PCR方法检测EHEC O157∶H7的rfb_(O157)、fliC_(H7)、hlyA、eaeA、stx2及其变种基因

Detection rfb_(O157)、fliC_(H7)、hlyA、eaeA stx2 and mutation of stx2 genes of EHEC O157∶H7 by PCR and meaning

目的:应用当前国际上常用的rfbO157、fliCH7、hlyA、eaeA、stx2及其变种5对引物,对45株疑似肠出血性大肠埃希菌O157∶H7(EHEC O157∶H7)检测鉴定,全面了解此类菌的致病力情况。方法:分纯并富集被鉴定菌株,首先以单克隆诊断血清学凝集,阳性者再经100℃水煮制成粗模板,应用PCR检测各菌株是否具有rfbO157、fliCH7、hlyA、eaeA及stx2及其变种5种基因,扩增结果经EB染色后在凝胶成像仪下观察特异性条带并拍照。结果:45株被检测菌经PCR扩增检测,5种基因全部阳性的有10株,4种基因出现阳性的只有1株,3种基因阳性的有6株,2种基因阳性的有17株,1种基因阳性的有10株,1株菌的5种基因检测均为阴性。45株菌中rfbO157阳性的有44株,fliCH7基因阳性的菌株有30株,hlyA阳性的菌株只有10株,eaeA阳性基因的菌株有21株,stx2及其变种阳性的也只有11株。结论:这次45株菌中有5株具有全部的5种基因,44株为EHEC O157,其中30株为EHEC O157∶H7,这次选择的5种基因不仅能给被检测菌株定性,而且能较全面地反应其毒力强度,适合于有条件的基层实验室开展。

Objective：To understand the pathogaicity of enteroheromrrhagic Escherichia coli O157：H7 by detecting 45 strains using five pairs of pmriers in common use on currently international of rfbO157, fliCH7, hlyA, eaeA, stx2 and stx2 mutation. Methods： Identified strains were enriched and isolate purely. Positive strains were executed in 100℃ water after monoclone diagnostic serum coagulation. Strains were detected whether they have rfbO157 fliCH7 ,hlyA,eaeA stx2 and mutation of stx2 five pairs of genes by PCR. Especial strips were observed in gel imaging present and taken photograph after spread result dyed in EB. Results： 10 strains from 45 strains detect by PCR were positive on five genes, four genes positive had only one. Six strains had 3 species positive genes. 17 strains had 2 species positive genes. 10 strains had 1 species gene. One strain was detected that it was negative all five genes. 44 strains were rfbO157 gene positive,30 strains were fliCH7 positive gene, 10 strains were hlyA positive gene,21strains were eaeA positive gene,only 11 strains were stx2 and stx2 mutation gene of 45 strains. Condusion：Five strains of 45 strains had all five genes. 44 strains were EHEC O157 and 30 strains were EHEC O157： H7. Five genes selected can not only determine strains but also reflect their virulent degree. This test can be applied in conditional base lab.