荧光定量PCR检测猪链球菌2型主要毒力因子方法的建立

Establishment of the Real-time q PCR method for Detection of Main Virulence Factors of Streptococcus suis Serotype 2

[目的]建立荧光定量PCR检测猪链球菌2型(Streptococcus suis serotype 2,SS2)、溶菌酶释放蛋白(Muramidase-released protein,MRP)和胞外因子(Extracellular protein factor,EF)3种主要毒力因子的方法。[方法]根据cps2j、mrp和ef基因的基因序列,分别设计并合成3对引物及相应的Taqman探针,其中cps2j和mrp的5'端标记FAM荧光发射基团,ef的5'端标记HEX荧光发射基团,3种基因的3'端都标记BHQ1淬灭荧光基团。通过优化反应体系和程序,建立了一种基于Taqman探针法的荧光定量PCR方法检测上述3种主要毒力因子,其中cps2j单独检测,mrp与ef的实行双重荧光PCR方法检测。[结果]cps2j、mrp和ef的最低检测限分别为12、51和51 CFU,灵敏度很高;与其他病原菌无交叉反应,重复性及特异性均较好;此外,整个检测过程在60 min内即可完成。[结论]该试验所建立的双重荧光定量PCR方法的敏感性、重复性及特异性均较好,可用于同时快速检测猪链球菌2型3种主要毒力因子。

[ Objective ] Taq Man real-time qPCR was established for detection of Streptococcus suis serotype 2（ SS2 ）, muramidase-released protein （MRP） and extracellular protein factor（EF）. [ Method] The double Taq Man real-time PCR assay was developed to simultaneously detect virulence genes cps2j,mrp and efof S. suis serotype 2. Three pairs of specific primers and fluorogenic-labeled probes were designed and synthesized in accordance with the above target genes. The 5＇-ends of probes for cps2j and mrp were labeled with FAM,while the 5＇-ends of ef were individually labeled with HEX, and their 3 ＇-ends were all labeled with quencher BHQ1. The reaction system and procedures were optimized. [ Result] The detection limits for purified recombinant plasmids of cps2j, mrp and ef were 12,51 and 51 C FU,respectively. There was no cross reaction between S. suis serotype 2 and other pathogens. The entire detection could be completed within 60 min. [ Conclusion] The double Taq Man real-time PCR assay developed in this study is fast, sensitive, repeatable and specific,which can be used for rapid detection of three kinds of virulence factors of S. suis serotype 2.