Evaluation of real time PCR assays for the detection and enumeration of enterohemorrhagic \u3ci\u3eEscherichia coli\u3c/i\u3e directly from cattle feces

Shiga toxin-producing Escherichia coli are a growing concern in the area of food safety, and the United States Department of Agriculture Food Safety and Inspection Service has identified the serotypes O26, O45, O103, O111, O121, O145, and O157 as adulterants in certain types of raw beef. The most relevant to human disease are the enterohemorrhagic E. coli (EHEC) strains that possess intimin (eae), Shiga toxin 1 and/or 2 (stx1–2), and in most cases the conserved pO157 or pO157 like virulence plasmid. Contamination of raw beefwith EHEC is likely to occur via the transfer of cattle feces on hides to the carcass. To detect EHEC directly from cattle feces,we evaluated the utility of a multiplex real time PCR assay that targets the EHEC associated gene target ecf1 in combination with eae and stx1–2. Our assay had an increased sensitivity and provided a reliable limit of detection (LOD) of 1.25× 103 colony-forming units permL(CFUs/mL) in an EHEC spiked fecal background. In addition,we evaluated the use of a duplex qPCR assay using ecf1 for the enumeration of total EHEC directly from cattle feces. The reliable limit of quantification (LOQ)was determined to be 1.25 × 103 CFUs/mL. Our assay requires minimal sample processing and provides LOD and LOQ of EHEC directly fromcattle feces that are the lowest reported. The application of this assay towards the identification of cattle shedding EHEC at a level above 1.25× 103 CFUs/mL could be a first line of defense in identifying cattle shedding these pathogens