Multiplex Real-time PCR Assay for Detection of Eight STEC Serotypes

Introduction: Shiga toxin-producing Escherichia coli (STEC) are strains of E. coli that cause serious illness, including fatal hemolytic uremic syndrome. This group consists of STEC serotypes O157, O26, O45, O103, O111, O121 and O145 that are generally associated with raw meat and are also responsible for more than 71% of infections.  Thus, in addition to STEC O157, the USDA FSIS also declared these six serotypes as adulterants.

Purpose: The aim of this study was to design a multiplex real-time PCR assay for detection of eight STEC serotypes.

Methods: Serotype-specific PCR primers were designed for the specific amplification of each STEC serotype. Final primer sets were selected based on amplicon T_m, reaction efficiency, and amplicon size. Two multiplex melt curve real-time PCR assays with an internal amplification control (IAC) were standardized for the detection of eight STEC serotypes. The applicability of the assays was tested using 11 different meat and produce samples.

Results: The first multiplex assay detected E. coli O145, E. coli O121, E. coli O104, E. coli O157 and IAC; while the second set targeted E. coli O26, E. coli O45, E. coli O103, E. coli O111 and IAC. Following an enrichment period of 6h, all targets of the multiplex assays could be detected in food samples contaminated with a cocktail of four STEC serotypes with a combined count of 10 CFU/25 g food. The assay showed a highly reproducible result for nine food samples tested in this study.

Significance: The assay developed in this study can be used for the detection of eight STEC serotypes and can be completed in less than 11h. Unlike other commercially available methods, it does not require fluorescent-labeled probes or immunomagnetic beads, making it one of the shortest and most commercially feasible methods available.