摘要
为建立小反刍兽疫病毒(PPRV)Ⅱ系与Ⅳ系毒株SYBR Green Ⅰ荧光RT-PCR的鉴别检测方法,针对PPRV-H基因的高变区域设计1对特异性引物,确定反应条件,分析其敏感性和特异性,并对临床样本进行验证。60℃退火、76℃收集荧光信号获得试验效果最好,熔解曲线为单一峰,Ⅱ系毒株熔解温度78.63℃,Ⅳ系毒株熔解温度80.08℃,易于区分;最低检测限为10拷贝/μL,批内、批间变异系数均小于0.5%,羊口疮病毒(ORFV)等4种病毒无扩增曲线产生;检测140份临床样本,与国家标准检测方法结果符合率100%。本研究建立的SYBR Green Ⅰ荧光RT-PCR检测方法可以快速、灵敏和特异地鉴别检测出PPRVⅡ系与Ⅳ系毒株,满足疫病快速诊断的需求。
To establish a SYBR Green Ⅰ real-time RT-PCR approach for the differential detection of peste des petits ruminants virus Ⅱ and Ⅳ strains.A pair of specific primers were designed for the hypervariable region of PPRV-H gene,the reaction conditions were determined,the sensitivity and specificity were analyzed,and the clinical samples were verified.The best test results was obtained by annealing at 60℃ and collecting fluorescence signal at 76℃,and the melting curve was a single peak,the melting temperature of strain Ⅱ was 78.63℃,and the melting temperature of strain Ⅳ was 80.08℃,which was easy to distinguish.The minimum detection limit was 10 copies/μL,and the intra-and inter-coefficients of variation were less than 0.5%.There was no amplification curve with ORFV and other four viruses.One hundred and forty clinical samples were detected,and the coincidence rate with the national standard detection method was 100%.The SYBR GreenⅠreal-time RT-PCR detection method established in this study can quickly,sensitively and specifically identify and detect strains of the strains of peste des petits ruminants virus Ⅱ and Ⅳ,meeting the needs of rapid disease diagnosis.
作者
董帅
杨哲
孟卫芹
陈金龙
刘建钗
沈志强
王金良
DONG Shuai;YANG Zhe;MENG Weiqin;CHEN Jinlong;LIU Jianchai;SHEN Zhiqiang;WANG Jinliang(Shandong Binzhou Animal Science&Veterinary Medicine Academy,Binzhou,Shandong 256600,China;College of Life Science and Food Engineering,Hebei University of Engineering,Handan,Hebei 056000,China;College of Animal Science and Technology,Shihezi University,Shihezi,Xinjiang 832003,China)
出处
《中国兽医学报》
CAS
CSCD
北大核心
2023年第12期2433-2438,共6页
Chinese Journal of Veterinary Science
基金
山东省羊产业技术体系岗位专家资助项目(SDAIT-10-07)。
