基于内参的大肠埃希氏菌O157∶H7实时荧光定量PCR快速检测方法的建立

Development of Real-Time Quantitative PCR Assay for the Detection of E. coli O157:H7 Based on Internal Amplification Reference

针对大肠埃希氏菌O157∶H7 rfb E和Flic靶基因保守序列,设计特异性引物和探针,优化反应体系,并加入内参(internal amplification control,IAC),建立能够实时监控反应过程的荧光定量聚合酶链式反应(polymease chain reaction,PCR)检测方法。结果表明,该方法对E.coli O157∶H7基因组DNA的最低检测限为1 pg/μL;对含有靶基因质粒的最低检测限为103 copies/μL;对细菌的最低检测限为5×103 CFM/m L;Ct值与模板拷贝数均呈良好的线性关系(R2=0.999)。人工污染实验结果表明,在初始菌量为7 CFM/25 g时,采用水洗加试剂盒法提取DNA,E.coli O157∶H7在增菌6 h后即可检出;而采用水煮法提取DNA,则在增菌10 h后方可检出。建立的E.coli O157∶H7实时荧光定量PCR方法,既能有效检测食品中O157∶H7,又能实时监测PCR反应过程,有效防止"假阴性"的发生。

Based on the rfb E and Flic genes of Escherichia coli O157：H7, the specific primers and probes were designed, and a real-time fluorescence quantitative PCR（RT-q PCR） was developed. An internal amplification control（IAC） was added to the reaction system to monitor the performance of reaction system. The assay could be used reliably to detectE. coli O157：H7 genomic DNA with a sensitivity of 1 pg/μL. For the plasmid with rfb E and Flic, the limit of detection（LOD） reached 103 copies/μL. The LOD for E. coli O157：H7 was 5 × 103 CFM/m L using the DNA extracted by water boiling as the template. Through the standard curves of rfb E and Flic, the quantifi cation was linear between Ct values and the copy number of template（R2 = 0.999）. For artificially contaminated meat samples with an initial bacterial concentration of 7 CFΜ/25 g, the E. coli O157：H7 could be detected after 6 hours of culture using the DNA extracted by a commercial kit. Using the DNA extracted through water boiling, the E. coli O157：H7 could be detected after 10 hours of culture. The fluorescence quantitative PCR assay could be applied to detect E. coli O157：H7 in food samples and monitor the PCR reaction process without false negative results. Furthermore, the comparison results of two different DNA extraction methods were helpful to standardize the RT-q PCR method for E. coli O157：H7.