大肠埃希菌O157:H7环介导等温扩增可视化检测方法的建立  

Visual detection of enterohemorrhagic Escherichia coli O157:H7 by loop-mediated isothermal amplification with hydroxynaphthol blue dye

作　　者：吕恒[1] 张锦海[1] 陈凤娟[1] 谭维国[1] 顾海涛[1] 王平[1] 王长军[1] 

机构地区：[1]南京军区疾病预防控制中心,江苏南京210002

出　　处：《中国病原生物学杂志》2013年第4期289-292,309,共5页Journal of Pathogen Biology

基　　金：国家重大传染病专项课题(No.2013ZX10004-103;No.2013ZX10004-218);江苏省科级支撑计划(社会发展)项目(No.BE2012609);全军后勤科研"十二五"重大项目(No.AWS11C001;No.AWS11C009);南京军区重点课题(No.10Z038;11Z040)

摘　　要：目的建立肠出血性大肠埃希菌O157︰H7环介导等温扩增(LAMP)可视化快速检测方法。方法针对肠出血性大肠埃希菌(EHEC)O157︰H7编码脂多糖rfbE基因保守区设计LAMP引物及环引物,反应体系加入染料羟基萘酚蓝(HNB)作为LAMP扩增的指示剂,结合LAMP浊度仪优化扩增条件,根据HNB的颜色变化进行结果判定,评价LAMP方法的特异性和灵敏性。结果建立的LAMP法对O157︰H7rfbE基因的最低检出限约为100copy/反应管,检测肠产毒性大肠埃希菌(ETEC)、肠致病性大肠埃希菌(EPEC)、肠侵袭性大肠埃希菌(EIEC)、肠聚集性大肠埃希菌(EAEC)、宋内志贺菌、福氏志贺菌均为阴性。体系的扩增效率较高,可在40min内出结果。结论建立的基于颜色判定的EHEC O157︰H7LAMP检测方法具有特异、灵敏,设备要求简单等特点,适用于EHEC O157︰H7的快速检测。Objective A loop-mediated isothermal amplification （LAMP） assay was developed for rapid visual detection of enterohemorrhagic Escherichia coli O157 ： HT. Methods A set of specific primers specific to conserved regions of rfbE gene was designed. The reaction was performed in a single tube with the addition of hydroxynaphthol blue （HNB） dye prior to amplification. A loopamp reattime turbidimeter was used to ascertain amplification conditions. The result was determined by a color change in HNB, and the sensitivity and specificity of LAMP were evaluated. Results Results showed that the detection limit of the LAMP assay was 100 copies per tube. Test results for ETEC, EPEC, EIEC, EAEC, S. sonnei, and S. flexneri were all negative. The LAMP reaction was completed within 40 min. Conclusion The LAMP assay with HNB dye has a high level of specificity and sensitivity and requires only simple laboratory equip- ment. It is suitable for the rapid detection of Enterohemorrhagic E. coli O157 ： H7.

关 键 词：肠出血性大肠埃希菌(EHEC)O157 H7 可视化检测 环介导等温扩增(LAMP) 羟基萘酚蓝(HNB) 

分 类 号：R378.21[医药卫生—病原生物学]