【摘要】 为建立肠出血性大肠埃希菌O157:H7的多重PCR检测方法,针对O157菌特异性eaeA基因、fliC基因、志贺毒素基因(stx1和stx2)及rfbE基因设计5对引物,构建多重PCR反应体系,优化反应的引物浓度和温度检测其特异性和敏感性,并对牛、猪、鸡及犬等不同动物来源的样品进行了大肠埃希菌O157检测。结果显示,该方法具有良好的特异性和敏感性,敏感性达到5×10~3CFU。从检测阳性样本中均分离到目的菌。应用建立的方法在确定样品中是否含有大肠埃希菌O157的同时,还能对菌株毒力进行初步判定。成功建立了肠出血性大肠埃希菌O157:H7多重PCR检测方法,为该病原菌感染的预防和监测提供了简便、快速的技术手段,具有良好的应用前景。更多还原

【Abstract】 To establish a rapid and specific multiple PCR method for detection of Enterohemorrhagic Escherichia coli O157:H7.Five specific genes of E.coli O157 were selected,such as eaeA gene,fliC gene,the shiga-like toxin gene(stx1,stx2)and rfbE gene.Concentration of primers and annealing temperature were optimized.Specificity and sensitivity of the PCR assay were detected.Practical samples,such as feces,rectal swabs,chicken,beef,pork and rectal swabs of diarrhea pig,were detected.The multiplex PCR assay showed good sensitivity and specificity.Sensitivity of the assay was 5 000 CFU.Strains of EHEC O157 were isolated from the positive samples.The method made an initial estimation of the virulence and whether or not enterohemorrhagic Escherichia coli O157:H7 was in the samples.The multiplex PCR method for detection of Enterohemorrhagic Escherichia coli O157:H7 was estabilished successfully.It provided a rapid and simple detecting method for prevention and monitoring of enterohemorrhagic Escherichia coli O157:H7 with an excellent prospect of application.更多还原