鸭坦布苏病毒SYBR Green qPCR检测方法的建立  

Establishment of a SYBR Green qPCR detection method for duck Tembusu virus

作　　者：陈国强 李永旺 王凯民[4] 陈雷[4] 钱伟[2] 朱婷 陆春华 时长军 CHEN Guoqiang;LI Yongwang;WANG Kaimin;CHEN Lei;QIAN Wei;ZHU Ting;LU Chunhua;SHI Changjun(Guangdong Provincial Key Laboratory of Technical Measures of Animal,Plant and Food Import and Export,Guangzhou 510623,China;Suzhou Entry/Exit Inspection and Quarantine Bureau,Suzhou 215021,China;Suzhou Xishan Biotechnology Inc.,Suzhou 215123,China;Jiangsu Entry/Exit Inspection and Quarantine Bureau,Nanjing 210001,China)

机构地区：[1]广东省动植物与食品进出口技术措施研究重点实验室,广东广州510623 [2]苏州出入境检验检疫局,江苏苏州215021 [3]苏州西山生物技术有限公司,江苏苏州215123 [4]江苏出入境检验检疫局,江苏南京210001

出　　处：《畜牧与兽医》2018年第9期78-81,共4页Animal Husbandry & Veterinary Medicine

基　　金：质检总局科研项目(2016IK132);广东省动植物与食品进出口技术措施研究重点实验室开放课题(IQTC201602)

摘　　要：通过BLAST比较已公布的鸭坦布苏病毒核酸序列,在其E基因保守区域设计了4对引物,并用鸭坦布苏病毒培养物核酸筛选出最佳的一对引物建立了SYBR Green qPCR。用构建的重组质粒建立标准曲线,扩增效率为90.5%,在2.0×10~1~2.0×10~8copies/μL范围内具有良好的线性关系,最低检测限为100拷贝/反应。该qPCR方法可以检测5个鸭坦布苏病毒分离株以及WF100活疫苗,灵敏性比文献已发表的其他3对引物高10倍;且特异性很好,不与其他常见禽类病毒交叉反应。由此表明本研究新建了一种高度灵敏和特异的鸭坦布苏病毒的SYBR Green qPCR检测方法。Following comparison of nucleic acid sequences of duck Tembusu virus（DTMUV） by BLAST,four pairs of primers were designed in the conservative region of the E gene. The best primer pair was selected for establishing a SYBR Green qPCR by screening with nucleic acid of DTMUV culture. The standard curve of this qPCR method was established with recombinant plasmid,showing an amplification efficiency at 90. 5% and a lower limit of detection at 100 copies/reaction with a good linear relationship from 2. 0×10~1 to 2. 0×10~8 copies/μL.This qPCR method could detect the live DTMUV vaccine of WF100 and five DTMUV isolates,with higher sensitivity than the other three published primer pairs,and of good specificity without crossing reaction with other avian viruses. These results suggested that a highly sensitive and specific SYBR Green qPCR method was established in this study for detection of duck Tembusu virus.

关 键 词：鸭坦布苏病毒 qPCR 灵敏性 特异性 

分 类 号：S852.6[农业科学—基础兽医学]