多重PCR快速鉴定鼠疫耶尔森氏菌

Rapid identification of Yersinia pestis by multiplex-PCR

目的 建立鼠疫耶尔森氏菌多重PCR鉴定系统 ,用于鼠疫耶尔森氏菌的快速鉴定。方法 针对分别存在于pFra、pRst质粒上毒力基因caf1和pla ,以及一段 2 76bp染色体序列 3a分别设计引物。采用多重PCR技术 ,同时检测caf1、pla、3a三个靶序列。结果 应用该多重PCR反应体系 ,对我国鼠疫耶尔森氏菌 17个生态型及新发现的青海田鼠鼠疫疫源地菌株的多重PCR扩增结果表明 ,实验菌株中除 2株分别缺失pFra、pPst质粒而扩增出 2条产物带外 ,其余 5 2株均扩增出预期的 3条产物带 ,相关菌株均阴性 ,其检测灵敏度为 1× 10 -4ngDNA。 结论 该方法用于鼠疫耶尔森氏菌的鉴定简便、快捷 ,具有很高的特异性和敏感性 。

Aim To develop a multiplex PCR method for rapid identification of Y.pestis strains.Methods This procedure was based upon the use of a single polymerase chain reaction with multipe pairs of primers directed to three Y.pestis specific sequences.pEra plasmid located gene(caf1) encoding Y.pestis specific capsular antigen fraction.pPst plasmid located gene(pla) encoding plasminogen activator.3a primer pair designed by using highly Y.pestis specific region on chrosmosomal DNA.Results The selected primers amplified a 171 bp fragment from caf1 gene,a 276 bp 3a gene and a 480 bp pla gene.No PCR product was generated for closely related becteria,inclusing Y.pseudotuberculosis,Y.enterocolitica and others in Enterobacteriaceae.Conculsion This multiplex PCR system is specific for rapid identification of Y.pestis among pathogenic Yersinia species and other Enterbacteriaceae having antigens common to Y.pestis. Since this method is simple and accurate,it will be useful to identify Y.pestis in case of emergency and for the surveillance of epidemics.