BTV和PPRV双重荧光定量RT-PCR检测方法的建立  

Establishment of duplex TaqMan real-time RT-PCR for detection of BTV and PPRV

作　　者：勾倩倩 杜吉革 李启红[2] 李倩琳 赵洋 钱莺娟[1] 印春生[2] 郑龙三 GOU Qian-qian;DU Ji-ge;LI Qi-hong;LI Qian-lin;ZHAO Yang;QIAN Ying-juan;YIN Chun-sheng;ZHENG Long-san(MOE Joint International Research Laboratory of Animal Health and Food Safety/Key Laboratory of Animal Bacteriology,Ministry of Agriculture/College of Veterinary Medicine,Nanjing Agricultural University,Nanjing 210095,China;China Institute of Veterinary Drug Control,Beijing 100081,China)

机构地区：[1]南京农业大学动物医学院教育部动物健康与食品安全国际合作联合实验室农业部动物细菌学重点实验室,江苏南京210095 [2]中国兽医药品监察所,北京100081

出　　处：《中国兽医科学》2022年第3期285-291,共7页Chinese Veterinary Science

基　　金：国家“十三五”重点研发计划资助项目(2017YFD0502306)。

摘　　要：为建立一种能够同时检测蓝舌病病毒(BTV)和小反刍兽疫病毒(PPRV)的快速检测方法,本研究选取蓝舌病病毒的NS3基因和小反刍兽疫病毒的N基因作为靶基因,参照GenBank上公布的两种基因序列,通过序列比对后选取一段保守性较高的区域,利用Beacon designer 8设计引物和探针并优化反应体系及反应条件,建立了双重荧光定量RT-PCR检测方法。结果显示,该方法检测BTV和PPRV阳性质粒标准品的最低检出量分别为1.7 copies/μL和1.2 copeis/μL;与山羊痘病毒、羊口疮病毒、口蹄疫病毒、羊腐败性梭菌、布氏杆菌及弓形虫无交叉反应,具有良好的特异性;其组间重复性和组内重复性的变异系数均小于2%,表明该方法具有较好的重复性。结果表明,建立的双重荧光定量RT-PCR检测方法适用于BTV和PPRV的快速检测。In order to establish a rapid detection method that can simultaneously detect the blue tongue virus(BTV)and the peste des petits ruminants virus(PPRV).In this study,the NS3 gene of BTV and the N gene of PPRV were selected as target genes.According to sequence aligment of NS3 of BTV and N of PPRV published on Gen Bank,a highly conservative region was selected to design primers and probes by Beacon designer 8.The duplex real-time RT-PCR assay was established after optimizing reaction system and the reaction conditions.The sensitivity results showed that the detection limitations of BTV and PPRV positive plasmids were 1.7 copies/μL and 1.2 copies/μL.The specificity of the assay was high,without any cross reactions with goatpox virus,orf virus,foot-and-mouth disease virus,clostridium septicum,Brucella and Toxoplasma gondii.The coefficient variation between inter-group and in tra-group is less than 2%,indicating this method has good repeatability.The results above indicated that duplex Taq Man real-time RT-PCR is suitable for the rapid detection of bluetongue virus and petit pestis ruminant virus.

关 键 词：蓝舌病病毒 小反刍兽疫病毒 双重荧光定量RT-PCR 

分 类 号：S852.659.4[农业科学—基础兽医学]