非洲猪瘟病毒野毒株与基因缺失株三重荧光定量PCR鉴别检测方法的建立及应用  

Development and application of a triplex real-time PCR method for detection and differentiation of gene-deleted and wide-type African swine fever virus

作　　者：郭振华[1] 邢广旭[1] 翁茂洋 金前跃[1] 乔松林[1] 张改平[1,2,3] GUO Zhenhua;XING Guangxu;WENG Maoyang;JIN Qianyue;QIAO Songlin;ZHANG Gaiping(Henan Provincial Key Laboratory of Animal Immunology,Henan Academy of Agricultural Sciences,Zhengzhou 450002,China;College of Veterinary Medicine,Henan Agricultural University,Zhengzhou 450002,China;Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses,Yangzhou,Jiangsu 225009,China)

机构地区：[1]河南省农业科学院动物免疫学重点实验室,河南郑州450002 [2]河南农业大学动物医学学院,河南郑州450002 [3]江苏高校动物重要疫病与人兽共患病防控协同创新中心,江苏扬州225009

出　　处：《中国兽医学报》2022年第4期619-625,共7页Chinese Journal of Veterinary Science

基　　金：河南省重点研发与推广专项基金资助项目(202102110248);河南省农业科学院优秀青年科技基金资助项目(2020YQ15);河南省生猪产业技术体系资助项目(S2012-06)。

摘　　要：针对非洲猪瘟病毒B646L、MGF505-2R和CD2v基因分别设计了引物和探针,经过条件优化,建立了基于TaqMan探针技术的三重荧光定量PCR检测方法。结果显示,本研究建立的三重荧光定量PCR检测方法与猪场常见病毒的核酸不发生交叉反应,具有良好的特异性;敏感性分析显示,针对B646L、MGF505-2R和CD2v基因的最低检测下限分别为6.5,8.0和14.0 copies/μL;并且该方法在10~10copies/μL模板范围内具有良好的线性关系,组间变异系数为0.05%~2.68%,组内变异系数为0.10%~1.17%,稳定性良好。进一步针对临床核酸样品的检测显示,本方法和OIE推荐的检测方法具有良好的一致性。本研究成功建立了非洲猪瘟病毒野毒株和基因缺失株的荧光定量PCR鉴别检测方法,为临床非洲猪瘟病毒的监测诊断提供了良好的技术支撑。Primers and probes based on the B646 L,MGF505-2R and CD2v genes of ASFV were designed.Then,a triplex real-time PCR method was established by optimizing the conditions.The triplex real-time PCR can specifically detect B646 L,MGF505-2R and CD2 v genes or simultaneously detect all three genes without cross-reaction with other porcine viruses tested.The limit of detection was 6.5,8.0 and 14.0 copies/μL for the standard plasmids containing gene of B646 L,MGF505-2R and CD2 v,respectively.The reproducibility of intra-and inter-assay displayed that the coefficient of variations was 0.05%-2.68% and 0.10%-1.17%,respectively.Clinical nucleic acid samples were tested in parallel by the triplex real-time PCR and detection method recommended by OIE.The detection results of these two assays against B646 L gene were well consistent.In conclusion,a triplex TaqMan real-time PCR method was successfully established,which can effectively distinguish the wide-type and MGF505 or CD2v gene-deleted ASFVs.It would be a useful tool for the clinical diagnosis and control of ASF.

关 键 词：非洲猪瘟病毒 荧光定量PCR B646L基因 MGF505-2R基因 CD2v基因 

分 类 号：S852.65[农业科学—基础兽医学]