弥散黏附性大肠埃希菌多重PCR检测方法的建立及其在感染性腹泻患者中的流行情况  

Establishment of multiplex polymerase chain reaction assay for detection of diffuse adherent Escherichia coli in diarrhea patients

作　　者：阳波[1] 张网[1] 郭秋生 白向宁[1] 许彦梅[1] 熊衍文[1] 罗成旺[1] 

机构地区：[1]中国疾病预防控制中心传染病预防控制所传染病预防控制国家重点实验室,北京102206 [2]河南省睢县疾病预防控制中心,河南睢县476900

出　　处：《疾病监测》2017年第5期423-427,共5页Disease Surveillance

基　　金：国家自然科学基金(No.81371762)~~

摘　　要：目的建立一种快速检测弥散黏附性大肠埃希菌(DAEC)的普通多重PCR方法,了解DAEC在腹泻患者中的流行情况。方法根据DAEC黏附基因afa B、afa C、afa D、daa E及16S rRNA基因rrs,建立4重PCR反应体系,对PCR引物、反应体系和反应条件进行优化,评价敏感性和特异性,并应用于389份腹泻患者粪便标本的筛查。结果 4重PCR反应可以特异性扩增DAEC菌株afa B/C、afa D、daa E基因片段,除2株肠集聚性大肠埃希菌(EAEC)外,检测引物对其他致泻性大肠埃希菌及常见肠道病原菌均无特异性扩增,此多重PCR方法的检测下限可达3.20×101CFU/反应(8.01×103CFU/ml)。利用本研究建立的多重PCR方法对腹泻患者粪便标本进行检测,发现DAEC菌株分离率为6.2%(24/389)。结论本研究建立的多重PCR方法可用于DAEC菌株的快速鉴别,也可用于人粪便标本DAEC的初步筛查。Objective To establish a rapid and specific multiplex polymerase chain reaction assay for the detection of diffuse adherent Escherichia coli （DAEC） and understand the prevalence of DAEC infection in diarrhea patients in China. Methods The multiplex PCR assay was established based on four adhesion-associated genes, i. e. afaB, afaC, afaD, daaE and 16S rRNA gene was used as control. The assay was evaluated with other E. coli strains and enteric pathogens. A total of 389 stool samples from diarrhea patients were screened by the multiplex PCR assay. Results The multiplex PCR assay could amplify the related genes and the sensitivity was 3.20 × 10^1 CFU/reaction or 8.01 × 10^3 CFU/ml for pure culture. The detection rate of DAEC in diarrhea patients was 6. 2%. Conclusion The established multiplex PCR assay is specific suitable for screening and detection of DAEC.

关 键 词：致泻性大肠埃希菌 弥散黏附性大肠埃希菌 多重PCR 黏附基因 

分 类 号：R440[医药卫生—诊断学] R574.62[医药卫生—临床医学]