LAMP技术应用于志贺菌及侵袭性大肠埃希菌ipaH基因快速检测  

Rapidly detecting ipaH gene of Shigella and EIEC through improved Loop-mediated isothermal amplification technology

作　　者：朱水荣[1] 徐宝祥[1] 余昭[1] 章海文[2] 张政[1] 梅玲玲[1] 

机构地区：[1]浙江省疾病预防控制中心,杭州310051 [2]浙江省乐清市疾病预防控制中心,浙江乐清325600

出　　处：《中国卫生检验杂志》2010年第8期1843-1846,共4页Chinese Journal of Health Laboratory Technology

摘　　要：目的:建立一种适合基层实验室快速检测志贺菌及侵袭性大肠埃希菌(EIEC)的实验方法。方法:根据志贺菌及EIEC共同含有的侵袭性质粒抗原ipaH基因序列设计4条引物,应用环介导等温扩增(Loop-mediated isothermal am-plification)方法,分别对8株背景明确的不同血清型志贺菌标准株、34株志贺菌地方分离株、1株EIEC实验标准株、7株大肠埃希菌本室保存标准株、3株大肠埃希菌暴发株、10株其他肠道实验对照株进行检测,验证该方法的特异性及最低检测限,通过肉眼目测与电泳检测比较结果。结果:本方法中共计42株志贺菌、1株EIEC及1株44147大肠埃希菌,经用LAMP方法检测,均为绿色并电泳有相应阶梯状条带为阳性结果,而9株其他大肠埃希菌及10株其他肠道实验对照株均呈橙色并电泳无相应阶梯状条带为阴性结果。至于1株44147大肠埃希菌为何也检测出ipaH基因,我们怀疑该菌株要么是EIEC中的一种,要么是在长期进化过程中从别处获得该基因,这尚待今后做进一步探讨。本方法具有较好的特异性,其最低检测限为53 cfu/反应,自然光下通过肉眼即可判断结果,从菌株核酸的提取至检测完成仅需1.5 h左右。应用本方法对24份不同临床病人样本进行实际考核,3份痰液、1份胸积水、2份粪便及18份尿样经检测ipaH基因均为阴性,该结果与分离培养/普通PCR检测结果相符。结论:本次建立的LAMP方法,能快速、特异地检测志贺菌/EIEC,较适合基层实验室应用。Objective：To set up a experimental method to detect ipaH gene of Shigella and Enteroinvasive Escherichia coli（EIEC） rapidly,which is suitable for the laboratories at basic level.Methods：Four primers（two inside and,two outside） designed according to the enteroinvasive plasmid antigen of both Shigella and EIEC,LAMP improved method used,to detect 8 Shigella standard strains,which the serotypes were different and the backgrounds were clear,34 Shigella strains,which were separated in different areas,1EIEC experimental standard strain,7 Escherichia coli standard strain stored in our laboratory,3 Escherichia coli strain which were collected in outbreak,10 other controlled strains of intestinal bacteria.To validate the specificity of the method and the minimum detection limit,observed by naked eye and electrophoresis.Results：42 Shigella,strains,1 EIEC coli strain and 1 Escherichia coli strain（44147） revealed green color with scalariform stripe in electrophoresis,which could be confirmed as positive result.While 9 other Escherichia coli strains and 10 other controlled strains of intestinal bacteria revealed orange color without scalariform stripe in electrophoresis,which could be confirmed as negative result.It was doubtful that 1 Escherichia coli strain（44147） was detected carrying ipaH gene.We concluded that the strain was one species of EIEC,or the one obtained the gene during the anagenesis course.This should be discussed in future.The LAMP method showed good specificity.The minimum detection limit was 53 cfu/reaction.The result could be judged by the naked eyes in white light.It took about 1.5 hours from the nucleic acid distillation to the end.The method was used to test 24 specimens of different patients which included 3 pieces of phlegm,1 piece of hydrothorax,2 pieces of stool and 18 pieces of urine.The results were all negative which were as same as the ones detected by isolated culture or PCR method.Conclusion：Shigella and EIEC could be detected rapidly and specifically through the LAMP m

关 键 词：志贺菌 侵袭性大肠埃希菌 LAMP IPAH基因 鉴定 

分 类 号：R115[医药卫生—公共卫生与预防医学]