Verification of a Commercial Real-Time PCR Shiga-toxin Producing E. coli (STEC) Detection Methods on Individually Quick-Frozen Vegetable Matrices
Erica Miller 1, Christopher Crowe 2, and J. David Legan 3
Eurofins Microbiology Laboratories, Inc., 1 Louisville, KY, 2 Salinas, CA, 3 Madison, WI
ABSTRACT
Introduction
With increasing concerns and rising numbers of product recalls associated with pathogens in produce, most major PCR testing platforms are validated for detection of STEC in produce. However, validations for other vegetable matrices are sparse, particularly for frozen matrices which can severely reduce initial temperatures of enrichment broths.
Purpose
To verify the ability to detect STEC in frozen vegetable matrices utilizing real-time PCR.
Methods
Twenty-three different frozen vegetable matrices were tested, including cauliflower, carrots, various vegetable medleys, and chopped spinach: each with seven replicates (125g) inoculated with Escherichia coli O111 at ca. 30 cfu/sample. All samples were enriched with 1,125 mL of BACGro™ modified tryptic soy broth (mTSB) and were incubated within the AOAC validated enrichment time window of 10-24 hrs. Following incubation, samples were tested for STEC using BACGene™ real-time PCR kits according to the manufacturer’s instruction. All proprietary media and tests kits were supplied by Gold Standard Diagnostics™
Results
All seven replicates returned a presumptive positive result for STEC for each of the 23 matrices, giving a total of 161 presumptive positive PCR results. The FDA microbiological method validation guidelines require 7/7 (or 19/20) presumptive results to consider a method extended to a new matrix, with no statistical analysis necessary. This standard was met for all 23 matrices in this study.
Significance
These data demonstrate that this method can reliably detect STEC organisms in a wide range of frozen vegetable matrices.
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