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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。
According to Gen Bank published sequence of E. coli O157: H7 bacterial antigen gene rfb E, flagella antigen gene fli C, hemolysin gene hly A, compact adhesin gene eae A and shiga-like toxin genes stx1 and stx2, homology After comparison, six pairs of specific primers and corresponding Taqman probes were designed by selecting conservative sequences. The 5 ’end of rfb E / eae A, Stx1 / hly A and fli C / Stx2 probes were labeled as FAM, HEX, CY5 fluorescent reporter The 3 ’end of the group is labeled as the BHQ1 fluorescence quencher. By optimizing the reaction system and procedure, two triplex real-time PCR methods were established to detect E. coli O157: H7 and its four major virulence genes rapidly and specifically. The results showed that the sensitivity of this method was high, and the detection limits of stx1, rfb E, fli C, eae A, hly A and stx2 were 20,30,20,20,30 and 40 copies / μL, respectively. The bacteria and other species no cross-reaction; good repeatability, the coefficient of variation of less than 2%; The detection process takes about 1 h. The established fluorescence quantitative PCR system was applied to artificial pigmentation simulated pork samples. The minimum detectable limit of E. coli O157: H7 was 200 cfu / m L and 1 cfu / m L. The above results showed that the triplex fluorescence quantitative PCR method established in this study was more sensitive, reproducible and specific and could be used as a rapid and rapid method for simultaneous detection of enterohemorrhagic Escherichia coli O157: H7 and its virulence genes.