Design and Evaluation of Two-stage Multiplex Real-time PCR Method for Detecting O157:H7 and Non-O157 STEC Strains from Beef Samples

Introduction: E. coli O157:H7 was first recognized as a human pathogen in 1982 and until recently was the only E. coli strain mandated for testing by the USDA. In June 2012, the USDA declared six additional Shiga Toxin-producing E. coli serogroups (O26, O45, O103, O111, O121, and O145) as adulterants in ground beef and beef trim, if they also contain virulence genes for Shiga Toxin 1 and/or 2 (stx1, stx2) and intimin (eae).  

Purpose: To develop a complete workflow, including a two-stage real-time PCR method that meets USDA regulations to detect E. coli O157:H7 and the “big six” non-O157 STEC serogroups.

Methods: Using Applied Biosystems™ assay design software, TaqMan™ real-time PCR assays were designed against each of the 6 non-O157 STEC O-antigen genes and the virulence factors stx1, stx2, and eae.  Each assay was tested against 132 STEC inclusion strains and 283 exclusion strains to determine assay sensitivity and specificity. Assays demonstrating 100% specificity and sensitivity were multiplexed with the MicroSEQ™ E. coli O157:H7 assay and optimized across two PCR reactions.  The final optimized assays were tested against 375-g ground beef samples spiked with as low as 7 CFU of representative E. coli isolates and enriched with TSB for 10 and 15 h. Real-time PCR was performed on the 7500 Fast real-time PCR system using RapidFinder™ Express software.

Results: All assays detected all inclusion strains and showed no cross-reactivity to any of the exclusion strains tested. The stx assays detected all known variants of stx1 and stx2, including stx2f and stx2g. The optimized workflow showed equivalent detection to the USDA MLG reference method 5B.05. 

Significance: The multiplex real-time PCR assays developed are part of a complete food testing solution for routine and rapid detection of E. coli O157:H7 and the "big six" non-O157 STEC strains in beef samples.