多重PCR方法鉴定六类致泻性大肠埃希菌和志贺菌

目的 建立一种快速检测和鉴定六类致泻性大肠埃希菌和志贺菌的多重PCR方法。方法 根据六类致泻性大肠埃希菌和志贺菌的毒力基因eae、stx1、stx2、elt、estIa、estIb、aggR、ipaH、afaD及16S rRNA基因rrs建立多重PCR反应体系,优化引物浓度、PCR反应条件等,评价该方法的特异性和灵敏度,并用该方法鉴定本实验室保存的174株致泻性大肠埃希菌和24株志贺菌,将结果与单重PCR、本实验室已建立的鉴定五类致泻性大肠埃希菌和志贺菌的多重PCR方法结果进行对比。结果 10对PCR引物均可特异性扩增相应基因片段。该反应体系对174株致泻性大肠埃希菌和24株志贺菌的鉴定结果与单重PCR检测结果一致,本研究的多重PCR检测下限可达4.56 101 CFU/反应(1.14 104 CFU/ml)。结论 本研究采用毒力基因及内参照基因建立的多重PCR方法可在一个反应体系中鉴定和区分致泻性大肠埃希菌和志贺菌,为临床快速检测、疾病预防控制实验室筛查、腹泻的暴发溯源等提供了方便快捷的技术支持。

Multiplex polymerase chain reaction for identification of six diarrheagenic Escherichia coli and Shigella

Objective To develop a rapid and specific multiplex polymerase chain reaction (PCR) system for the identification of six diarrheagenic Escherichia coli and Shigella. Methods The multiplex PCR system was established based on 9 virulence genes eae, stx1, stx2, elt, estIa, estIb, aggR, ipaH, afaD and 16S rRNA gene rrs was used as control. The multiplex PCR system was optimized and evaluated by using 174 diarrheagenic E. coli strains, 24 Shigella strains and 44 other enteric pathogen strains. Results The multiplex PCR could amplify the related virulence genes, and the results were 100% consistent with those of the simplex PCR. The sensitivity of the multiplex PCR was 4.56 101 CFU/reaction or 1.14 104 CFU/ml for pure culture. Conclusion The established multiplex PCR system is specific for screening and detection of six diarrheagenic E. coli pathovars and Shigella.