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

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afaB、afaC、afaD、daaE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afaB/C、afaD、daaE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 x 101 CFU/reaction or 8.01 x 103 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.