre-enrichment protocol is given in Table 1: Table 1 Distribution per tested category and type Categories Raw beef meats, ready-toreheat and ready-to-eat meat products (including poultry) Heat treated milks and dairy products TOTAL Types Positive Negative samples samples Total - Ground beef, beef trim Cooked delicatessen Ready-to-reheat foods Total Milk powders and infant formula without and with probiotic - Fermented milk and yoghurts Pasteurized milks Total ADRIA Développement 7/75 March 25, 2014 (Pathatrix Plate Summary Report Version 0)

8 Number of samples Life Technologies Artificial contamination of samples Artificial contaminations were performed by spiking. The strains were stressed using various injury protocols. The injury efficiency was evaluated by comparing enumeration s onto selective and non selective agars (respectively and TSYEA). 73 samples were artificially contaminated, using 25 different strains and 8 injury protocols. 62 samples gave a positive. Most of the spiking inoculations, after injury protocols on the inoculum, were lower or equal to 5 CFU/sample (See figures 1 and 2). No more than 5 positive s were obtained within the same strain. Figure 1 Inoculation levels used for spiking Figure 2 Inoculated and positive samples according to the level of contamination All inoculated samples Positive samples x 5 CFU/sample 5<x 10 CFU/sample x>10 CFU/sample Only one product was naturally contaminated. ADRIA Développement 8/75 March 25, 2014 (Pathatrix Plate Summary Report Version 0)

9 Indiviidual samples Pooled samples Life Technologies Confirmation protocols The presumptive positive colonies were confirmed by latex test directly from selective agar plates Test s Raw data per category are given in Appendix 3. The paired s per category are given in the following tables. Table 2 All samples Response Reference method positive (R+) Reference method negative (R-) Alternative method Positive agreement (A+/R+) Positive deviation (R-/A+) positive (A+) PA = 55 PD = 2 Alternative method Negative deviation (A-/R+) Negative agreement (A-/R-) negative (A-) ND = 6 (PPND = 0) NA = 90 (PPNA = 0) Alternative method Positive agreement (A+/R+) Positive deviation (R-/A+) positive (A+) PA = 57 PD = 3 Alternative method Negative deviation (A-/R+) Negative agreement (A-/R-) negative (A-) ND = 4 (PPND = 0) NA = 89 (PPNA = 0) PP: positive presumptive non confirmed samples PD = positive deviation (R-/A+) ND = negative deviation (A-/R+) ADRIA Développement 9/75 March 25, 2014 (Pathatrix Plate Summary Report Version 0)

10 Indiviidual samples Pooled samples Indiviidual samples Pooled samples Life Technologies Results per category of samples Table 3 Raw beef meats, ready-to-reheat and ready-to-eat meat products (including poultry) Response Reference method positive (R+) Reference method negative (R-) Alternative method Positive agreement (A+/R+) Positive deviation (R-/A+) positive (A+) PA = 29 PD = 1 Alternative method Negative deviation (A-/R+) Negative agreement (A-/R-) negative (A-) ND = 2 (PPND = 0) NA = 42 (PPNA = 0) Alternative method Positive agreement (A+/R+) Positive deviation (R-/A+) positive (A+) PA = 30 PD = 1 Alternative method Negative deviation (A-/R+) Negative agreement (A-/R-) negative (A-) ND = 1 (PPND = 0) NA = 42 (PPNA = 0) Table 4 Heat treated milks and dairy products Response Reference method positive (R+) Reference method negative (R-) Alternative method Positive agreement (A+/R+) Positive deviation (R-/A+) positive (A+) PA = 26 PD = 1 Alternative method Negative deviation (A-/R+) Negative agreement (A-/R-) negative (A-) ND = 4 (PPND = 0) NA = 48 (PPNA = 0) Alternative method Positive agreement (A+/R+) Positive deviation (R-/A+) positive (A+) PA = 27 PD = 2 Alternative method Negative deviation (A-/R+) Negative agreement (A-/R-) negative (A-) ND = 3 (PPND = 0) NA = 47 (PPNA = 0) ADRIA Développement 10/75 March 25, 2014 (Pathatrix Plate Summary Report Version 0)

11 Individual samples Pooled samples Individual samples Pooled samples Life Technologies Results per protocol Table 5 Protocol 1 Response Reference method positive (R+) Reference method negative (R-) Alternative method Positive agreement (A+/R+) Positive deviation (R-/A+) positive (A+) PA = 29 PD = 1 Alternative method Negative deviation (A-/R+) Negative agreement (A-/R-) negative (A-) ND = 2 (PPND = 0) NA = 42 (PPNA = 0) Alternative method Positive agreement (A+/R+) Positive deviation (R-/A+) positive (A+) PA = 30 PD = 1 Alternative method Negative deviation (A-/R+) Negative agreement (A-/R-) negative (A-) ND = 1 (PPND = 0) NA = 42 (PPNA = 0) Table 6 Protocol 2 Response Reference method positive (R+) Reference method negative (R-) Alternative method Positive agreement (A+/R+) Positive deviation (R-/A+) positive (A+) PA = 26 PD = 1 Alternative method Negative deviation (A-/R+) Negative agreement (A-/R-) negative (A-) ND = 4 (PPND = 0) NA = 48 (PPNA = 0) Alternative method Positive agreement (A+/R+) Positive deviation (R-/A+) positive (A+) PA = 27 PD = 2 Alternative method Negative deviation (A-/R+) Negative agreement (A-/R-) negative (A-) ND = 3 (PPND = 0) NA = 47 (PPNA = 0) ADRIA Développement 11/75 March 25, 2014 (Pathatrix Plate Summary Report Version 0)

12 Table 7 Calculation of relative accuracy (AC), relative sensitivity (SE) and relative specificity (SP) Per category of samples Category PA NA ND PD N Raw beef meats, readyto-reheat and ready-toeat meat products (including poultry) Heat treated milk and dairy products Relative accuracy AC (%) [100x(PA+NA])/N] N+ PA + ND Relative sensitivity SE (%) [100xPA]/N+] N- NA + PD Relative specificity SP (%) [100xNA]/N-] Total products Category PA NA ND PD N Raw beef meats, readyto-reheat and ready-toeat meat products (including poultry) Heat treated milk and dairy products Relative accuracy AC (%) [100x(PA+NA])/N] N+ PA + ND Relative sensitivity SE (%) [100xPA]/N+] N- NA + PD Relative specificity SP (%) [100xNA]/N-] Total products Table 8 Calculation of relative accuracy (AC), relative sensitivity (SE) and relative specificity (SP) Per protocol Protocol 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-] Total products Protocol 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-] Total products ADRIA Développement 12/75 March 25, 2014 (Pathatrix Plate Summary Report Version 0)

13 3.1.5 Calculation of relative accuracy (AC), relative sensitivity (SE) and relative specificity (SP) The alternative method percentage values are: Alternative method Relative accuracy : AC 94.8 % 95.4 % Relative specificity : SP 97.8 % 96.7 % Relative sensitivity : SE 90.2 % 93.4 % Sensitivity of both methods, when the positive deviations of the alternative method are considered, is presented below: Alternative method Alternative method 90.5 % 93.8 % Reference method 96.8 % 95.3 % The alternative method percentage per protocol values are: Alternative method Protocol 1 Protocol 2 Protocol 1 Protocol 2 Relative accuracy : AC 95.9 % 93.7 % 97.3 % 93.7 % Relative specificity : SP 97.7 % 98.0 % 97.7 % 95.9 % Relative sensitivity : SE 93.5 % 86.7 % 96.8 % 90.0 % Analysis of discordants 6 negative deviations (See Table 9) observed with the pooling protocol, 4 with the individual protocol. 2 positive deviations (See Table 10) were observed for the pooled samples; one additional positive deviation was obtained when the individual samples were analyzed (n 3612). ADRIA Développement 13/75 March 25, 2014 (Pathatrix Plate Summary Report Version 0)

14 Category Protocol Sample Raw beef meats, ready-toreheat and ready-to-eat meat products (including poultry) Heat treated milks and dairy products Table 9 Negative deviations n Category Protocol Sample Raw beef meats, ready-toreheat and ready-to-eat meat products (including poultry) Heat treated milks and dairy products Contamination (level contamination) S. Anatum 6140 (2.4) / 2616 S. Typhimurium 702 (5.0) S. Typhimurium 702 (5.0) S. Ohio Ad 1482 (0.6) S. Ohio Ad 1482 (0.6) 3496 S. Montevideo 510 (0.6) S. Montevideo 510 (0.6) 3611 S. Tennessee Ad 1171 (7.4) S. Tennessee Ad 1171 (7.4) 3613 S. Mbandaka Ad 1722 (1.0) / Total 6 4 Table 10 Positive deviations n Contamination (level contamination) Natural Natural S. Anatum Ad 298 (9.2) S. Anatum Ad 298 (9.2) 3612 / S. Mbandaka Ad 1722 (1.0) Total 2 3 According to the Annex F of the EN ISO standard: Y = PD + ND = = 8 m = PD = 2 M = 0 m > M, the two methods are not different at < Y = PD + ND = = 7 m = PD = 3 M = 0 m > M, the two methods are not different at < The s conclude to show no statistical difference between the alternative method (pooled and single workflows) and the reference method. ADRIA Développement 14/75 March 25, 2014 (Pathatrix Plate Summary Report Version 0)

15 3.1.7 Confirmations The latex test was used to confirm the presumptive colonies onto and Agar. For individual samples analyses, differences were observed between the selective agar plates for 9 samples; they are listed below: Sample n Contamination 3216 S. Bredeney M 3221 S. Panama M 3222 S. Bredeney / S. Dublin Ad M S. Dublin Ad / S. Dublin Ad M S. Mbandaka Ad p 3613 S. Mbandaka Ad (3) - no typical colonies m: minoritary level of target analyte 1/2: 50% level of target analyte M: majoritary level of target analyte st: plate without any colony P: pure culture level of target analyte (x): number of typical colonies For 5 samples, no typical colony was observed on plates while colonies were observed on Agar. For sample n 3613, only few colonies recovered on Agar. For samples n 3605, 3607 and 3608, no typical colony was observed on Agar; these samples were all inoculated with Dublin which can produce white or plate magenta colonies on C8 esterase agar plates. ADRIA Développement 15/75 March 25, 2014 (Pathatrix Plate Summary Report Version 0)

16 3.1.8 Selective enrichment broth storage at 2-8 C for 32 h The positive samples enrichment broths of the alternative method were stored at 2-8 C for 32 h and analyzed a second time. Table 11 Sample n T0 Upper storage 32 h at 2 8 C Final Agreement Final Agreement PA - ND PD - NA The discordant s became: ND = = 5 PD = 3 1 = 2 Y = PD + ND = = 7 m = PD = 2 M = 0 m > M, the two methods are not different at < ADRIA Développement 16/75 March 25, 2014 (Pathatrix Plate Summary Report Version 0)

17 3.2 Relative detection level The relative detection level is the smallest number of culturable microorganisms that can be detected in the sample in 50% of occasions by the alternative and reference methods 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 s. Detection limits were defined by analyzing the different matrix/strain pairs. Four levels were tested. Six replicates of each combination were prepared. In the method workflow, the pooled samples are firstly analyzed as a screening step. If positive s are observed, the samples are analyzed individually in order to identify the contaminated one. This part of the study was run in the opposite flow in order to optimize and facilitate the experimental process. The following matrices were tested: - Ground beef, inoculated with Typhimurium A00C060 (Protocol 1), - Probiotic infant formula, inoculated with Anatum Ad 298 (Protocol 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: The inoculation s levels were the following: - 0 CFU/ g or ml, - level required to get 0 to 50 % positive samples, - level required to get 50 to 75 % positive samples. - level required to get 75 to 100 % positive samples. The samples were analyzed by both methods, and the background microflora were enumerated. ADRIA Développement 17/75 March 25, 2014 (Pathatrix Plate Summary Report Version 0)

18 3.2.3 Results Raw data are given in Appendix 4. Detection levels are presented in the table 12. Table 12 Relative detection level s Relative detection level (CFU / 25 g) according to Spearman-Kärber test 1 Strain / matrix pairs Reference method Alternative method Ground beef / Typhimurium A00C060 (Protocol 1) [0.368; 1.042] [0.321; 1.101] [0.321; 1.101] Probiotic infant formula / Anatum Ad 298 (Protocol 2) [0.487; 1.467] [0.350; 0.863] [0.350; 0.863] Conclusion The relative detection limit for the reference method is comprised between 0.4 and 1.5 log CFU/25 g; the relative detection limit for the alternative method for the 2 tested protocols (pooled and single workflows) is comprised between 0.3 and 1.1 log CFU/25 g. The relative detection limits of the reference and the alternative methods are similar. 1 "Hitchins A. Proposed Use of a 50 % Limit of Detection Value in Defining Uncertainty Limits in the Validation of Presence-Absence Microbial Detection Methods, Draft 10th December, 2003". ADRIA Développement 18/75 March 25, 2014 (Pathatrix Plate Summary Report Version 0)

19 3.3 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 Test protocols Inclusivity strain cultures were performed in BHI medium at 37 C. Dilutions were done in order to inoculate 10 cells/225 ml in pre-warmed BPW + Brilliant Green (0.002%) (Protocol 2). The broths were incubated for 18 h at 37 C before performing the alternative method protocols. The target strains were tested by adding BPW + Brilliant Green in a volume that will mimic the pooling (50 ml). Exclusivity Negative strains cultures were performed in BHI at 37 C. Dilutions were generated in order to inoculate 10 5 cells/ml BPW. The broths were incubated for 22 h at 37 C. The alternative method were then performed Results Raw data are given in Appendix 5. Inclusivity 51 strains were tested and gave a negative. The study was repeated by adding 25 ml UHT milk in the enrichment broth; all the strains grown then on selective plates. For 2 strains ( Paratyphi A ATCC 9150, Rissen 39), it was necessary to inoculate the broth at a higher level in order to obtain colonies on the plates. Exclusivity 30 strains were tested; no typical colonies were observed Conclusion The Pathatrix Auto spp. method is specific and selective. ADRIA Développement 19/75 March 25, 2014 (Pathatrix Plate Summary Report Version 0)

20 3.4 Practicability The alternative method practicability was evaluated according to the AFNOR criteria relative to method comparison study. Packaging and reagents - Sterile filter bag (pre-sterilized sample and elution Vessel Packs, presterilized capture phase packs, pre-sterilized flat cap lids) - Anti- ssp. antibody-coated paramagnetic beads (2.5 ml 50 tests): ZBSQCAP500 for pooled sample, ZBSQCA for individual sample. - Optional: * 10- pool kit straws (254 mm) and syringes (10 ml) * foam Storage conditions and shelf-life Specific equipment Reagents - PBS 10 X ph 7.4 The storage temperature for beads is 2 8 C. The storage temperature for the other parts is room temperature. All the reagents must be stored at the temperature mentioned on the package. The shelf-life is given on the package. - Pathatrix Auto Instrument, including sample Vessel Holder and Elution Vessel Holder - Magnetic-particle concentrator (Dynamag, 2 magnets or magnetic rack) Training One day is required for technician with microbiology background. ADRIA Développement 20/75 March 25, 2014 (Pathatrix Plate Summary Report Version 0)

21 Workflow (in minutes) for 20 samples Steps Negative samples Reference method 20 individual samples Alternative method 2 pooled samples (2 x 10) Sampling Stomach RVS and MKTTn subcultures 40 / IMS step / 30 Streaking on selective agar plates 65 5 Reading 25 1 Total for negative samples analyses Total/negative sample Steps Presumptive samples or positive samples Reference method 20 individual samples Alternative method 20 individual samples IMS step / 50 Streaking on selective agar plates / 15 Reading / 15 Strike on nutrient agar 20 / Confirmation tests Total for positive samples Total/positive sample For negative and positive sample analysis, the Pathatrix Auto 10-pooling method requires less time than the reference method. ADRIA Développement 21/75 March 25, 2014 (Pathatrix Plate Summary Report Version 0)

22 Time to Steps Reference method Alternative method Technician background Common step with the reference method Traceability of the s Maintenance Negative samples Pre-enrichment Day 0 Day 0 Enrichment Day 1 / IMS pooled / Day 1 Streaking onto selective agar plates Day 2 Day 1 Reading Day 3 Day 2 Steps Reference method Alternative method Presumptive positive or positive s Pre-enrichment Day 0 Day 0 Enrichment Day 1 / IMS pooled / Day 1 Streaking onto selective agar plates Day 1 Reading / Day 2 Latex test / Day 2 IMS individual / Day 2 Streaking onto selective agar plates Day 2 Day 2 Reading Day 3 Day 3 Latex test / Day 3 Confirmatory tests Day 4 to Day 6 / Technician qualified in microbiology No common step None There is no required maintenance for the Pathatrix Auto system. Other than routine cleaning/decontamination the Pathatrix Auto does not have a specific planned maintenance schedule. Cartridges used on the Pathatrix Auto system are considered consumables and may need to be replaced depending on the usage routine. Any performance or operational errors should be addressed by Life Technologies Technical Support, Field Applications, or Service depending on the nature of the issue. Consult the instrument manual or maintenance guides for further information. The Pathatrix Auto spp. 10-pooling protocol linked to selective agar plates allows the screening of the negative samples within 2 days, while 3 days are required for the positive samples. ADRIA Développement 22/75 March 25, 2014 (Pathatrix Plate Summary Report Version 0)

23 4 INTERLABORATORY STUDY ORGANISATION AND RESULTS 4.1 Study organisation Samples were sent to 15 laboratories. The study was done with ground beef samples contaminated with Typhimurium A00C060. The inoculation levels were as follows: - 0 CFU/25 ml, CFU/25 ml, CFU/25 ml. The samples were inoculated individually. 8 replicates were provided per level to each laboratory. The total viable count microflora was analysed with a supplementary sample. Another sample s flask contained a sensor in order to follow the temperature during the travel and at reception. Blind coded samples were placed in isothermal boxes, which contained cooling blocks, and express-shipped to the different laboratories. A temperature control flask containing a sensor was added to the package in order to register the temperature profile during the transport, the package delivery and storage until analyses. Samples were shipped in 24 h to 72 h to the involved laboratories. The temperature conditions had to stay lower or equal to 8.4 C during transport, and between 0 C 8.4 C in the labs. Collaborators and ADRIA Développement carried out the analyses with the alternative and reference methods on Tuesday 15 th October or Wednesday 16 th October Samples for the reference and the alternative method were analysed at the same time. The collaborative study instructions were sent on September 27, ADRIA Développement 23/75 March 25, 2014 (Pathatrix Plate Summary Report Version 0)

24 4.2 Experimental parameters control Contamination level before inoculation, levels obtained after the artificial contaminations of the samples Before inoculation In order to detect spp., the ISO 6579 method was performed on five test portions (25 g) before the inoculation. All the s were negative. Sample stability Sample stability was checked by inoculating the matrix at 100 CFU/g and 5 CFU/g. Enumerations were performed for the high contamination level and detection analyses were performed for the low contamination level. Triplicata were analysed, and the s were the following: Table 13 Reference method (research) CFU/g () Aerobic Day mesophilic flora Sample 1 Sample 2 Sample 3 Sample 1 Sample 2 Sample 3 (CFU/g) Day Day Day No evolution was observed during the storage at 4 C. ADRIA Développement 24/75 March 25, 2014 (Pathatrix Plate Summary Report Version 0)

25 Contamination levels The contamination levels and the confidence intervals were: Table 14 Level Level 0 Low level High level Samples Theoretical target level (b/25 g) True level (b/25 g sample) Low limit / 25 g sample High limit / 25 g sample 0 / / / Logistic conditions Temperature conditions are given below: Laboratories Temperature measured by the sensor ( C) Table 15 - Sample temperatures at receipt Temperature measured at receipt ( C) Receipt date and time Analyse date A /10/ h00 15/10/ h20 B /10/ h15 15/10/ h00 C /10/ h00 17/10/2013 / D /10/ h43 16/10/ h00 E /10/ h30 15/10/ h30 F /10/ h00 16/10/ h00 G /10/ h00 16/10/2013 / H /10/ h50 15/10/ h00 I /10/ h00 15/10/ h00 J /10/ h30 15/10/ h00 K /10/ h24 15/10/ h00 L This lab. was unable to process the ring trial M /10/ h39 15/10/ h00 N / /10/ h00 16/10/ h00 O /10/ h15 16/10/ h30 ADRIA Développement 25/75 March 25, 2014 (Pathatrix Plate Summary Report Version 0)

26 4.2.3 Conclusion All the temperatures during transport and at receipt were correct. Lab C measured a temperature at receipt above 8.4 C (10.0 C), but the probe indicated that the temperature was 7.0 C. 4.3 Results analysis Aerobic mesophilic flora enumeration Depending on the lab s, the enumeration levels varied from to CFU/g Expert lab s The raw data are given in Appendix 6. Table 16 Results obtained by the expert Lab. Level Reference method Alternative method L0 0/8 0/8 0/8 L1 8/8 8/8 8/8 L2 8/8 8/8 8/ Collaborator lab s Samples were delivered to 15 Labs: - Lab L was unable to proceed to the analyses, - Lab C started the analyses at day 3 (17/10/2013), - Lab K discarded a part of the enrichment broths before proceeding to individual sample analyses, - Lab O didn't use the protocol correctly, the PBS solution was not used diluted (1/10 dilution) as mentioned in the protocol. ADRIA Développement 26/75 March 25, 2014 (Pathatrix Plate Summary Report Version 0)

27 Finally, the interpretation was done with 11 labs: A, B, D, E, F, G, H, I, J, M and N. All the raw data are given in Appendix 7. Table 17 Results obtained by the collaborator Labs. Laboratories Reference method L0 L1 L2 A 0/8 8/8 8/8 B 0/8 7/8 8/8 Analyses at D3 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 K 0/8 8/8 8/8 M 0/8 8/8 8/8 N 0/8 7/8 8/8 O 1/8 8/8 8/8 Laboratories Alternative method- L0 L1 L2 A 0/8 8/8 8/8 B 0/8 8/8 8/8 Analyses at D3 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 7/8 I 0/8 8/8 8/8 J 0/8 8/8 8/8 K 0/8 8/8 8/8 M 0/8 8/8 8/8 N 0/8 6/8 8/8 Protocol not respected O??? Laboratories Alternative method- L0 L1 L2 A 0/8 8/8 8/8 B 0/8 8/8 8/8 Analyses at D3 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 Samples discarded K 0/4 4/4 4/4 M 0/8 8/8 8/8 N 0/8 6/8 8/8 Protocol not respected O??? ADRIA Développement 27/75 March 25, 2014 (Pathatrix Plate Summary Report Version 0)

28 4.4 Results interpretation Specificity and sensitivity for each method For the L0 level and for each method, specificity percentages are calculated according to: FP SP 1 x 100% N with: N- = total number of all L0 assays FP = number of false positive s For each contamination level and each method, the sensitivity percentages are calculated according to: TP SE x 100% N with : N+ = total number of all L1 or L2 assays TP = number of true positive s Results (see Appendix 8 for raw data) are reported in Table 18. Table 18 Interpretation Alternative method Reference method Level SP/SE % LCL% SP/SE % LCL% SP/SE % LCL% Lo(SP) L1(SE) L2(SE) L1+L2(SE) LCL: confidence interval ADRIA Développement 28/75 March 25, 2014 (Pathatrix Plate Summary Report Version 0)

29 4.4.2 Relative accuracy (AC) Results for all levels (See Appendix 9) are given below: Table 19 - Paired s of the alternative and reference methods Pooled Samples Individual samples Alternative method Reference method + - Total + PA = 172 PD = ND = 2 NA = Total N+ = 174 N- = 90 N = PA = 173 PD = ND = 1 NA = Total N+ = 174 N- = 90 N = 264 Relative accuracy (AC) (in %) is calculated according to: ( PA NA) AC x 100% N with : N = number of samples analysed PA = number of positive agreement NA = number of negative agreement The alternative method accuracy values with regard to the reference method are: Table 20 Interpretation Level AC % LCL % AC % LCL % L L L L1 + L Total ADRIA Développement 29/75 March 25, 2014 (Pathatrix Plate Summary Report Version 0)

30 4.4.3 Discordant s The discordant s observed are: Pooled sample protocol ND = 2 (N18, H5) PD = 1 (B6) Y = 3 Note that for sample H5, it was asked to the Lab to proceed to a second analysis (IMS and PCR) and the was then positive (Ct = 24.0) Individual sample protocol ND = 1 (N18) PD = 1 (B6) Y = 2 For the two protocols Y < 6, no statistical test is available. The reference method and the two alternative protocols are considered equivalent. ADRIA Développement 30/75 March 25, 2014 (Pathatrix Plate Summary Report Version 0)

31 4.5 Interpretation 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 21. Table 21 - Alternative method values calculated during the comparative and inter-laboratory studies Inter-laboratory study Method comparison study Pooled Individual Pooled Individual samples samples samples samples Relative accuracy (AC) 98.9% 99.2% 94.8% 95.4% Sensitivity (SE) 98.3% 100.0% 90.2% 93.4% Specificity (SP) 100.0% 100.0% 97.8% 96.7% Sensitivity of both methods, when the positive deviations of the alternative method are considered, is presented below: Table 22 Sensitivity Reference method 99.4% Alternative method 98.9% 99.4% Accordance (DA) Accordance values for both methods are found in Table 23 below (see Appendix 10 for raw data): Table 23 Interpretation Level Reference method (DA) Alternative method (DA) L % 100.0% 100.0% L1 96.0% 96.6% 96.6% L % 98.0% 100.0% ADRIA Développement 31/75 March 25, 2014 (Pathatrix Plate Summary Report Version 0)

32 4.5.3 Concordance Both methods concordance values are found in Table 24 below (see Appendix 11 for raw data): Table 24 Interpretation Level Reference method Alternative method L % 100.0% 100.0% L1 95.5% 95.5% 95.5% L % 97.7% 98.8% Odds Ratio (COR) The odds ratio value is determined according to: Both method odds ratio values are: Accordance x 100 condorcance COR Concordance x (100 accordance ) Table 25 Interpretation Level Reference method Alternative method (COR) (COR) L L L ADRIA Développement 32/75 March 25, 2014 (Pathatrix Plate Summary Report Version 0)

33 5 CONCLUSION The method comparison study conclusions are: The Pathatrix Auto spp. 10-pooling protocol linked to selective agar plates shows satisfying relative accuracy, specificity and sensitivity s: the statistical tests conclude to equivalence between the reference method and the two protocols (pooled and individual samples analyses) of the alternative method for the studied scope: - Raw beef meats, ready-to-reheat and ready-to-eat meat products (including poultry), - Heat treated milk and dairy products. The relative detection limits of the alternative method and the ISO standard are similar. The alternative method shows satisfying inclusivity and exclusivity s. The Pathatrix Auto spp. 10-pooling protocol linked to selective agar plates allows the screening of the negative samples within 2 days, while 3 days are required for the positive samples. The scope of the validation is the following: Categories Types Food items (some examples) Sterilized or pasteurized Fermented/acidified milks and yoghurts milk and dairy products Milks, desserts, ice cream, etc (UHT, canned or pasteurized) Pasteurized cheeses, creams, butters Heat treated milk and dairy products Raw beef meats, readyto-reheat and ready-to-eat meat products (including poultry) Dry Raw beef meats Ready-to-eat meat products Ready-to-reheat meat products (canned, pasteurized or cooked) Milk powders Powders for dairy desserts Infant formula with and without probiotic Beef trim Ground beef Steak Fermented and cured meat products: dried sausages, cured ham, chorizo, salami, etc. Heat treated meat products: cooked ham, pâté, deliturkey, corned beef, Frankfurt sausages, etc Heat treated meat preparations: lasagnes, goulash, moussaka, bœuf bourguignon, etc ADRIA Développement 33/75 March 25, 2014 (Pathatrix Plate Summary Report Version 0)

34 The inter-laboratory study conclusions are: The observed data and s confirmed that the alternative method and reference method show equivalent performances (accordance, concordance, odds ratio). ADRIA Développement 34/75 March 25, 2014 (Pathatrix Plate Summary Report Version 0)

35 Appendix 1 Reference method: NF EN ISO 6579: 2002: Microbiology of food and animal feeding stuffs Horizontal method for the detection of spp. 25g of sample 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 and ASAP 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 (latex test applied during the ring trial) ADRIA Développement 35/75 March 25, 2014 (Pathatrix Plate Summary Report Version 0)

36 Appendix 2 Method alternative protocols Enrichment Protocol 1: Sample 1/10 dilution in (37 C ± 1 C) pre-warmed BPW Protocol 2: Sample 1/10 dilution in pre-warmed BPW + Brilliant Green (0.002%) (25 g) Stomaching For potentially acidic and alkaline samples, leave to stand for 60 ± 5 min at room temperature Determine the ph and if necessary, adjust to 6.8 ± 0.2 Store enrichment broth at 2 8 C up to 32 h Pre-warm at 37 C Transfer 37 C ± 1 C, 20h ± 2 h 10 x 5 ml enrichment broth (pooled samples) 10 ml enrichment broth (individual sample) into a Sample Vessel (Use a foam filter for highly particulate samples) Add 35 ml 1xPBS in the Elution Vessel Add 50 µl bead suspension into the spout on the lid of the Sample Vessel using the appropriate beads for each sample type: APS 50: individual samples APS 500: pooled samples Immuno-magnetic separation in the Pathatrix Auto Instrument Place the Elution Tube(s) in the Pathatrix magnetic Vessel Holder for 1 min Remove the 1xPBS (leaving the Elution Tube in the Pathatrix magnetic Vessel Holder) Add 120 µl of fresh 1xPBS and fully resuspend the bead pellet Place the Elution Tube in the Pathatrix magnetic Vessel Holder for 1 min Remove the 1xPBS Add 120 µl Nuclease-free water and fully resuspend the bead pellet Streak 10 µl of the beads onto and h at 37 C Confirmatory tests on typical colonies by latex test (OXOID) ADRIA Développement 36/75 March 25, 2014 (Pathatrix Plate Summary Report Version 0)

37 Appendix 3 Relative accuracy: raw data Legend: Bold typing: artificially inoculated samples m: minoritary level of target analyte M : majoritary level of target analyte P: pure culture level of target analyte 1/2 : 50% level of target analyte -: no typical colonies but presence of background microflora st: plate without any colony (x) number of typical colonies PA: positive agreement NA: negative agreement ND: negative deviation PD: positive deviation PPNA: positive presumptive negative agreement PPND : positive presumptive negative deviation ADRIA Développement 37/75 March 25, 2014 (Pathatrix Plate Summary Report Version 0)

38 N Sample 2037 Product (French name) Blanquette de veau (RTR) 2038 Couscous (RTR) Filet de poulet à la Normande (RTR) Bœuf bourguignon (RTR) Fricadelles sauce tomate (RTR) 2042 Paella (RTR) Navarin d'agneau (RTR) Langue de boeuf sauce piquante (RTR) Tomate farcie (RTR) Poulet basquaise (RTR) Blanquette de veau (RTR) 2048 Couscous (RTR) Filet de poulet à la Normande (RTR) Bœuf bourguignon (RTR) Fricadelles sauce tomate (RTR) 2052 Paella (RTR) 2053 Navarin d'agneau (RTR) Product (English name) Veal cooked with a white wine and cream based dressing Couscous (Maghreb speciality with vegetables, meat and spices) Poultry with a cream based dressing Beef with a red wine based dressing Sausages with a tomatoes based dressing Spanish speciality with poultry, spices, rice Sheep meat with dressing containing vegetables Beef tongue with a spicy dressing Cooked tomatoes with cooked minced meat Poultry meat with a tomatoes and paprika based dressing Veal cooked with a white wine and cream based dressing Couscous (Maghreb speciality with vegetables, meat and spices) Poultry with a cream based dressing Beef with a red wine based dressing Sausages with a tomatoes based dressing Spanish speciality with poultry, spices, rice Sheep meat with dressing containing vegetables Enrichment broth Global RVS broth RAW BEEF MEATS, READY-TO-REHEAT AND READY-TO-EAT MEAT PRODUCTS (INCLUDING POULTRY) ISO 6579 method MKTTn broth ASAP ASAP Result N positive sample N negative samples Pathatrix spp method After enrichment incubation Enrichment broth storage 32h at 2-8 C Latex test global Reference method confirmatory Protocol 1 + +P +P +P +P to m ni +M PA +M +M + + PA +M +P + + PA Protocol 1 + +P +P +P +P to m +M PA +M +P + + PA +m +P + + PA Protocol 1 + +P +P +P +P to /2 +M PA +M +P + + PA +P +P + + PA Protocol 1 + +P +P +m +m to m ni PA +m ni +m ni + + PA +m ni +m ni + + PA Protocol 1 + +P +P +M +M to / / - ND +m +m ni + + PA St +m ni + + PA Protocol 1 + +P +P +P +M to m +M PA +P +P + + PA +P +P + + PA Protocol 1 + +P +P +M +P Protocol 1 + +P +P +M +P Protocol 1 + +P +P +P +P Protocol 1 + +P +P +P +P to to to to tests Final Agree- ment Latex and reference confirmatory tests Final Agree- ment Latex test global +m ni +M PA +M +M + + PA +M +M + + PA +m +M PA +M +M + + PA +P +M + + PA +m +M PA +P +P + + PA +P +P + + PA +1/2 +M PA +M +M + + PA +P +M + + PA Protocol 1 - St St / / / - NA - - / - NA Protocol 1 - St St St St - / / / - NA - - / - NA Protocol / / / - NA - - / - NA Protocol St / / / - NA - - / - NA Protocol / / / - NA - - / - NA Protocol St - / / / - NA - - / - NA Protocol / / / - NA - - / - NA Final Agree- ment Analysis performed according to the COFRAC accreditation ADRIA Développement 38/75 March 25, 2014 (Pathatrix Plate Summary Report Version 0)

39 N Sample 2054 Product (French name) Langue de boeuf sauce piquante (RTR) Product (English name) Beef tongue with a spicy dressing Enrichment broth ADRIA Développement 39/75 March 25, 2014 (Pathatrix Plate Summary Report Version 0) Global RVS broth RAW BEEF MEATS, READY-TO-REHEAT AND READY-TO-EAT MEAT PRODUCTS (INCLUDING POULTRY) ISO 6579 method MKTTn broth ASAP ASAP Result N positive sample N negative samples Pathatrix spp method After enrichment incubation Enrichment broth storage 32h at 2-8 C Protocol / d d Latex test global Reference method confirmatory tests - (Serratia liquefaciens) Final Agree- ment - NA - d 2055 Tomate farcie Cooked tomatoes with (RTR) cooked minced meat Protocol 1 - St St St St - / / / - NA - - / - NA 2056 Poultry meat with a Poulet basquaise tomatoes and paprika (RTR) based dressing Protocol St St - / / / - NA - - / - NA 2608 Jambon cuit Cooked ham Protocol to / / - NA - - / - NA - - / - NA 2609 Saucisses de Francfort Sausages Protocol St to / / - NA - St / - NA - St / - NA 2610 Saucisson cuit Cooked sausage Protocol 1 + +P +P +P +P to m + 1/ PA +M +M + + PA +M +m + + PA 2611 Pâté de campagne Pâté Protocol 1 + +P +P +P +P to m + 1/ PA +1/2 +M + + PA +m +M + + PA 2612 Andouille Chitterling Protocol 1 + +P +P +P +P to m +M PA +M +M + + PA +M +M + + PA 2613 Saucisson sec Dry sausage Protocol 1 + +P +P +P +P to m +M PA +P +P + + PA +M +M + + PA 2614 Saucisse sèche Dry sausage Protocol 1 + +P +P +P +P to m +M PA +M +M + + PA +P +P + + PA 2615 Saucisson sec Dry sausage Protocol 1 + +M +P +P +P to m (3) ni + 1/ PA +M +M + + PA +P +P + + PA 2616 Chorizo Cooked sausage Protocol 1 + +P +P +P +P to / / - ND St St / - ND st st / - ND 2617 Saucisse sèche Dry sausage Protocol 1 + +m +P +P +P to m +M PA +M +M + + PA +P +P + + PA 2618 Jambon cuit Cooked ham Protocol St / / / - NA - - / - NA 2619 Saucisses de Francfort Sausages Protocol 1 - St St St St - / 2619 St St / / - NA St St / - NA 2620 Saucisson cuit Cooked sausage Protocol 1 - St St St St - / / / - NA - - / - NA 2621 Pâté de campagne Pâté Protocol 1 - St St / / / - NA - - / - NA 2622 Andouille Chitterling Protocol 1 - St St / / / - NA - - / - NA 2623 Saucisson sec Dry sausage Protocol 1 - St St St St - / 2623 St St / / - NA St St / - NA 2624 Saucisse sèche Dry sausage Protocol 1 - St St St St - / 2624 St St / / - NA St St / - NA 2625 Saucisson sec Dry sausage Protocol St - / St / / - NA - St / - NA 2626 Chorizo Cooked sausage Protocol 1 - St St St St - / St / / - NA - St / - NA 2627 Saucisse sèche Dry sausage Protocol St St - / St / / - NA - St / - NA 3073 Rosette Cooked sausage Protocol St St - / / / - NA - - / - NA 3074 Saucisse sèche Dry sausage Protocol 1 - St St / 3074 St St / / - NA St St / - NA 3075 Pâté de campagne poivre vert Pâté Protocol St - - / / / - NA - - / - NA 3076 Poitrine rôtie au four Cooked ham Protocol 1 - St St St St - / / / - NA - - / - NA 3077 Cervelas Cooked sausage Protocol 1 - St St St St - / 3077 St - / / - NA St - / - NA 3078 Saucisse de Morteau Cooked sausage Protocol 1 - St St St St - / 3078 St St / / - NA St St / - NA 3079 Jambon cuit Cooked ham Protocol 1 - St St St St - / / / - NA - - / - NA 3080 Rôti de porc Cooked ham Protocol 1 - St St St St - / / / - NA - - / - NA 3081 Andouille de vire Chitterling Protocol 1 - St St St St - / / / - NA - - / - NA 3082 Saucisse de Francfort Cooked sausage Protocol 1 + +p +p +p +p to m ni +M PA +p +p + + PA +p +M + + PA Latex and reference confirmatory tests d (Serratia liquefaciens) Final Agree- ment - NA Latex test global Final Agree- ment

40 N Sample Product (French name) Product (English name) Enrichment broth Global RVS broth RAW BEEF MEATS, READY-TO-REHEAT AND READY-TO-EAT MEAT PRODUCTS (INCLUDING POULTRY) ISO 6579 method MKTTn broth ASAP ASAP Result N positive sample N negative samples Pathatrix spp method After enrichment incubation Enrichment broth storage 32h at 2-8 C 3083 Poitrine rôtie au four Cooked ham Protocol 1 + +p +p +p +p to m ni +1/ PA +p +p + + PA +p +M + + PA 3211 Rumsteak Beef trim Protocol 1 + +m +p +M +p to / PA 1/2 ni +M + + PA +m ni +M + + PA 3212 Onglet Beef trim Protocol 1 + +m +1/2 +M +M to m (1) ni PA +m ni +m + + PA +m ni +m ni + + PA 3213 Gite de noix Beef trim Protocol 1 + +M +M +M +p to m ni +M PA +m +M + + PA +m +M + + PA 3214 Bavette Beef trim Protocol 1 + +m +M +M +M to m ni PA +m ni +m + + PA +m +m ni + + PA 3216 Tranche en tournedos Beef trim Protocol 1 + +m +p +M +M to m +M PA - +M + + PA - +M + + PA 3217 Gite de noix Beef trim Protocol 1 + +m +M +1/2 p to M PA +m ni +M + + PA +m ni +m + + PA 3218 Basse côtes Beef trim Protocol 1 + +m +M +M +p to m ni PA +m ni +m + + PA +m d +m ni + + PA 3219 Steak haché pur boeuf Ground beef Protocol /2 +M +M +M to m ni +M PA +m +M + + PA + m +M + + PA 3220 Steak haché Ground beef Protocol 1 + +m +p +M +M to m +M PA +1/2 +M + + PA +m +M + + PA 3221 Steak haché Ground beef Protocol 1 + +m +M +M +p to m ni +m PA - +M + + PA +m ni +1/2 + + PA 3222 Steak haché Ground beef Protocol 1 + +M +M +1/2 +M to / PA - +1/2 + + PA +m (1) ni +m + + PA 3223 Steak haché Ground beef Protocol to / / - NA - - / - NA - - / - NA 3224 Steak haché Ground beef Protocol to m (1) ni +1/ PD +m ni +m + + PD +m (1) ni +1/2 + + PD 3225 Steak haché Ground beef Protocol to / / - NA - - / - NA - - / - NA 3226 Steak haché Ground beef Protocol / / / - NA - - / - NA 3227 Steak haché Ground beef Protocol / / / - NA - - / - NA 3228 Steak haché Ground beef Protocol / / / - NA - - / - NA 3229 Steak haché Ground beef Protocol / / / - NA - - / - NA 3230 Rumsteak Beef trim Protocol / / / - NA - - / - NA 3231 Onglet Beef trim Protocol / / / - NA - - / - NA 3232 Gite de noix Beef trim Protocol / / / - NA - - / - NA 3233 Faux filet Beef trim Protocol / / / - NA - - / - NA 3234 Tranche en tournedos Beef trim Protocol / / / - NA - - / - NA Latex test global Reference method confirmatory tests Final Agree- ment Latex and reference confirmatory tests Final Agree- ment Latex test global Final Agree- ment ADRIA Développement 40/75 March 25, 2014 (Pathatrix Plate Summary Report Version 0)