TaqMan-MGB探针法快速检测对虾Taura综合征病毒的研究  

TaqMan-MGB probe real-time fluorescence quantitative RT-PCR for rapid detection of taura syndrome virus in shrimps

作　　者：韦信贤[1,2] 童桂香[1] 谢宗升[1] 黎小正[1] 吴祥庆[1] 廖永志[1] 黄国秋[1] 

机构地区：[1]广西渔业病害防治环境监测和质量检验中心,广西南宁530021 [2]上海海洋大学水产与生命学院,上海201306

出　　处：《海洋科学》2011年第9期37-42,共6页Marine Sciences

基　　金：广西壮族自治区直属公益性科研院所基本科研业务费专项资金项目(2060302GXIF-2009-05)

摘　　要：为建立Taura综合征病毒(Taura syndrome virus,简称TSV)的TaqMan-MGB探针荧光定量RT-PC方法,根据TSV衣壳蛋白基因保守序列设计引物和TaqMan-MGB探针,扩增产物长度为118bp。将PCR产物克隆到pGM-T载体,重组质粒经筛选鉴定、SalⅠ单酶切,得到线性化转录模板DNA,经体外转录获得标准品RNA。以体外转录的RNA作为模板,进行一步法荧光定量RT-PCR反应,根据RNA模板拷贝数与Ct(Cycle threshold,Ct)值制备了标准曲线,制作的标准曲线在2×101拷贝/μL≤TSV RNA≤2×107拷贝/μL的范围内具良好的线性关系,可以用于TSV的定量分析,检测灵敏度为20个拷贝数。特异性、重复性试验结果表明,该方法对SPF对虾组织RNA没有扩增反应,表现出良好的特异性;组内和组间实验变异系数分别为0.32%～1.84%和0.79%～2.71%。利用TaqMan-MGB探针建立检测TSV的一步法荧光定量RT-PCR方法可用于对虾TSV的检测。To establish a TaqMan-MGB probe real-time fluorescence quantitative RT-PCR for detection of TSV, primers and probe were designed based on the conserved region of the TSV capsid protein genome with 118 bp target products. The PCR products were cloned into the pGM-T vector, than the positive linearized plasmids were used to transcript RNA in vitro. The RNAs were used as standard quantitative templates to make the standard curve, which was generated based on the linear relationship between the amount of RNA （log X） and cycle threshold （Ct）. The real-time RT-PCR assay can exactly detect and quantify TSV from 2×10^1copies/μL to 2×10^7copies/μL, and had a detection limit of 20 RNA copies. The method is specific for TSV and did not amplify SPF shrimp RNA. The coefficient variation （CVs） is 0.32%-1.84% within assay and 0.79%-2.71% among assays. The results showed that the one step real-time RT-PCR is useful for TSV detection.

关 键 词：TSV TAQMAN-MGB探针 一步法荧光定量 RT-PCR 

分 类 号：S945.4[农业科学—水产养殖]