Automated DNA extraction and multiplexed PCR assay to identify Escherichia coli O157:H7 from ground beef and kitchen surface swab samples

Abstract

Escherichia coli O157:H7 is a common pathogen found in processed beef trimmings and ground beef that has lead to many infectious outbreaks associated with undercooked meat. Only one strain of E. coli is tested for in ground beef, however, legislation is being considered for identifying up to 6 additional strains. We have developed a testing method that uses the Automated Sample Preparation (ASP) instrument, with a multiplexed PCR assay containing HyBeacons, to easily identify three genes in the O157:H7 strain. HyBeacons probes are hybridization probes that simultaneously identify multiple genes via melt curve analysis. In this study, samples of ground beef inoculated with O157:H7, and swabs from inoculated surfaces that had been in contact with ground beef, were cultured at 37°C for 24 hours. Following incubation, 200 μl from each sample was placed into an ASP cartridge for DNA extraction and purification. DNA from each sample was used in a multiplexed PCR assay that identifies stx 1, stx 2, and eae gamma genes of O157:H7. DNA yields from beef culture and swab samples were measured on a NanoDrop (Thermofisher) and yielded an average of 26ng/ul and 17.2 ng/ul respectively. All gene targets were successfully amplified and confirmed via high resolution melt curve analysis. The multiplexed assay would be ideally suited for expansion to include additional O157:H7 genes or even identification of multiple strains of E. coli.