Detection of Shiga Toxin-producing Escherichia coli, Seven stx Subtypes and Salmonella via a Two-tiered Multiplex Real-time PCR

Introduction: Shiga Toxin-producing Escherichia coli (STEC) are strains producing Shiga toxin type 1, type 2 or both, leading to hemorrhagic colitis and hemolytic uremic syndromes. E. coli O157:H7 and non-O157 serotypes O26, O45, O103, O111, O121 and O145 are currently considered as adulterants in non-intact beef. Further, Salmonella leads to the second highest number of foodborne infections in the United States. These two pathogens continue to be a major food safety challenge worldwide.

Purpose: The aim of this study was to design a two-tiered multiplex real-time PCR assay for the detection of seven STEC serotypes, stx₁, stx₂ genes and virulent strains of Salmonella.

Methods: PCR primers were designed for the specific amplification of each target. Two multiplex real-time PCR melt curve assays with an internal amplification control (IAC) were standardized for the detection of seven STEC serotypes and Salmonella. The applicability of the assays was tested using ground beef and beef trims. 

Results: The first multiplex assay detected E. coli O121, E. coli O145, E. coli O157, stx₁, stx₂ and IAC; while the second set targeted E. coli O26, E. coli O111, E. coli O103, E. coli O45, Salmonella, and IAC. Following an enrichment period of 8 h, all targets of the multiplex assays could be detected in 325 g of food samples inoculated with 10-20 CFU of each pathogen. The assay showed a reproducible result for beef products with different fat content. 

Significance:  These assays, which do not rely on the use of fluorescent-labeled probes or immunomagnetic beads, can detect seven STEC serotypes, seven of the most common stx gene subtypes and Salmonella, and can be completed in less than 11 h, making them highly suitable for industrial application.