Design and Evaluation of a 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 late 2011, the USDA announced it will declare 6 additional Shiga toxin-producing E. coli serotypes as adulterants, namely O26, O45, O103, O111, O121, and O145. These 6 non-O157 STECs will be classified as adulterants if they also contain the virulence genes, eae and stx₁ and/or stx₂.

Purpose: To design a real-time PCR assay for detecting STEC adulterants including O157:H7 and the 6 non-O157 STEC bacteria using multiplex designs to reduce the number of tests to one screening assay and one confirmation assay.

Methods: A minimum of four TaqMan® real-time PCR assays were designed against each of the 6 non-O157 STEC serotypes using Applied Biosystems assay design software. Additional assays were also designed against virulence factors stx₁, stx₂, and eae. Each assay was tested against an inclusion/exclusion panel of 242 E. coli strains and 31 non-E. coli bacteria to determine assay specificity and sensitivity. The inclusion panel included all known E. coli O-types, strains belonging to various STEC serogroups, and 62 of the big six non-O157 STEC strains. High throughput screening by real-time PCR was performed on the 7900 real-time PCR system and multiplex optimization was performed on the 7500 Fast real-time PCR system using standard cycling conditions (95 °C for 10 min, followed by 40 cycles at 95 °C for 15 seconds and 60 °C for 60 seconds).

Results: Multiple assays for each of the 6 non-O157 STECs detected all inclusion strains within the targeted serotype, and no exclusion strains from other serotypes. The stx assays detected all variants of stx₁ and stx₂ tested including stx_2f and stx_2g. The assays were combined into two separate multiplex assays and optimized using statistical methods based on Design of Experiments. The assays will be evaluated for detecting STEC in ground beef and beef trim.

Significance: Regulations are moving toward increased testing for foodborne pathogens. Multiplex real-time PCR can combine up to four PCR tests into a single reaction by using four different fluorescent dyes, reducing time and costs.