肠出血性大肠埃希菌O157：H7多重PCR检测方法的建立  

Establishment of Multiplex PCR for Detecting Enterohemorrhagic Escherichia coli O157:H7

作　　者：纪雪[1] 张雪[1] 孙洋[1] 孙诗雯 祝令伟[1] 刘军[1] 姜海艳[2] 周伟[1] 梁冰[1] 郭学军[1] 刘彦晶[2] 

机构地区：[1]军事医学科学院军事兽医研究所,吉林省人兽共患病预防与控制重点实验室,吉林长春130122 [2]长春中医药大学附属医院,吉林长春130021

出　　处：《动物医学进展》2017年第7期1-6,共6页Progress In Veterinary Medicine

基　　金：吉林省自然科学基金项目(20150101110JC,20140101032JC);武汉市科技计划项目(2014060101010051)

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。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 er 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.

关 键 词：肠出血性大肠埃希菌O157:H7 多重PCR 检测 rfbE基因 

分 类 号：S852.612[农业科学—基础兽医学]