AOAC-RI ERP Book Micro Jan 19 2017

1.0 OBJECTIVE The objective of this study was to validate the 3M™ Molecular Detection Assay (MDA) for detection of Escherichia coli O157 in ground beef with an 18 hr enrichment time. The Food Safety Net Services (FSNS) San Antonio Laboratory worked to validate the MDA system, considered here as the ‘candidate method’, using a 325 g sample size for ground beef with an 18 hr enrichment. The study followed the format for the validation of a candidate method using the experimental setup and Probability of Detection (POD) statistical analysis that are provided in the 2012 version of the AOAC International Methods Committee Guidelines for Validation of Microbiological Methods for Food and Environmental Surfaces document (1). For the purposes of this study, the candidate method results were compared with the analysis of 325 g sample sizes for ground beef performed by reference methods outlined via the 2015 version of the United States Department of Agriculture Food Safety and Inspection Service Microbiological Laboratory Guidebook (USDA FSIS MLG) (2). 2.1 Acquisition and Initial Analysis of Ground Beef As mentioned in Section 1.0, ground beef was tested in the validation of the MDA. The FSNS San Antonio Laboratory acquired raw intact whole muscle beef from either commercial grocers or producers. Multiple lots and producers were sourced, whereupon the products were mixed in order to make an independent lot. The raw intact whole muscle beef was ground in-house at the FSNS San Antonio Laboratory. All samples were stored at refrigeration temperatures until the inoculation and testing commenced. Prior to inoculation, one 25 g sample was removed and analyzed for E. coli O157 according to the USDA FSIS MLG methods for E. coli O157 (2), as outlined in Section 2.4. 2.2 Preparation of E. coli O157:H7 Inoculum As stated, the objective of this study was to determine the ability of the MDA to detect the presence of E. coli O157:H7 in ground beef. The inoculum used for this validation study consisted of the following strain of E. coli O157:H7: Prior to each day of the experiment, an individual culture was prepared by streaking loopfuls of culture from - 80˚C freezer stocks onto Tryptic Soy Agar (TSA; Becton, Dickinson and Company, Franklin Lakes, NJ) and incubating aerobically for 24 h at 35 ± 2°C. One isolated colony from each TSA plate was then transferred into Tryptic Soy Broth (TSB; Becton, Dickinson and Company) and incubated aerobically at 35 ± 2°C for 24 h to achieve stationary phase growth. Cells were 2.0 METHODS AND MATERIALS  Escherichia coli serotype O157:H7 ATCC 35150

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