【摘要】 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。更多还原

【Abstract】 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.更多还原