鉴别非洲猪瘟病毒I177L基因缺失株的双重荧光PCR方法的建立

Establishment of a duplex real-time PCR method for differentiation of African swine fever virus I177L gene-deleted strains

非洲猪瘟病毒I177L基因缺失疫苗是当前非洲猪瘟(African swine fever,ASF)减毒活疫苗研发的重点方向之一。为有效鉴别非洲猪瘟病毒(African swine fever virus,ASFV)野毒株和I177L基因缺失株,本试验根据ASFV B646L和I177L基因分别设计特异性引物和探针,经过筛选和优化,建立了双重荧光PCR检测方法。结果显示,该方法特异性强,可同时检测ASFV B646L和I177L基因,与猪圆环病毒2型、塞内卡病毒、猪瘟病毒、口蹄疫病毒、猪繁殖与呼吸综合征病毒等无交叉反应;灵敏度高,该法对重组质粒pUC57-B646L和pUC-I177L的检出限均为1×10^(3)拷贝/mL;重复性好,批内和批间变异系数(coefficient of variation,Cv)均小于4%。应用该双重荧光PCR方法和WOAH推荐的荧光PCR法对122份猪肉及猪肉制品进行比对检测,2种方法对B646L基因检测结果符合率为100%,且所建立方法阳性结果均出现2条扩增曲线。本研究建立的方法可用于ASFV I177L基因缺失株检测,为ASF监测和流行病学调查提供了技术支持。

African swine fever virus(ASFV)I177L gene deletion vaccine is one of the key directions of African swine fever(ASF)live attenuated vaccine research and development.In order to effectively distinguish between the wild-type ASFV strain and the I177L gene-deleted strain,specific primers and probes were designed based on ASFV B646L and I177L genes,respectively.After screening and optimization,a duplex real-time PCR method was developed that can simultaneously detect these two genes.The results showed that ASFV B646L and I177L genes were detected specifically and simultaneously by the method developed without cross-reactions with porcine circovirus type 2,Seneca virus A,classical swine fever virus,foot-and-mouth disease virus,porcine respiratory and reproductive syndrome virus.The detection limits of the duplex real-time PCR for recombinant plasmids pUC57-B646L and pUC57-I177L were 1×10^(3) copies/mL.The intra-and inter-assay coefficients of variation were less than 4%,respectively.Detection of 122 pork and pork products using the duplex real-time PCR developed and the real-time PCR recommended by WO AH showed that the coincidence rates of the two methods for B646L gene detection was 100%with two amplification curves appeared in the positive results of the established methods.The method established in this study can be used for the detection of ASFV I177L gene deletion strains,which provides technical support for ASF surveillance and epidemiological investigation.