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CONFIDENTIAL INFORMATION
Page 2 of 13
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).
2.0 METHODS AND MATERIALS
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:
Escherichia coli
serotype O157:H7 ATCC 35150
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