Validation of a Test System for Detecting Non-O157 Shiga Toxin-producing Escherichia coli Strains in Beef

Introduction:  Shiga toxin-producing Escherichia coli (STEC) strains other than serotype O157 represents some of the most important causes of foodborne infections worldwide, but their occurrence is probably underestimated because of the lack of an integrated detection method for these serotypes. 

Purpose:  To validate a test system based on multiplex PCR (mPCR) and lateral flow immunoassay (LFI) for detecting the “top six” non-O157 STEC serotypes, and to evaluate its performance on artificially inoculated beef trim.

Methods:  About 3 to 6 CFU of each serotype (O26, O45, O103, O111, O121 and O145) were spiked into 375 g of beef trim, stored overnight at 4°C, and then enriched with mTSB + 8 ug/ml vancomycin.  Sample aliquots were taken after 9 and 12 h incubation at 42°C, and analyzed by using multiplex PCR and LFI.  Results were confirmed using the USDA-FSIS MLG 5B.01 method.

Results: A multiplex PCR assay for screening and detecting non-O157 STEC using intimin, Shiga-toxin, and serotype-specific O-antigen flippase genes as targets, was developed and validated.  The detection limit of the multiplex PCR and LFI methods were 10⁴ cells/ml and 10⁵cells/ml, respectively.  The performance of the test system was assessed on 20 replicates of artificially inoculated beef samples and compared with the MLG 5B.01 method.  Positive PCR signals were detected as early as after 9 h enrichment, and all confirmed positives were detected after 12 h. Both the multiplex PCR and LFI methods gave 100% concordance with the MLG 5B.01 results. 

Significance:  The test system effectively detects six non-O157 STEC serotypes from spiked meat samples at a shorter incubation time (12 h) than the MLG 5B.01 method (15-22 h).