Application of Duplex Real-time PCR for Detection of Escherichia coli O157 by Using MGB Fluorescence Probe

Abstract:

A duplex real-time PCR, based on Taqman hybridization probes technology, were developed and applied to detect enterohemorrhagic Escherichia coli O157.Two pairs of primer and two probes were designed for detection rfbE and stx2 genes .the rfbE probe was 5′end labeled with FAM) and 3′end labeled with Taqman-MGB. The stx2 probe was 5′end labeled with VIC and 3′end labeled with Taqman-MGB. The detection limits of the sensitivity assays were 101 copies/uL of DNA; the qualitative consensus PCR assay indicated all Escherichia coli O157 were found rfbE positive and did not detect DNA from non- O157 isolates. In the duplicated experiment, coefficients of variation intra-assay and inter-assay over the dynamic range of the MGB probe assays were lower than 70% and 80%, respectively. These results show that this duplex real-time PCR can detect the Escherichia coli O157 and stx2 gene from samples rapidly, and that the virulence of the strains were known.

Key words:Escherichia coli O157, Duplex real-time PCR, MGB probe,rfbE,stx2