塞内卡病毒A和脑心肌炎病毒双重TaqMan荧光定量PCR检测方法的建立与应用

Establishment and application of dual TaqMan fluorescent quantitative PCR assay for Senecavirus A and encephalomyocarditis virus

为建立同时快速鉴别检测塞内卡病毒A(SVA)和脑心肌炎病毒(EMCV)的方法,根据SVA和EMCV高度保守的3D基因区域,分别设计了2对特异性引物和带2种不同发光基团标记的TaqMan探针,建立同时检测这2种病毒核酸的双重TaqMan荧光定量PCR方法,并对反应条件进行优化。结果显示,该检测方法对SVA和EMCV的最低检测量分别为760 copies/μL和98 copies/μL,并且能够特异性检测出SVA和EMCV,而对猪繁殖和呼吸障碍综合征病毒(PRRSV)、猪流行性腹泻病毒(PEDV)、猪瘟病毒(CSFV)等检测结果均为阴性。本方法中所建立的标准曲线呈现良好的线性关系,重复性试验组内和组间变异系数均低于5%,说明该检测方法重复性高。本研究所建立的双重荧光定量PCR具有方便、快速、特异性好、灵敏度高、重复性好等优点,能够用于SVA和EMCV的同时检测。

In order to establish a method for rapid differential identification of Senecavirus A(SVA)and encephalomyocarditis virus(EMCV),two pairs of corresponding specific primers were designed based on the highly conserved 3D genes of SVA and EMCV.And two different fluorescent labeled TaqMan probes were used to establish a dual TaqMan real-time PCR method for simultaneous detection of these two viruses,and we also optimize the reaction conditions.The results showed that the minimum detection of the method was 760 copies/μL and 98 copies/μL for SVA and EMCV,respectively,and it can specifically detect SVA and EMCV,and there was no cross reaction with CSFV,PRRSV and PEDV.The established standard curves showed good linear relationship.Repeated experimental group and inter-group coefficient of variation were less than 5%.The results indicated that the dual-quantitative PCR established in this study has the advantages of convenience,rapidity,good specificity,high sensitivity and good repeatability,and can be used for simultaneous detection of SVA and EMCV.