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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。
In order to establish a multiplex PCR assay for detection of enterohemorrhagic Escherichia coli O157: H7, five pairs of primers were designed according to the eaeA gene, fliC gene, shigotoxin gene (stx1 and stx2) and rfbE gene of O157. The specificity and sensitivity of the system were optimized by primer concentration and temperature. The samples of different animal sources including cattle, pigs, chickens and dogs were tested for E. coli O157. The results show that the method has good specificity and sensitivity, the sensitivity of 5 × 10 ~ 3CFU. The target bacteria were isolated from the positive samples tested. Application of the established method to determine whether the sample contains Escherichia coli O157 at the same time, but also preliminary determination of virulence strains. The successful establishment of a multiplex PCR method for detection of enterohemorrhagic Escherichia coli O157: H7 provided a simple and rapid technical means for the prevention and monitoring of the infection of the pathogenic bacteria and has good application prospects.