肠出血性大肠杆菌(O157:H7)的特异性荧光探针检测方法的建立

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

目的:建立一种real-time PCR,快速准确检测肠出血性大肠杆菌O157:H7。方法:以肠出血性大肠杆菌0157:H7 rfbE为待检靶基因,设计一对引物和一条Taqman探针,探针5'端用FAM基团标记,3'端用TAMRA标记。通过重组质粒的构建,建立并优化了大肠杆菌0157:H7的荧光定量PCR检测方法。结果:在人工污染样本无需富集的情况下,检测的最低DNA浓度是10拷贝/反应(3CFU/mL);特异性检测实验中,0157菌株检测结果均为rfbE阳性,而非0157:H7菌株检测结果均为阴性;重复性实验中,批内、批间变异系数均小于3%。结论:实验结果显示此荧光定量PCR方法特异性、灵敏度高,重复性好,可对分离的可疑大肠杆菌0157:H7菌株进行快速鉴定。

Objective： To develop a specific real-time PCR assay for the detection of Eschefichia Coli O157：H7 in food. Methods： Probes and primers for a TaqMan quantitative PCR were designed and synthesized according to the conserved gene sequence rtbE of Enterohemorrhagic Escherichia coli （EHEC） O157： H7, available in GenBank. The rfoE 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 O 157：H7 were found rfbE positive and did not detect DNA from non-O 157：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.