DuPont Qualicon BAX System

1 Copyright 2009 DuPont . All rights reserved. The DuPont Oval, DuPont , The miracles of science and BAX are registered trademarks or trademarks of DuPont or its affiliates. BAX System real-time PCR assays and Q7 instruments are sold under licensing arrangement Applied Biosystems for food testing. Use for research and development, quality assurance and quality control testing under supervision of technically qualified persons. Not approved for clinical use. Please read the limitation of warranty and liability before use. 23A-054-0610. WHITE PAPER DuPont Qualicon ESL Bldg 400, PO Box 80400 Wilmington, DE 19880 USA Tel: 800-863-6842 or 302-695-5300 Fax: 302-695-5301 Europe: 00 800 3876 6838 Singapore: +65 6586 3635 DuPont Qualicon puts necessary info at your fingertips for USDA FSIS micro method audits In November 2009, the National Meat Association and the Southwest Meat Association sponsored a joint seminar on upcoming changes to FSIS audit procedures.

2 Dr. William K. Shaw, Jr., USDA FSIS Office of Policy and Program Development, presented a proposed supplement to Directive , which describes evaluation procedures that FSIS inspectors should follow when auditing beef plants. The draft supplement contains nine broad questions that focus additional attention on pathogen testing methods. Beef plants would be required to not only answer these questions but also supply supporting evidence. At first glance, responding to these questions may seem like a daunting task. However, customers of DuPont Qualicon will quickly discover that the information they need concerning BAX System PCR assays is fully documented and readily available: Package inserts are included in each kit User Guides and Ready References are included on the CD with each software release AOAC study reports and technical bulletins are available upon request To illustrate this point, we ve taken each of the nine questions and provided brief answers, including examples of the documentation available from DuPont Qualicon to help you during an audit by FSIS of your testing methods.

3 For this example, we are using the BAX System Real-Time PCR Assay for E. coli O157:H7 in beef. Question 1. Does the establishment provide documentation necessary to understand the methods used and the scientific basis for their selection? Yes. Supporting documentation available from DuPont Qualicon : Package insert gives instructions for use and a description of the scientific principles behind the assay. Ready reference provides a graphical flowchart of the protocol, which can be displayed in the lab. User Guide provides description of entire System with scientific principles, instructions for individual assays, tips and troubleshooting.

4 DuPont Qualicon BAX System REAL-TIME PCR ASSAY for E. coli O157:H7 White Paper Page 2 USDA FSIS Micro Method Audit Information Question 2. Is the method designed to detect the lowest possible level of injured pathogens from product? Yes. Supporting documentation available from DuPont Qualicon : Package insert provides enrichment instructions to nurture bacterial growth from a theoretical single cell per sample to levels detectable with the BAX System . For E. coli O157:H7, ground beef is enriched at least 9 hours and beef trim at least 10 hours in pre-warmed BAX System MP broth. Analytic sensitivity report provides in-house validation of detection limit, showing that the BAX System successfully detects E.

5 Coli O157:H7 when enrichment concentrations reach 104 cfu/mL. AOAC-RI method comparison report provides in-house and independent lab validation of BAX System performance as equivalent to the USDA FSIS method for detecting the target in low-spike studies (diagnostic sensitivity). For E. coli O157:H7, the real-time assay was consistent with single cell detection of the target pathogen in ground beef and beef trim, with <1% false positives or false negatives. AOAC-RI inclusivity/exclusivity report details BAX System capabilities for detecting only the target bacteria (inclusivity) and none of the non-target bacteria (exclusivity).

6 The real-time assay detected 100% of diverse strains, including atypical strains, from an inclusivity panel of E. coli O157:H7 that originated from human clinical, veterinary and food sources. The assay detected none of the non-target bacteria in the exclusivity panel. Question 3. Do the sample collection procedures support the establishment s hazard analysis decisions resulting in an effective food safety System ? Your company would typically provide the standard operating procedures with scientific support for the sample collection scheme from the sampling plan in your HACCP program. Question 4. Does the establishment use a test portion equivalent to the comparable FSIS method?

7 Yes. Supporting documentation available from DuPont Qualicon : AOAC-RI method comparison report validates BAX System performance on industry-standard analytical portions with <1% false results: o Raw ground beef 65g samples homogenized in pre-warmed (42 C) MP broth and enriched at least 9 hours at 42 C (appropriate for 325g ground beef test portion as five 65g samples). o Beef trim 375g samples homogenized in pre-warmed (45 C) MP broth and enriched at least 10 hours at 42 C (appropriate for 325-375g test portions). o Note: These validated BAX System enrichment protocols differ from MLG 5, which uses mTSB+n as enrichment medium, and incubates all homogenates 15-22 hours at 42 C.

8 White Paper Page 3 USDA FSIS Micro Method Audit Information Question 5. Does the screening and confirmation method detect the same target pathogen as the FSIS method? Yes. Supporting documentation available from DuPont Qualicon : AOAC-RI inclusivity/exclusivity report shows that the BAX System detected 100% of the E. coli O157:H7 strains tested and returned negative results for 100% of the exclusivity strains tested. The E. coli O157:H7 strains used for inclusivity testing were obtained from the Pennsylvania State University Department of Veterinary Science E. coli Reference Center, Michigan State University National Food Safety & Toxicology Center Microbial Evolution Laboratory, USDA collaborators and the DuPont Qualicon culture collection.

9 Strains used for exclusivity testing were obtained from the DuPont Qualicon culture collection. Package insert describes confirmation according to the FSIS procedures, with additional step to accommodate atypical strains that may not be detected with immunomagnetic separation. Question 6. Could additional detection or confirmatory procedures affect interpretation of the sample result? Probably not. Supporting documentation available from DuPont Qualicon : Sampling distribution effects technical bulletin describes the probability of getting different results due to random distribution for single versus duplicate testing schemes.

10 Package insert sample preparation protocol does not include a cell concentration (IMS) step because IMS may not capture atypical or rough strains, if present. Sufficient time is specified in the enrichment protocol for bacterial growth of all E. coli O157:H7 strains to reach detectable levels. Recommended confirmation includes both the FSIS-specified IMS step and a separate plate from the enrichment without IMS to confirm atypical or rough strains. Question 7. Is the method used by a regulatory body or properly validated by an independent body? Yes. Supporting documentation available from DuPont Qualicon : AOAC-RI certificate #031002 certifies this method as Performance TestedSM by the AOAC Research Institute for 65g ground beef enriched in MP broth at least 9 hours and 375g beef trim enriched in MP broth at least 10 hours.