无乳链球菌(Streptococcus agalactiae) 三重PCR 快速检测方法的建立与应用

参照GenBank发表的无乳链球菌sip、cpsE这两个高度保守基因设计两组特异性引物,应用已报道的无乳链球菌cpsL基因高度保守序列的引物作为阳性对照引物,经反应条件和反应体系参数优化,建立一种检测无乳链球菌的三重PCR方法,并应用本三重PCR方法检测40尾人工感染罗非鱼样本和22尾临床上收集的疑似无乳链球菌感染鱼样本。结果表明,所建立的三重PCR方法具有良好特异性,检测无乳链球菌DNA样本时出现三条扩增条带,检测其他7种供试菌株DNA则不出现出任何扩增条带,对无乳链球菌DNA样本的最低检测浓度为0.32ng/μl,最适Mg2+浓度为37.5mmol/L,整个检测过程耗时100min左右。40尾人工感染罗非鱼的肝脏、肾脏组织DNA样本阳性检出率分别为100%,22尾疑似无乳链球菌感染鱼的肝脏、肾脏组织DNA样本阳性检出率分别为72.7%,以上两批样本检测结果与常规细菌鉴定方法结果一致。

THE DEVELOPMENT AND APPLICATION OF A TRIPLE PCR METHOD FOR RAPID DETECTION OF STREPTOCOCCUS AGALACTIAE

A pair of specific primers were designed based on the two well conserved genes of sip and cpsE of Streptococcus agalactiae published on the GenBank,respectively.The primer of cpsL gene well conserved in S.agalactiae was chosen to be the positive control.After the optimization of the reaction conditions and reaction system parameters,the triple PCR method for S.agalactiae detection was developed.The method was evaluated by examining samples of forty tilapia with artificial infection and twenty two suspected fish collected clinically.Results showed that the triple PCR method was well specific.The detection result of S.agalactiae DNA showed three bands,and those of other seven strains showed no bands.The minimum detection concentration of the S.agalactiae DNA was 0.32ng/μl,the optimization value of Mg 2+ concentration was 37.5mmol/L,and it took about 100min to perform the examination.The positive rates of forty liver DNA samples and forty kidney DNA samples from the tilapia with artificial infection were 100%,respectively,and those of the twenty two liver DNA samples and twenty two kidney DNA samples from the suspected fish were 72.7%,respectively.Detection results of the two groups of samples were consistent with the results using general bacterial identification method.