Application of a Novel Four-Plex Quantitative PCR Assay for Quantification of Escherichia coli O157 on Cattle Hide and Carcass

Introduction: We have previously developed and validated a multiplex (four-plex) quantitative PCR (mqPCR) to detect and quantify E. coli O157 in cattle feces.  The assay targets genes that code for serogroup specific O157 antigen (rfbE_O157) and three major virulence factors, Shiga Toxins 1 and 2 (stx1 and stx2) and intimin (eae).  Cattle hides are the major source of E. coli O157 contamination on carcasses at the time of slaughter.  Although prevalence data have been reported, concentration data are an important component of quantitative microbial risk assessments.  Although several real-time PCR assays have been developed to quantify E. coli O157, none have included rfbE in combination with stx1, stx2, and eae.

Purpose: Our objective was to validate a mqPCR for the quantification of E. coli O157 in cattle hide and carcass samples.

Methods: Pure culture sensitivity of the assay was determined with extracted DNA from serial ten-fold dilutions of E. coli O157 strain (ATCC 43894), positive for all four genes, cultured in Luria-Bertani broth.  Cattle hide and carcass sponge samples (n = 78) were collected at a slaughter plant.  Samples, determined to be negative for E. coli O157 by PCR were inoculated with ten-fold serial dilutions of the same strain.  Sensitivity of the mqPCR assay from spiked hide and carcass samples before and after six-hour enrichment was then determined.

Results: In pure culture, the minimum detection limit of the mqPCR assay was 2.2 x 10³ CFU/ml.  The detection limit of the assay for E. coli O157 with DNA extracted from cattle hide and carcass samples was identical to pure culture sensitivity (2.2 x 10³ CFU/ml) for both sample matrices.  After a six-hour enrichment, sensitivity increased to 2.2 x 10⁰ CFU/m for carcass and hide samples.

Significance: The assay targeting the four genes is a sensitive and high-throughput method to quantify E. coli O157 in cattle hide and carcass samples.