多重实时荧光PCR检测三种致泻性大肠埃希菌和志贺菌方法的建立  

Development of a multiplex real-time fluorescence PCR assay for the identification of three diagrrheagenic Escherichia coli and Shigella spp.

作　　者：金玉娟[1] 陈应坚[1] 刘渠[1] 甘莉萍[1] 杨慧[1] 

机构地区：[1]广东省深圳市龙岗区疾病预防控制中心,广东深圳518172

出　　处：《中国卫生检验杂志》2013年第1期103-108,共6页Chinese Journal of Health Laboratory Technology

基　　金：2009年深圳市科技计划项目(医疗卫生类)(200903236)

摘　　要：目的:建立一种多重实时荧光PCR同时检测EPEC、EIEC、EHEC三种致泻性大肠埃希菌和志贺菌的方法。方法:于GenBank数据库中查找三种致泻性大肠埃希菌及志贺菌毒力基因stx1、stx2、eaeA及ipaH基因序列并使用软件进行比对后设计四套引物及探针,建立四重实时荧光PCR检测EPEC、EIEC、EHEC和志贺菌的方法,并对方法反应体系及反应条件进行优化,对方法的特异性、敏感性及重复性进行分析。结果:显示该方法具有检测灵敏度高、特异性强,结果与细菌培养结果完全一致,均为100%。结论:四重实时荧光PCR检测体系可同时检测EPEC、EIEC、EHEC和志贺菌,具有快速、特异性强、灵敏度高等特点,可广泛应用于临床检验、卫生检验、食品检验等领域,对快速有效的实施针对治疗,预防食物中毒,保障公众健康具有重大意义。Objective：To develop a multiplex real-time PCR assay for the simultaneous identification of three diagrrheagenic Escherichia coli and Shigella spp.Methods： The sequences of virulence genes stx1,stx2,eaeA and ipaH of three diagrrheagenic Escherichia coli and Shigella spp.were searched and blasted,the four sets of primers and probes were designed.A quadruple real-time fluorescence PCR assay was developed to detect EPEC,ETEC,EIEC and Shigella spp.The reaction system and conditions were modified.The specificity,sensitivity and repeatability were analyzed.Results： The results showed that this method possessed high sensitivity and specificity.The results were the same（100%） as those by strain cultures.Conclusion： This quadruple real-time fluorescence PCR assay can simultaneously,rapidly,sensitively and specifically identify EPEC,ETEC,EIEC and Shigella spp.It can be widely used in clinical,sanitary and food examinations.It is helpful for the rapid treatment,preventing food poisoning and protecting public health.

关 键 词：实时荧光PCR 致泻性大肠埃希菌 肠致病性大肠埃希菌 肠侵袭性大肠埃希菌 肠出血性大肠埃希菌 志贺菌 

分 类 号：R378.2[医药卫生—病原生物学]