MGB(Minor Groove Binder)复合探针二重实时定量PCR检测大肠杆菌O157

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

摘要:

以大肠杆菌O157rfbE和stx2为待检靶基因，设计两对引物和两条MGB探针，rfbE和stx2探针5′端分别用FAM和VIC基团标记，3′端均用Taqman-MGB标记。建立并优化了检测大肠杆菌O157的二重荧光定量PCR方法，可检测的最低DNA浓度是10拷贝/μL；实验中O157菌株检测结果均为rfbE阳性,而非O157菌株检测结果均为阴性；重复性实验中,批间差异小于80%,批内差异小于70%。实验结果显示此二重荧光定量PCR方法可对分离的可疑大肠杆菌O157菌株进行快速鉴定,同时得知菌株是否携带stx2毒力基因，有利于菌株毒力强弱的判定。

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.