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CONFIDENTIAL INFORMATION
Page 2 of 17
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).
2.0 METHODS AND MATERIALS
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:
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
harvested by centrifuging aliquots of the stationary phase TSB culture at maximum