Establishment of Specific Fluorescent Quantitative PCR Assay for Detection of the Enterohemorrhagic Escherichia coli O157:H7

Objective: To develop a specific real-time PCR assay for the detection of Escherichia Coli O157:H7 in food.Methods: Probes and primers for a TaqMan quantitative PCR were designed and synthesized according to the conserved gene sequence rfbE of Enterohemorrhagic Escherichia coli(EHEC) O157: H7,available in GenBank.The rfbE probe was 5' end labeled with FAM and 3' end labeled with TAMRA.Then reaction parameters were optimized to develop a TaqMan-quantitative PCR assay.Results: The real-time PCR assay was applied to samples artificially contaminated by Escherichia Coli O157: H7,the detection limits of the sensitivity assays were 10 copies/reaction(3CFU/mL) of DNA.The qualitative consensus PCR assay indicated all Escherichia coil O157:H7 were found rfbE positive and did not detect DNA from non-O157:H7 isolates.In the duplicated experiment,coeficients of variation intra-assay and inter-assay over the dynamic range of the TaqMan probe assays were lower than 3%.Conclusion: This study shows that the real-time PCR is a repeatable,specific,sensitive and rapid method for the detection of EHEC O157: H7.