Detection of viable shiga toxin-producing Escherichia coli by multiple-timepoint quantitative competitive polymerase chain reaction

Shiga toxin-producing Escherichia coli (STEC) has emerged as a threat to public health. E. coli O157:H7 is the most prominent member of this group. Most E. coli O157:H7 infections are foodborne, and its infective dose is very low (less than 10 cells). Rapid and sensitive methods to detect STEC are needed in the food industry.; A quantitative competitive polymerase chain reaction (QC-PCR) assay was developed to detect and quantify E. coli O157:H7. A 401 bp fragment in the A subunit of the stx2 gene was chosen as a target sequence and a 275 bp competing DNA sequence for use in the QC-PCR assay was constructed from the stx2 sequence using composite primer PCR. The amplification efficiency of the competitor sequence was determined to be the same as that of the target sequence. DNA extracted from broth or milk containing 103 to 108 CFU/ml E. coli O157:H7 can be detected and quantified. The cell densities determined by plate counts and QC-PCR were highly correlated from broth (R2 = 0.99) or skim milk (R2 = 0.99). These results indicated that QC-PCR has potential for quantitative detection and enumeration of pathogenic bacteria in foods.; A detection procedure was developed including enrichment, immunomagnetic separation (IMS) and the established QC-PCR assay. Viable E. coli O157:H7 cells were inoculated into cooked ground beef at levels of 0.1, 1,10 CFU/g in 25 g ground beef, and enriched with 225 ml of modified EC broth supplemented with novobiocin at 37°C with shaking at 200 rpm. IMS was an effective step to isolate and concentrate cells from ground beef enrichments. The detection limit (∼103 CFU/ml enrichment) from ground beef enrichments by IMS-QC-PCR was two logs lower than that (∼10 5 CFU/ml enrichment) by QC-PCR. Timewise, detection of viable E. coli O157:H7 cells from ground beef enrichments by IMS-QC-PCR saved 2 h compared to QC-PCR without IMS. Cell viability was confirmed by the quantitative increase in signals of target bands in the multiple QC-PCRs. E. coli O157:H7 inoculated at 0.20 CFU/g ground beef (25 g meat +225 ml mEC+novo) was detected and confirmed as viable in less than 15 h by the application of enrichment and multiple-timepoint IMS-QC-PCR.