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

Transcription

1 ACCREDITATION N PORTEE DISPONIBLE SUR LIFE TECHNOLOGIES Route de l Orme des Merisiers Immeuble Le Discovery Parc Technologique F 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 and up to 10-pooling linked to MicroSEQ spp Detection Kit in raw beef meats, readyto-reheat and ready-to-eat meat products (including poultry), heat treated milk and dairy products Qualitative method This report includes 77 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 Artificial contamination of 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 4 INTERLABORATORY STUDY ORGANISATION AND RESULTS Study organisation Experimental parameters control Contamination level before inoculation, levels obtained after the artificial contaminations of the Logistic conditions Conclusion Results analysis Aerobic mesophilic flora enumeration Expert lab s Collaborator lab s 26 ADRIA Développement 2/77 March 25, 2014 (Pathatrix PCR Summary Report Version 0)

3 4.4 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 47 Appendix 5 Inclusivity and exclusivity: raw data 50 Appendix 6 Results obtained by the Expert Laboratory 55 Appendix 7 Results obtained by each Collaborator 56 Appendix 8 Specificity and sensitivity 70 Appendix 9 Paired s of the alternative and reference methods for each level 71 Appendix 10 Accordance 72 Appendix 11 Concordance 75 ADRIA Développement 3/77 March 25, 2014 (Pathatrix PCR 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 realized 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 and up to 10-pooling linked to MicroSEQ spp Detection Kit 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 4/77 March 25, 2014 (Pathatrix PCR Summary Report Version 0)

5 1 AIM OF THE STUDY The Pathatrix Auto spp. for individual and up to 10-pooling linked to MicroSEQ spp Detection Kit 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 ). Thus, the developed validation protocol is based on the analyses on pooled. The pooling can be done between 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. Analysis performed according to the COFRAC accreditation ADRIA Développement 5/77 March 25, 2014 (Pathatrix PCR Summary Report Version 0)

6 After a RIMS step, detection is done by real-time PCR using the MicroSEQ spp. Detection Kit. Pathatrix protocols can be performed on individual, and on pooled (up to 10 per pool). A specific protocol design is proposed in this ISO validation study. Based on the background microflora level of the tested, 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 milks 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, 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 are provided in Appendix 3 in raw data tables. During the study, it was never necessary to adjust it before incubation. The confirmations are done by recovering the strains on and TM Agar, followed by a latex test (Oxoid). The protocols are described in Appendix 2. ADRIA Développement 6/77 March 25, 2014 (Pathatrix PCR 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 A total of 153 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 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 7/77 March 25, 2014 (Pathatrix PCR Summary Report Version 0)

8 Number of Life Technologies Artificial contamination of 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 were artificially contaminated, using 25 different strains and 8 injury protocols. 63 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 29,5% 59,7% x 5 CFU/sample 5<x 10 CFU/sample Figure 2 Inoculated and positive according to the level of contamination All inoculated Positive x 5 CFU/sample 5<x 10 CFU/sample x>10 CFU/sample Only one product was naturally contaminated. ADRIA Développement 8/77 March 25, 2014 (Pathatrix PCR Summary Report Version 0)

9 Indiviidual Life Technologies Confirmation protocols Samples identified as PCR positive were confirmed by plating retained unlysed Pathatrix beads (streaking 10 µl onto and TM Agar). 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 Response Reference method positive (R+) Reference method negative (R-) Alternative method Positive agreement (A+/R+) Positive deviation (R-/A+) positive (A+) PA = 54 PD = 3 Alternative method Negative deviation (A-/R+) Negative agreement (A-/R-) negative (A-) ND = 7 (PPND = 0) NA = 89 (PPNA = 0) Alternative method Positive agreement (A+/R+) Positive deviation (R-/A+) positive (A+) PA = 56 PD = 3 Alternative method Negative deviation (A-/R+) Negative agreement (A-/R-) negative (A-) ND = 5 (PPND = 0) NA = 89 (PPNA = 0) PP: positive presumptive non confirmed PD = positive deviation (R-/A+) ND = negative deviation (A-/R+) ADRIA Développement 9/77 March 25, 2014 (Pathatrix PCR Summary Report Version 0)

10 Indiviidual Indiviidual Life Technologies Results per category of 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 = 27 PD = 1 Alternative method Negative deviation (A-/R+) Negative agreement (A-/R-) negative (A-) ND = 4 (PPND = 0) NA = 42 (PPNA = 0) 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) 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 = 27 PD = 2 Alternative method Negative deviation (A-/R+) Negative agreement (A-/R-) negative (A-) ND = 3 (PPND = 0) NA = 47 (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 10/77 March 25, 2014 (Pathatrix PCR Summary Report Version 0)

11 Individual Individual 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 = 27 PD = 1 Alternative method Negative deviation (A-/R+) Negative agreement (A-/R-) negative (A-) ND = 4 (PPND = 0) NA = 42 (PPNA = 0) 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) 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 = 27 PD = 2 Alternative method Negative deviation (A-/R+) Negative agreement (A-/R-) negative (A-) ND = 3 (PPND = 0) NA = 47 (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) PCR inhibitions were observed for 2 pooled (n 3076 and 3218) and for 4 individual. The s were obtained by proceeding to a 1/6 dilution of the DNA extract. For sample n 3078, a 1/10 dilution was necessary to obtain the PCR. ADRIA Développement 11/77 March 25, 2014 (Pathatrix PCR Summary Report Version 0)

12 Table 7 Calculation of relative accuracy (AC), relative sensitivity (SE) and relative specificity (SP) Per category of 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 Individual 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 Individual 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 12/77 March 25, 2014 (Pathatrix PCR 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 Individual Relative accuracy : AC 93.5 % 94.8 % Relative specificity : SP 96.7 % 96.7 % Relative sensitivity : SE 88.5 % 91.8 % Sensitivity of both methods, when the positive deviations of the alternative method are considered, is presented below: Alternative method Individual Alternative method 89.1 % 92.2 % Reference method 95.3 % 95.3 % The alternative method percentage values per protocol are: Alternative method Individual Protocol 1 Protocol 2 Protocol 1 Protocol 2 Relative accuracy : AC 93.2 % 93.7 % 95.9 % 93.7 % Relative specificity : SP 97.7 % 95.9 % 97.7 % 95.9 % Relative sensitivity : SE 87.1 % 90.0 % 93.5 % 90.0 % ADRIA Développement 13/77 March 25, 2014 (Pathatrix PCR Summary Report Version 0)

14 3.1.6 Analysis of discordants Table 9 Negative deviations Category Protocol Sample n Individual Contamination PCR Confirmation PCR Confirmation (level contamination) Raw beef meats, / / S. Anatum 6140 (2.4) ready-to-reheat S. Typhimurium 702 (5.0) and ready-to-eat meat products / / S. Panama 8 (9.4) (including poultry) /+/+ + S. Bredeney 396 (7.6) Heat treated milks S. Ohio Ad 1482 (0.6) and dairy products S. Montevideo 510 (0.6) S. Tennessee Ad 1171 (7.4) Total negative deviations were observed with the pooling protocol, 5 with the individual protocol. For 3, the confirmatory tests concluded to the presence of spp. in the enrichment broth; the detection level was probably not reached in these cases. For 4, the discordant s were probably due to sampling heterogeneity in this unpaired data study (enrichment broth different for the reference and the alternative method). Table 10 Positive deviations Category Protocol Sample n Contamination (level contamination) Individual Raw beef meats, readyto-reheat Natural Natural and ready-to- eat meat products (including poultry) Heat treated milks and S. Anatum Ad 298 (9.2) S. Anatum Ad 298 (9.2) dairy products 3612 S. Mbandaka Ad 1722 (1.0) S. Mbandaka Ad 1722 (1.0) Total positive deviations were observed for the pooled protocol and for the individual protocol. ADRIA Développement 14/77 March 25, 2014 (Pathatrix PCR Summary Report Version 0)

15 According to the Annex F of the EN ISO standard: Y = PD + ND = = 10 m = PD = 3 M = 1 m > M, the two methods are not different at < Individual Y = PD + ND = = 8 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 individual workflows) and the reference method Confirmations The positive PCR s on individual were confirmed by streaking Pathatrix bead suspension onto and Agar, then a latex test was realized on presumptive colonies. Differences were observed between the selective agar plates for 9 ; they are listed below. Sample n Contamination 2617 S. Typhimurium 702 St + M 3216 S. Bredeney M 3221 S. Panama M 3222 S. Bredeney / S. Dublin Ad P S. Dublin Ad P S. Dublin Ad P S. Mbandaka Ad P 3613 S. Mbandaka Ad (3 colonies) - 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 ADRIA Développement 15/77 March 25, 2014 (Pathatrix PCR Summary Report Version 0)

16 For 6, no typical colony was observed on plates while a lot of colonies were observed on Agar plates. This was not the case for the n 3613 and 3863, for which only few colonies were observed. For the n 3605, 3607 and 3608, no typical colony was observed on Agar; these were all inoculated with Dublin which can produce white or pale magenta colonies on C8 esterase Agar plates Selective enrichment broth storage at 2-8 C for 32 h The positive enrichment broths of the alternative method were stored at 2-8 C for 32 h and analyzed a second time. Sample n Individual T0 Individual After storage 32 h at 2 8 C Final Agreement Final Agreement ND + PA PD - PPNA PA - PPND 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 16/77 March 25, 2014 (Pathatrix PCR 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 are firstly analyzed as a screening step. If positive s are observed, the 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, - level required to get 50 to 75 % positive. - level required to get 75 to 100 % positive. The were analyzed by both methods, and the background microflora were enumerated. ADRIA Développement 17/77 March 25, 2014 (Pathatrix PCR Summary Report Version 0)

18 3.2.3 Results Raw data are given in Appendix 4. PCR inhibitions were observed for 2 pooled (n 3296 and raw beef matrix). The PCR s were obtained by proceeding to a 1/6 dilution of the DNA extract. Detection levels are presented in the table 11 Table 11 Relative detection level s Relative detection level (CFU / 25 g) according to Spearman-Kärber test 1 Strain / matrix pairs Reference method Alternative method Individual Ground beef / Typhimurium A00C (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 two tested protocols (pooled and individual) 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 18/77 March 25, 2014 (Pathatrix PCR 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 protocol. 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 PCR. The study was repeated by adding 25 ml UHT milk in the enrichment broth; all the strains gave then a positive. For 3 strains ( arizonae 51:z4,z23:-, Paratyphi A ATCC 9150 and Rissen 39), it was necessary to inoculate the broth at a higher level in order to obtain positive PCR tests. Exclusivity 30 strains were tested; all gave a negative PCR test Conclusion The Pathatrix Auto spp. method is specific and selective. ADRIA Développement 19/77 March 25, 2014 (Pathatrix PCR 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. - MicroSEQ spp. Detection kit (part 1 of 2) - Pathogen Detection Negative Control (part 2 of 2) - Optional: foam filters, 10- Pool kit straws (254 mm) and syringes (10 ml) Storage conditions and shelf-life The storage temperature for beads, MicroSEQ detection kit, Pathogen Detection, lysis buffer 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. Specific equipment - Heating block 97 C ± 2 C - Pathatrix Auto Instrument, including sample Vessel Holder and Elution Vessel Holder - Magnetic-particle concentrator (Dynamag, 2 magnets or magnetic rack) - Applied Biosystems 7500 Fast Real-Time PCR system Reagents - PBS 10 X ph Nuclease free water - Lysis buffer Training One day is required for technician with microbiology and molecular biology background. ADRIA Développement 20/77 March 25, 2014 (Pathatrix PCR Summary Report Version 0)

21 Workflow (in minutes) for 20 Steps Negative Reference method 20 individual Alternative method 2 pooled (2x10) Sampling Stomach RVS and MKTTn subcultures Streaking on selective agar plates 40 / 65 / Reading 25 / IMS step / 30 Extraction / 2 PCR / 5 Total for negative analyses Total/negative sample Steps Presumptive or positive Reference method 20 individual Alternative method 20 individual IMS step / 50 Extraction / 15 PCR / 30 Streaking on selective agar plates / 15 Reading / 15 Strike on nutrient agar 20 / Confirmation tests Total for positive Total/positive sample For negative sample analysis, the Pathatrix Auto 10-pooling method requires less time than the reference method. For positive sample analysis, the Pathatrix Auto 10-pooling method requires a little less time than the reference method. ADRIA Développement 21/77 March 25, 2014 (Pathatrix PCR Summary Report Version 0)

22 Time to Steps Reference method Alternative method Negative Pre-enrichment Day 0 Day 0 Enrichment Day 1 / IMS pooled - Extraction PCR Streaking onto selective agar plates / Day 1 Day 2 / Reading Day 3 / Steps Reference method Alternative method Technician background Common step with the reference method Traceability of the s Maintenance Presumptive positive or positive s Pre-enrichment Day 0 Day 0 Enrichment Day 1 / IMS pooled Extraction PCR IMS individual Extraction PCR Streaking onto selective agar plates / Day 1 / Day 1 to Day 2 Day 2 Day 1 to Day 2 Reading Day 3 Day 2 to Day 3 / Day 2 to Day 3 Confirmatory tests Day 4 to Day 6 / Technician qualified in microbiology and molecular biology No common step All data obtained with the alternative method are traced over time by computer. 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. The Applied Biosystems 7500 FAST Real-time PCR system should be calibrated by a Life Technologies Field Service Engineer once per year, or any time that the system is moved. Consult the instrument manual or maintenance guides for further information. The Pathatrix Auto spp. 10 pooling protocol linked to MicroSEQ spp Detection Kit allows the screening of negative within one day, while 2 or 3 days are required for the positive. ADRIA Développement 22/77 March 25, 2014 (Pathatrix PCR 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 contaminated with Typhimurium A00C060. The inoculation levels were as follows: - 0 CFU/25 ml, CFU/25 ml, CFU/25 ml. The 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 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 methods were analyzed at the same time. The collaborative study instructions were sent on September 27, ADRIA Développement 23/77 March 25, 2014 (Pathatrix PCR Summary Report Version 0)

24 4.2 Experimental parameters control Contamination level before inoculation, levels obtained after the artificial contaminations of the 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 analyzed, and the s were the following: Table 12 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. Contamination levels The contamination levels and the confidence intervals were: Table 13 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 / / / ADRIA Développement 24/77 March 25, 2014 (Pathatrix PCR Summary Report Version 0)

25 4.2.2 Logistic conditions Temperature conditions are given below: Laboratories Temperature measured by the sensor ( C) Table 14 - 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/ h 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. ADRIA Développement 25/77 March 25, 2014 (Pathatrix PCR Summary Report Version 0)

26 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 15 Results obtained by the expert Lab. Level Reference method Alternative method Individual 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. 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. ADRIA Développement 26/77 March 25, 2014 (Pathatrix PCR Summary Report Version 0)

27 Table 16 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-individual 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 27/77 March 25, 2014 (Pathatrix PCR 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 6. Table 17 Interpretation Alternative method- Reference method Level Individual SP/SE % LCL% SP/SE % LCL% SP/SE % LCL% Lo(SP) L1(SE) L2(SE) L1+L2(SE) LCL: confidence interval ADRIA Développement 28/77 March 25, 2014 (Pathatrix PCR Summary Report Version 0)

29 4.4.2 Relative accuracy (AC) Results for all levels (See Appendix 9) are given below: Table 18 - Paired s of the alternative and reference methods Samples Individual 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 analysed PA = number of positive agreement NA = number of negative agreement The alternative method accuracy values with regard to the reference method are: Table 19 Interpretation Level Individual AC % LCL % AC % LCL % L L L L1 + L Total ADRIA Développement 29/77 March 25, 2014 (Pathatrix PCR Summary Report Version 0)

30 4.4.3 Discordant s The discordant s observed are: 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 30/77 March 25, 2014 (Pathatrix PCR 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 20. Table 20 - Alternative method values calculated during the comparative and inter-laboratory studies Inter-laboratory study Method comparison study Individual Individual Relative accuracy (AC) 98.9% 99.2% 93.5% 94.8% Sensitivity (SE) 98.3% 100.0% 88.5% 91.8% Specificity (SP) 100.0% 100.0% 96.7% 96.7% Sensitivity of both methods, when the positive deviations of the alternative method are considered, is presented below: Table 21 Sensitivity Reference method 99.4% Alternative method 98.9% Individual 99.4% Accordance (DA) Accordance values for both methods are found in Table 22 below (see Appendix 10 for raw data): Table 22 Interpretation Level Reference method (DA) Alternative method (DA) Individual L % 100.0% 100.0% L1 96.0% 96.6% 96.6% L % 98.0% 100.0% ADRIA Développement 31/77 March 25, 2014 (Pathatrix PCR Summary Report Version 0)

32 4.5.3 Concordance Both methods concordance values are found in Table 23 below (see Appendix 11 for raw data): Table 23 Interpretation Level Reference method Alternative method Individual 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 24 Interpretation Level Reference method Alternative method (COR) (COR) Individual L L L ADRIA Développement 32/77 March 25, 2014 (Pathatrix PCR Summary Report Version 0)

33 5 CONCLUSION The methods comparative study conclusions are: The Pathatrix Auto spp. 10-pooling protocol linked to MicroSEQ spp Detection Kit 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 analysis) 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 MicroSEQ spp Detection Kit allows the screening of negative within one day, while 2 or 3 days are required for the positive. ADRIA Développement 33/77 March 25, 2014 (Pathatrix PCR Summary Report Version 0)

34 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 The interlaboratory 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 34/77 March 25, 2014 (Pathatrix PCR 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 ( applied during the ring trial) ADRIA Développement 35/77 March 25, 2014 (Pathatrix PCR Summary Report Version 0)

36 Appendix 2 Method alternative protocol 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%) Store enrichment broth at 2 8 C up to 32h Pre-warm at 37 C Streak 10 µl of the beads onto and h at 37 C Confirmatory tests on typical colonies by latex test (OXOID) Transfer (25 g) Stomaching For potentially acidic and alkaline, leave to stand for 60 ± 5 min at room temperature Determine the ph and if necessary, adjust to 6.8 ± C ± 1 C, 20h ± 2 h 10 x 5 ml enrichment broth (pooled ) 10 ml enrichment broth (individual sample) into a Sample Vessel (Use a foam filters for highly particulate ) 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 APS 500: pooled Immuno-magnetic separation in the Pathatrix Auto Instrument Place the Elution Vessel in the Pathatrix magnetic Vessel Holder for 1 min Remove the 1xPBS (leaving the Elution Vessel in the Pathatrix magnetic Vessel Holder) Add 120 µl of fresh 1xPBS and fully resuspend the bead pellet Place the Elution Vessel in the Pathatrix magnetic Vessel Holder for 1 min Remove the 1xPBS Add 120 µl Nuclease-free water and fully resuspend bead pellet Add 10 µl of 1 x Lysis Buffer to a 1.5 ml centrifuge tube Transfer 90 µl of resuspended sample into a 1.5 ml microcentrifuge tube containing 10 µl of Lysis Buffer Heat treatment at 97 C ± 2 C for 10 min Cool 1 min at room temperature Place the 1.5 ml microcentrifuge tubes in the magnetic rack for 1 min Remove 30 µl of the clear lysate from the tube without disturbing the bead pellet Add the 30 µl to a lyophilized MicroSEQ spp. assay well. PCR MicroSEQ spp. ADRIA Développement 36/77 March 25, 2014 (Pathatrix PCR Summary Report Version 0)

37 Appendix 3 Relative accuracy: raw data Legend: Bold typing: artificially inoculated 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 i: PCR inhibition PA: positive agreement NA: negative agreement ND: negative deviation PD: positive deviation PPNA: positive presumptive negative agreement PPND : positive presumptive negative deviation *: PCR after dilution 1/6 **: PCR after dilution 1/10 ADRIA Développement 37/77 March 25, 2014 (Pathatrix PCR Summary Report Version 0)

38 N Sample Product 2037 Blanquette de veau (RTR) 2038 Couscous (RTR) Product Veal cooked with a white wine and cream based dressing Couscous (Maghreb speciality with vegetables, meat and spices) RAW BEEF MEATS, READY-TO-REHEAT AND READY-TO-EAT MEAT PRODUCTS (INCLUDING POULTRY) Enrichment broth Global RVS broth ASAP ISO 6579 method MKTTn broth ASAP Result N positive sample Pathatrix spp method After enrichment incubation Individual Individual N negative MicroSEQ spp Detection Kit MicroSEQ spp Detection Kit Result Ct Result Ct Confirmatory tests and reference confirmatory tests Protocol 1 + +P +P +P +P to M +M + + PA + PA Protocol 1 + +P +P +P +P to M +P + + PA + PA 2039 Filet de poulet à la Normande (RTR) Poultry with a cream based dressing Protocol 1 + +P +P +P +P to M +P + + PA + PA 2040 Bœuf bourguignon (RTR) Beef with a red wine based dressing Protocol 1 + +P +P +m +m to m ni +m ni + + PA + PA 2041 Fricadelles sauce tomate (RTR) Sausages with a tomatoes based dressing Protocol 1 + +P +P +M +M to /-/-/-* / m +m ni + - ND + PA 2042 Paella (RTR) Spanish speciality with poultry, spices, rice Protocol 1 + +P +P +P +M to P +P + + PA + PA 2043 Navarin d'agneau (RTR) Sheep meat with dressing containing vegetables Protocol 1 + +P +P +M +P to M +M + + PA + PA 2044 Langue de boeuf sauce piquante (RTR) Beef tongue with a spicy dressing Protocol 1 + +P +P +M +P to M +M + + PA + PA 2045 Tomate farcie (RTR) Cooked tomatoes with cooked minced meat Protocol 1 + +P +P +P +P to P +P + + PA + PA 2046 Poulet basquaise (RTR) Poultry meat with a tomatoes and paprika based 2050 to Protocol 1 + +P +P +P +P dressing M +M + + PA + PA 2047 Blanquette de veau (RTR) Veal cooked with a white wine and cream based Protocol 1 - St St / / - / - - / - NA - NA 2048 Cousous (RTR) dressing Couscous (Maghreb speciality with vegetables, meat and spices) Protocol 1 - St St St St - / / - / - - / - NA - NA 2049 Filet de poulet à la Normande (RTR) Poultry with a cream based dressing Protocol / / - / - - / - NA - NA 2050 Bœuf bourguignon (RTR) Beef with a red wine based dressing Protocol St / / - / - - / - NA - NA 2051 Fricadelles sauce tomate (RTR) Sausages with a tomatoes based dressing Protocol / / - / - - / - NA - NA 2052 Paella (RTR) Spanish speciality with poultry, spices, rice Protocol St - / / / - NA - NA 2053 Navarin d'agneau (RTR) Sheep meat with dressing containing vegetables Protocol / / - / - - / - NA - NA 2054 Langue de boeuf sauce piquante (RTR) Beef tongue with a spicy dressing Protocol / / - / - d d (Serratia. liquefaciens) Final Agreement Final Agreement - NA - NA 2055 Tomate farcie (RTR) Cooked tomatoes with cooked minced meat Protocol 1 - St St St St - / / - / - - / - NA - NA 2056 Poulet basquaise (RTR) Poultry meat with a tomatoes and paprika based dressing Protocol St St - / / - / - - / - NA - NA 2608 Jambon cuit Cooked ham Protocol to / - / - - / - NA - NA 2609 Saucisses de Francfort Sausages Protocol St to / - / - St / - NA - NA 2610 Saucisson cuit Cooked sausage Protocol 1 + +P +P +P +P to M +M + + PA + PA 2611 Pâté de campagne Pâté Protocol 1 + +P +P +P +P to /2 +M + + PA + PA 2612 Andouille Chitterling Protocol 1 + +P +P +P +P to /2 +M + + PA + PA 2613 Saucisson sec Dry sausage Protocol 1 + +P +P +P +P to M +P + + PA + PA 2614 Saucisse sèche Dry sausage Protocol 1 + +P +P +P +P to P +M + + PA + PA 2615 Saucisson sec Dry sausage Protocol 1 + +M +P +P +P to M +M + + PA + PA 2616 Chorizo Cooked sausage Protocol 1 + +P +P +P +P to /i/i / -/i/i / St St / - ND - ND 2617 Saucisse sèche Dry sausage Protocol 1 + +m +P +P +P to St +M + + PA + PA 2618 Jambon cuit Cooked ham Protocol St / / - / +M - / - NA - NA 2619 Saucisses de Francfort Sausages Protocol 1 - St St St St - / / - / St St / - NA - NA 2620 Saucisson cuit Cooked sausage Protocol 1 - St St St St - / / - / - - / - NA - NA Analysis performed according to the COFRAC accreditation ADRIA Développement 38/77 March 25, 2014 (Pathatrix PCR Summary Report Version 0)

39 N Sample Product Product RAW BEEF MEATS, READY-TO-REHEAT AND READY-TO-EAT MEAT PRODUCTS (INCLUDING POULTRY) Enrichment broth Global RVS broth ASAP ISO 6579 method MKTTn broth ASAP Result N positive sample Pathatrix spp method After enrichment incubation Individual Individual N negative MicroSEQ spp Detection Kit MicroSEQ spp Detection Kit Confirmatory tests Result Ct Result Ct and reference confirmatory tests 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 - / / - / St St / - NA - NA 2624 Saucisse sèche Dry sausage Protocol 1 - St St St St - / / - / St St / - NA - NA 2625 Saucisson sec Dry sausage Protocol St - / / - / - St / - NA - NA 2626 Chorizo Cooked sausage Protocol 1 - St St St St - / / - / - St / - NA - NA 2627 Saucisse sèche Dry sausage Protocol St St - / / - / - St / - NA - NA 3073 Rosette Cooked sausage Protocol St St - / / - / - - / - NA - NA 3074 Saucisse sèche Dry sausage Protocol 1 - St St / / - / St St / - NA - 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 - / 3076 i/-* / - / - - / - NA - NA 3077 Cervelas Cooked sausage Protocol 1 - St St St St - / / i/-* / St - / - NA - NA 3078 Saucisse de Morteau Cooked sausage Protocol 1 - St St St St - / / i/i*/-** St St / - NA - 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 - / / i/-* / - - / - 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 p +p + + PA + PA 3083 Poitrine rôtie au four Cooked ham Protocol 1 + +p +p +p +p to p +p + + PA + PA 3211 Rumsteak Beef trim Protocol 1 + +m +p +M +p to /2 ni +M + + PA + PA 3212 Onglet Beef trim Protocol 1 + +m +1/2 +M +M to m ni +m + + PA + PA 3213 Gite de noix Beef trim Protocol 1 + +M +M +M +p to m +M + + PA + PA 3219 Steak haché pur boeuf Ground beef Protocol /2 +M +M +M to m +M + + PA + PA 3220 Steak haché Ground beef Protocol 1 + +m +p +M +M to /2 +M + + PA + PA 3223 Steak haché Ground beef Protocol to / - / - - / - NA - NA 3224 Steak haché Ground beef Protocol to m ni +m + + PD + PD 3225 Steak haché Ground beef Protocol to / - / - - / - 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 3214 Bavette Beef trim Protocol 1 + +m +M +M +M to /-/-/-* / m ni +m + - ND + PA 3216 Tranche en tournedos Beef trim Protocol 1 + +m +p +M +M to M + + PA + PA 3217 Gite de noix Beef trim Protocol 1 + +m +M +1/2 p to i/+* m ni +M + + PA + PA 3218 Basse côtes Beef trim Protocol 1 + +m +M +M +p to 3234 i/-*/-/- / -/+/ / m ni +m + - ND - ND 3221 Steak haché Ground beef Protocol 1 + +m +M +M +p to M + + PA + PA 3222 Steak haché Ground beef Protocol 1 + +M +M +1/2 +M to /2 + + PA + PA Final Agreement Final Agreement ADRIA Développement 39/77 March 25, 2014 (Pathatrix PCR Summary Report Version 0)