PRELIMINARY RESEARCH ON RAPID DETECTION OF ESCHERICHIA COLI O157 WITH DUPLEX REAL-TIME FLUORESCENCE PCR ASSAY

[Objective]To establish a duplex real-time PCR assay to detect rfbE gene and stx2 gene rapidly and synchronously.[Methods]Two combinations of primers and Taqman-based fluorescent probes were designed according to the sequences of rfbE gene and stx2 gene published by GenBank,and the reaction condition were optimized to set up a duplex Taqman-based real-time PCR assay.Simultaneously,the specificity,sensitivity and repetitiveness of the assay were assessed.[Results]The real-time PCR assay successfully distinguished the E.coli O157 serotype from non-E.coli O157 serotypes and other common enteric bacterial strains.All bacterial strains that lacked these genes were not detected by this assay.The quantitative ranges of the real-time PCR assays were linear for DNA concentrations of E.coli O157 in pure culture,ranging from 102 to 106 cfu/μl and the detection limit of the real-time PCR assay ranged from 102 to 106 cfu/μl for contaminated milk samples.The overall test could be finished within about 3 hours.[Conclusions]The duplex Taqman-based real-time PCR assay for rfbE gene and stx2 gene are proved to be a rapid,sensitive and specific test for the quantitative detection of E.coli O157 from the contaminated food samples.