猪链球菌2型主要毒力因子三重荧光定量PCR检测方法的建立

ESTABLISHMENT OF TRIPLE REAL-TIME QPCR FOR DETECTION OF VIRULENCE FACTORS OF STREPTOCOCCUS SUIS SEROTYPES 2

根据GenBank中猪链球菌2型三种毒力因子CP2SJ、MRP、EF基因序列,利用Primer Express 3.0软件分别设计3对特异性的引物及相应的Taqman探针。CP2SJ、MRP、EF探针5′端分别标记FAM、HEX、CY5荧光发射基团,3′端标记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探针法的三重荧光定量PCR,可同时检测上述三种毒力基因。该方法灵敏度高,CP2SJ、MRP、EF的最低检测限分别为90、40、60拷贝数/μL的质粒;特异性强,与其他病原菌无交叉反应;重复性好,变异系数均小于3%。整个检测扩增在60 min内完成。以上结果表明本试验所建立的三重荧光定量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型三种毒力因子。

In this study, triple TaqMan real-time PCR assay was developed to simultaneously detect virulence genes CPS2J, MRP and EF of Streptococcus suis serotype 2. Three pairs of specific primers and fluorogenic-labeled probes were designed and synthesized in accordance with the above target genes using software Primer Express 3.0. The 5′-ends of probes for CPS2J, MRP and EF were individually labeled with FAM, HEX and CY5 and their 3′-ends were all labeled with quencher BHQ1. The reaction system and procedures were optimized. The detection limits for purified recombinant plasmids of CPS2J, MRP and EF were 90, 40 and 60 copies/μL, respectively. There was no cross reaction between Streptococcussuis serotype 2 and other pathogens. The variation coefficient of the established method was less than 3%. The entire detection could be completed within 60 min. In conclusion, the triple TaqMan Real-time PCR assay developed in the present study was fast, sensitive, repeatable and specific.