摘要
目的建立一种快速、特异的检测肠出血性大肠埃希菌O157:H7菌株的多重PCR方法。方法针对O157:H7菌株的O157、H7抗原特异基因rfbEO157、fliCH7以及stx1、stx2、eaeA和hlyA四种毒力基因设计相应引物,在同一扩增体系中进行PCR反应,通过优化多重PCR反应条件和循环参数,建立检测O157:H7菌株的多重PCR方法,并测定其特异性和灵敏度。结果 6对特异性引物各自扩增相应的基因片段,检测结果与常规PCR获得的结果一致。细菌纯培养物的检测灵敏度为1.33×104CFU。结论该多重PCR方法能在一次检测中同时反映待测菌株是否为肠出血性大肠埃希菌O157:H7及其携带毒力基因的情况,可为O157:H7大肠埃希菌感染的诊断及流行病学调查提供一种简便、快速的检测手段。
Objective To develop a multiplex PCR assay for detecting enterohemorrhagic E.coli O157:H7.Methods Six pairs of primers were used to amplify the antigenic specific rfbEO157 and fliCH7 genes and virulence genes of stx1,stx2,eaeA and hlyA of E.coli O157:H7 in a single tube.The reaction conditions and thermal cycles were optimized.The specificity and sensitivity of the assay were evaluated.Results The multiplex PCR only amplified the corresponding regions of DNA of E.coli O157:H7.The resultant PCR products were concordant with those obtained by the conventional PCR.The sensitivity of the multiplex PCR was 1.33×104 CFU when the pure culture was used as the DNA templates.Conclusion The multiplex PCR may define the presence of E.coli O157:H7 and reveal the virulence genes simultaneously by one detection.The assay is simple and rapid,which may be used in the diagnosis of O157:H7.
出处
《临床检验杂志》
CAS
CSCD
北大核心
2010年第4期264-266,共3页
Chinese Journal of Clinical Laboratory Science
基金
国家科技部"973"项目(2005CB522904)