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 two different food matrices (sprouts and spinach). The Food Safety Net Services (FSNS) San Antonio Laboratory worked to validate the MDA system, considered here as the ‘candidate method’, a 25 g sample size for sprouts and a 200 g sample size for the spinach matrices. 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 200 g sample sizes for spinach and 25 g sample sizes for sprouts, performed by reference methods outlined via the 2014 version of the U.S. Food and Drug Administration Bacteriological Analytical Manual (FDA BAM) (2). 2.1 Acquisition and Initial Analysis of Food Matrices As mentioned in Section 1.0, three different food matrices were tested in the validation of the MDA. The FSNS San Antonio Laboratory acquired sprouts and spinach from either commercial grocers or producers. Multiple lots and producers were sourced for each matrix, whereupon the products were mixed in order to make an independent lot. All samples were stored at refrigeration temperatures until the inoculation and testing commenced. Prior to inoculation, one 25 g sample of each matrix was removed and analyzed for E. coli O157 according to the FDA BAM 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 various food matrices. 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 harvested by centrifuging aliquots of the stationary phase TSB culture at maximum 2.0 METHODS AND MATERIALS  Escherichia coli serotype O157:H7 ATCC 35150

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