Evaluations of Three Multiplex Real-time PCR Assays for the Detection of Six Shiga Toxin-producing Escherichia coli (STEC) Serogroups and the eae, stx1, and stx2 Genes in Ground Beef and Trim Enrichments

Introduction:  Regulations in the United States starting in June 2012 will require industry to begin monitoring for six Shiga toxin-producing E. coli (STEC) serogroups.  The regulations will specifically require detection of the so called “big-six” STEC (O26, O45, O103, O111, O121, and O145) as these have been most frequently associated with outbreaks of foodborne illnesses in the United States.  There is an additional requirement related to the detection of the virulence genes eae, stx_1/2. 

Purpose: The purpose of this study was to determine the feasibility and conduct initial evaluations of three separate, previously developed, multiplex PCR assays for the detection of the big-six serotypes of STEC and the eae, stx_1/2 genes in spiked food enrichments (ground beef and trim).      

Methods:  The three assay configurations were: Assay 1 – O26, O111, O121, and internal positive control (IPC); Assay 2 – O45, O103, O145, IPC; Assay 3 – eae, stx_1/2, IPC.   Both 65 g and 375 g ground beef and 375 g trim enrichments were diluted (1:10 or 1:5) in pre-warmed (44°C or 46°C) sterile tryptic soy broth (TSB) and/or TSB + 2 mg/l novobiocinand incubated for 9-24 h at 41 °C.  Twenty µl was removed for processing and testing by the BAX® System methods.

Results: For 65 g ground beef samples all spiked targets (n = 18) were detected appropriately in as little as 9 h of enrichment.  For 375 g ground beef samples all spiked targets were detected (n = 30) appropriately within 12 h of enrichment.  For 375 g trim samples all spiked targets (n= 18) were detected appropriately within 10 h of enrichment. 

Significance: These results demonstrate the feasibility of deploying a panel of three novel real-time PCR assay configurations for the detection and monitoring of STEC O groups as well as the virulence genes, eae, stx_1/2 in actual food enrichments.  Studies with other food types including produce are ongoing.