CAJ | 학술논문

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。
근거GenBank중저련구균2형삼충독력인자CP2SJ、MRP、EF기인서렬，이용Primer Express 3.0연건분별설계3대특이성적인물급상응적Taqman탐침。CP2SJ、MRP、EF탐침5′단분별표기FAM、HEX、CY5형광발사기단，3′단표기BHQ1쉬멸형광기단。통과우화반응체계화정서，건립료일충기우Taqman탐침법적삼중형광정량PCR，가동시검측상술삼충독력기인。해방법령민도고，CP2SJ、MRP、EF적최저검측한분별위90、40、60고패수/μL적질립；특이성강，여기타병원균무교차반응；중복성호，변이계수균소우3%。정개검측확증재60 min내완성。이상결과표명본시험소건립적삼중형광정량PCR방법적민감성、중복성급특이성균교호，가용우동시쾌속검측저련구균2형삼충독력인자。
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.