羊口疮病毒LAMP-LFD快速检测方法的建立与初步应用  

Rapid and sensitive diagnosis of Orf virus by loop-mediated isothermal amplification combined with a lateral flow dipstick

作　　者：杨倩 李召仁 鲜思美[1,2] 包涛涛 张友 梁倩 顾庆林 YANG Qian;LI Zhaoren;XIAN Simei;BAO Taotao;ZHANG You;LIANG Qian;GU Qinglin(College of Animal Science,Guizhou University,Guiyang550025,China;Institute of AnimalDisease Research of Guizhou,Guiyang 550025,China;Agriculture and Rural Bureau of Southeast Guizhou,Qiandongnan,Guizhou 556000,China)

机构地区：[1]贵州大学动物科学学院,贵州贵阳550025 [2]贵州省动物疫病研究所,贵州贵阳550025 [3]贵州省黔东南州农业农村局,贵州黔东南556000

出　　处：《中国兽医学报》2022年第3期507-513,共7页Chinese Journal of Veterinary Science

基　　金：贵州省大学生创新创业训练资助项目(S202010657099);贵州省科技计划资助项目(黔科合支撑[2018]2264);贵州省科学技术基金资助项目(黔科合基础[2019]1113号)。

摘　　要：根据羊口疮病毒(OrfV)的B2L基因设计4条环介导等温扩增(LAMP)特异性引物和1条异硫氰酸荧光素标记的DNA探针,再利用横向流动试纸条(LFD)检测扩增产物。通过对各反应条件包括反应温度、反应时间、内外引物浓度比、Bst DNA 2.0聚合酶浓度、dNTP Mix浓度、Mg^(2+)浓度以及探针结合温度等优化后,建立了检测OrfV的LAMP-LFD方法。结果显示,LAMP-LFD可特异性检测OrfV,而山羊痘病毒、口蹄疫病毒、小反刍兽疫病毒及丝状支原体山羊亚种等均未检出;LAMP-LFD对OrfV-B2L质粒DNA最低检测浓度为4.95×10^(-2)拷贝/μL,其灵敏度是以外引物建立PCR方法的1000倍。利用PCR和LAMP-LFD检测29份临床样品,两者检出符合率为100%。研究建立的LAMP-LFD检测方法可快速、特异地检测OrfV,可作为早期诊断和检测OrfV的有效手段。This study aims to establish an efficient and rapid detection method for Orf virus(OrfV).Four loop-mediated isothermal amplification(LAMP)specific primers and one fluorescein isothiocyanate-labeled DNA probe were designed based on the B2 L gene of OrfV,and then lateral flow dipstick(LFD)was used to detect amplification products.After optimizing the reaction conditions including reaction temperature,reaction time,internal and external primer concentration ratio,Bst DNA 2.0 polymerase concentration,dNTP Mix concentration,Mg^(2+)concentration and probe binding temperature,a LAMP-LFD method for detecting OrfV was established.The LAMP-LFD method could specifically detect OrfV,but not goatpox virus,foot-and-mouth disease virus,peste des petits ruminants virus and Mycoplasma mycoides subsp.capri.The lowest detection concentration of the LAMP-LFD for plasmid containing OrfV B2 L was 4.95×10^(-2)copies/μL.The sensitivity of this method was 1000 times higher than that of PCR with external primers.PCR and LAMP-LFD were used to detect 29 clinical samples,and the coincidence rate was 100%.The LAMP-LFD detection method established in this study can detect OrfV quickly and specifically,and can be used as an effective method for early diagnosis and detection of OrfV.

关 键 词：羊口疮病毒 B2L基因 环介导等温扩增技术 横向流动试纸条 

分 类 号：S852.65[农业科学—基础兽医学]