Purpose: While several Real-Time Polymerase Chain Reaction (RTi-PCR) assays are commercially available, the genetic targets of these assays are not disclosed making it more difficult to interpret indeterminate results. The purpose of this study was to develop a RTi-PCR assay to detect E. coli O157:H7 and Salmonella individually, or in combination, based on housekeeping gene sequences, in raw non-intact beef.
Methods: Conserved sequences within the housekeeping genes Z3276 and ttrC were chosen as molecular targets for primer and probe design to amplify E. coli O157:H7 and Salmonella specific sequences, respectively. Modifications of MLG 5B.03 were performed to decrease enrichment time and provide a more efficient DNA extraction using beef trim samples. Inclusivity and exclusivity panels of at least 25 isolates were investigated with conventional and real-time PCR. Exclusivity panels included closely related strains of E. coli O157:H7 (E. coli O55) and Salmonella (Proteus and Citrobacter).The use of a randomly generated sequence, not naturally occurring in nature, was used as an internal amplification control.
Results: E. coli O157:H7 primers detected all isolates in the inclusivity panel (n = 31) and none of the isolates in the exclusivity panel (n = 27). E. coli O157:H7 primers detected 4 E. coli O157:NM. Salmonella primers detected all inclusivity isolates (n = 32) and detected no isolates of the exclusivity panel (n = 25). Raw, non-intact beef was inoculated with 10 CFU/g of E. coli O157:H7 and incubated for 10h at 42°C in pre-warmed phosphate buffered tryptic soy broth. A 2 min microwave lysis and a quicker DNA extraction resulted in a RTi-PCR Ct value of 20.22 and 19498.22 dR (2547.55 dR threshold).
Significance: The shortened enrichment and DNA extraction protocol will facilitate rapid detection of E. coli O157:H7 and Salmonella in perishable foods. The RTi-PCR assay developed here can be adapted to different real-time chemistries and performed on an open system.