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 a frozen blueberry food matrix. The Food Safety Net Services (FSNS) San Antonio Laboratory worked to validate the MDA system, considered here as the ‘candidate method’, using a 25 g sample size for frozen blueberries. 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 a 25 g sample size for frozen blueberries, performed by reference method 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, the food matrix of frozen blueberries were tested in the validation of the MDA. The FSNS San Antonio Laboratory acquired blueberries from various sources, to be mixed prior to use. The frozen blueberries were thawed at refrigeration temperatures prior to inoculation. Prior to inoculation, a 25 g sample was removed and analyzed for E. coli O157 according to the FDA BAM for E. coli O157 (2, 3), 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 frozen blueberries. 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 the TSA plate was then transferred into Tryptic Soy Broth (TSB; Becton, Dickinson and Company) and incubated aerobically at 35 ± 2°C for 18-24 h to achieve stationary phase growth. A 1:9 dilution of the TSB culture was made in B utterfield’s Phosphate Buffer (BPB; Made In-House) and the transmittance at 560 nm was measured using a Spectronic TM 200 Spectrophotometer (Thermo Fisher Scientific, Inc., Waltham, MA). This suspension served as the inoculum for E. coli O157:H7 and the concentrations of 2.0 METHODS AND MATERIALS  Escherichia coli serotype O157:H7 ATCC 700599

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