Use of the Duplex TaqMan PCR System for Detection of Shiga-Like Toxin-Producing Escherichia coli O157

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Abstract
Real-time PCR assays have been applied for the detection and quantification of pathogens in recent years. In this study two combinations of primers and fluorescent probes were designed according to the sequences of the rfb Escherichia coli O157 and stx 2 genes. Analysis of 217 bacterial strains demonstrated that the duplex real-time PCR assay successfully distinguished the Escherichia coli O157 serotype from non- E. coli O157 serotypes and that it provided an accurate means of profiling the genes encoding O antigen and Shiga-like toxin 2. On the other hand, bacterial strains that lacked these genes were not detected by this assay. The quantitative ranges of the real-time PCR assay for these two genes were linear for DNA concentrations ranging from 10 3 to 10 9 CFU/ml of E. coli O157:H7 in pure culture and milk samples. The real-time PCR allowed the construction of standard curves that facilitated the quantification of E. coli O157:H7 in feces and apple juice samples. The detection sensitivity of the real-time PCR assay ranged from 10 4 to 10 9 CFU/g (or 10 4 to 10 9 CFU/ml) for feces and apple juice and 10 5 to 10 9 CFU/g for the beef sample without enrichment. After enrichment of the food samples in a modified tryptic soy broth, the detection range was from 10 0 to 10 3 CFU/ml. The real-time PCR assays for rfb E. coli O157 and stx 2 proved to be rapid tests for the detection of E. coli O157 in food matrices and could also be used for the quantification of E. coli O157 in foods or fecal samples.