鉴别非洲猪瘟病毒基因缺失株与野生株多重qPCR方法的建立  

Establishment of multiplex qPCR method for identification of gene deletion strains and wild strains of African swine fever virus

作　　者：卓玛 钱炳旭 薛峰[1,2] 戴建君[1] 吴晓东[3] Zhuoma;QIAN Bing-xu;XUE Feng;DAI Jian-jun;WU Xiao-dong(College of Veterinary Medicine,Nanjing Agricultural University,Nanjing 210000,China;Sanya Institute of Nanjing Agricultural University,Sanya 572000,China;China Animal Health and Epidemiology Center,Qingdao 266000,China)

机构地区：[1]南京农业大学动物医学院,江苏南京210000 [2]南京农业大学三亚研究所,海南三亚572000 [3]中国动物卫生与流行病学中心,山东青岛266000

出　　处：《中国兽医科学》2023年第9期1089-1094,共6页Chinese Veterinary Science

基　　金：国家重点研发计划项目(2021YFD1800500,2020YFA0910200);海南省科技专项(ZDYF2022XDNY248);江苏省农业科技自主创新基金项目[CX(21)2038];三亚市南京农业大学研究院指导基金项目(NAUSY-ZD08)。

摘　　要：为开发一种快速、灵敏的非洲猪瘟病毒(ASFV)野生型株与基因缺失型株的鉴别诊断方法,本研究通过对ASFV p72、CD2v、MGF110-9L基因保守序列设计特异性引物和探针,并对体系进行优化,建立了一种可以同时检测p72、CD2v、MGF110-9L基因的多重q PCR方法。该方法与其他常见猪病毒性病原均无交叉反应,特异性强;敏感性试验结果显示,该方法对p72、CD2v和MGF110-9L重组质粒标准品的最低检出限均为102copies/mL,敏感性好;组内变异系数为0.03%~0.5%,组间变异系数为0.04%~1.5%,表明该方法重复性好。利用本方法和普通qPCR方法分别对50份临床样品进行检测,结果显示,两种检测方法对ASFV核酸的检出率均为70%(35/50),符合率为100%。综上所述,本研究建立的多重qPCR方法为临床鉴别检测ASFV野毒株与基因缺失株提供了重要材料。To develop a convenient,rapid and sensitive identification technology between ASFV gene deletion strains and wild-type virus strains,three pairs of specific primers and probes were designed for ASFV p72(B646L)gene,CD2v(Ep402R)gene,and MGF110-9L gene.By optimizing each system,a multiplex qPCR system for simultaneousidentification of p72,CD2v,and MGF110-9L genes was constructed.This method has no cross-reactivity with other swine pathogens,Strong specificity;High reproducibility,the sensitivity of the three target genes of ASFV(p72,MGF110-9L,CD2v)is 102copies/mL;Highly repeatable,the intra-group variationcoefficientwas0.03%—0.5%,and the intergroup variation coefficient was 0.04%—1.5%;After verifying 50 clinical samples using the method established in this study and the common qPCR method,the results showed that the detection rate of ASFV by the two methods was 70%(35/50),and the consistency rate of 50 positive samples was 100%.In summary,the method established in this study provides important materials for the clinical identification of ASFV gene deletion strains and wild-type strains.

关 键 词：非洲猪瘟病毒 基因缺失型株 p72 MGF110-9L CD2v 

分 类 号：S852.659.1[农业科学—基础兽医学]