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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 multiplex PCR method for the rapid detection of diffuse adherent Escherichia coli (DAEC) to understand the prevalence of DAEC in patients with diarrhea. Methods According to DAEC adhesion gene afa B, afa C, afa D, daa E and 16S rRNA gene rrs, a 4-fold PCR reaction system was established. The PCR primers, reaction system and reaction conditions were optimized to evaluate the sensitivity and specificity. Screening of faecal specimens from 389 diarrhea patients. Results Four-step PCR reaction could amplify the afa B / C, afa D and daa E gene segments of DAEC strains. Except for two Escherichia coli intestine (EAEC) strains, other diarrhea-causing Escherichia coli There was no specific amplification of bacteria and common intestinal pathogens. The detection limit of multiplex PCR was up to 3.20 × 101CFU / reaction (8.01 × 103CFU / ml). Using the multiplex PCR method established in this study, diarrhea patients stool specimens were detected and found that the isolation rate of DAEC strains was 6.2% (24/389). Conclusion The multiplex PCR method established in this study can be used for rapid identification of DAEC strains and also for primary screening of human stool specimens DAEC.