中文摘要：

根据Gen Bank公布的大肠杆菌O157：H7的菌体抗原基因rfb E、鞭毛抗原基因fli C、溶血素基因hly A、紧密黏附素基因eae A和志贺样毒素基因stx1和stx2的序列,同源性比较后选择保守序列分别设计6对特异性的引物及相应的Taqman探针,rfb E/eae A、Stx1/hly A、fli C/Stx2探针5＇端分别标记为FAM、HEX、CY5荧光报告基团,3＇端均标记为BHQ1荧光淬灭基团。通过优化反应体系和程序,建立2个能够快速、特异性地检测大肠杆菌O157：H7及其4个主要毒力基因的三重荧光定量PCR方法。结果显示,该方法灵敏度高,stx1、rfb E、fli C、eae A、hly A、stx2的最低检测限分别为20、30、20、20、30和40拷贝数/μL;特异性试验证明,该菌与其他菌种无交叉反应;重复性好,变异系数均小于2%;检测过程耗时约1 h。将建立的荧光定量PCR体系应用到人工染菌模拟猪肉样品试验中,未富集或富集8 h后测得大肠杆菌O157：H7的最低检出限分别为200 cfu/m L与1 cfu/m L。以上结果表明,本试验所建立的三重荧光定量PCR方法的敏感性、重复性及特异性均较好,可作为同时快速检测肠出血性大肠杆菌O157：H7及其毒力基因的方法。

英文摘要：

According to the sequences of Escherichia coli O157： H7 O- antigen gene rfb E,H- antigen gene fli C,hemolysin gene hly A,intimin gene eae A,and shiga- like toxin genes stx1 and stx2 published on Gen Bank,and six pairs of specific primers and corresponding fluorogenic- labeled Taqman probes were designed based on conserved sequences. The 5＇- end of probes of rfb E / eae A,Stx1 / hly A,fli C / Stx2 were individually labeled with FAM,HEX and CY5 fluorescence reporting group,and 3＇- end of probes were all labeled with BHQ1 fluorescence_ quenching group. By optimizing of reaction system and procedures,two triple real- time fluorescence quantitative PCR methods were developed for rapid and specific detection of E. coli O157： H7 and four virulence genes. The results showed that the sensitivity of this method was high,the detection limits of five genes stx1,rfb E,fli C,eae A,hly A and stx2 were 20,30,20,20,30 and 40 copies / μL,respectively.Specificity test confirmed that there was no cross reaction with other strains. It had a good reproducibility,and the coefficients of variations in reproducibility test were less than 2%. The detection process took about one hour. In this study,the fluorescence quantitative PCR system was established and applied to the experiment of simulated pork samples. The lowest detection limits of Escherichia coli O157： H7 without enrichment or 8h enrichment were 200 cfu / m L and 1 cfu / m L,respectively. All the results above showed that,the sensitivity,re-producibility and specificity of the triple real- time fluorescence quantitative PCR established here were good,and could be used as a powerfulmethod for the rapid detection of E. coli O157： H7 and virulence genes.