鉴别检测非洲猪瘟野毒株和基因缺失疫苗株的三重荧光PCR的建立

Establishment of A Triplex Real ⁃ time PCR Assay for Detection andDifferentiation of Gene ⁃ deleted Vaccine Strains from Wild ⁃ type African SwineFever Virus Strains

非洲猪瘟是由非洲猪瘟病毒(African swine fever,ASFV)引起的猪的一种急性、高度接触性传染病。本研究根据ASFV B646L、MGF360⁃14L和CD2v基因设计了3套引物和探针,建立了可同时鉴别检测ASFV野毒株和基因缺失疫苗株的三重荧光PCR方法。结果表明,该法特异性强,可同时检测ASFV B646L、MGF360⁃14L和CD2v基因,与猪圆环病毒2型、猪瘟病毒、猪繁殖与呼吸综合征病毒、口蹄疫病毒、猪A型塞内卡病毒等无交叉反应;两份ASFV MGF360⁃505R与CD2v基因联合缺失疫苗株DNA三重荧光PCR法检测结果都仅出现一条B646L扩增曲线,与预期一致;灵敏度高,该法对重组质粒pUC⁃B646L、pUC⁃360⁃14L和pUC⁃CD2v的检出限分别为1.572×10^(3)拷贝/mL、1.934×10^(3)拷贝/mL和1.929×10^(3)拷贝/mL,灵敏度比世界动物卫生组织(World organization for animal health,OIE)推荐的荧光PCR高10倍;重复性好,对三种重组质粒检测的Ct值批内和批间变异系数均小于2%;应用该法及OIE推荐的荧光PCR对1215份样品进行平行检测,结果显示23份样品为阳性,1192份样品为阴性,两种方法检测结果一致。本研究建立的方法能鉴别检测ASFV野毒株和基因缺失疫苗株(缺失MGF360⁃505R和/或CD2v基因),对将来基因缺失疫苗的推广应用及ASF疫情防控具有重要的意义。

African swine fever(African swine fever,ASF)is an acute and highly contagious disease in swine,caused by ASF virus(ASFV).In this study,a triplex real⁃time PCR assay was developed to detect and differentiate the gene⁃deleted vaccine strains and wild⁃type ASFV strains.The results showed that ASFV B646L,MGF360⁃505R and CD2v genes were detected specifically and simultaneously by the assay developed without cross⁃reactions with porcine circovirus type 2,classical swine fever virus,porcine respiratory and reproductive syndrome virus,foot⁃and⁃mouth disease virus or senecavirus A.The detection results of two DNA samples of recombinant ASFV with deletion of MGF360⁃505R and CD2v genes by the triplex real⁃time PCR assay showed only one fluorescence amplification curves,which was consistent with expectations.The detection limits of the triplex real⁃time PCR were 1.572×10^(3),1.934×10^(3),and 1.929×10^(3) copies/mL for recombinant plasmids pUC⁃B646L,pUC⁃360⁃14L and pUC⁃CD2v,respectively.The sensitivity was 10 times higher than real⁃time PCR recommended by OIE.The intra⁃and inter⁃assay coefficients of variation of Ct values were less than 2%,respectively.A total of 1,215 field samples were tested in parallel by the triplex real⁃time PCR and the real⁃time PCR recommended by World organization for animal health(OIE),and results showed that 23 samples were positive and 1192 samples were negative.The results of the two methods were consistent.The triplex real⁃time PCR assay was successfully developed to identify pigs infected with wild⁃type ASFV strains or immunized with gene⁃deleted vaccine.This is important for future promotion and application of gene⁃deleted vaccines and prevention and control of ASF epidemic.