| 引用本文: | 韦信贤,童桂香,谢宗升,黎小正,吴祥庆,廖永志,黄国秋.TaqMan-MGB探针法快速检测对虾Taura 综合征病毒的研究[J].海洋科学,2011,35(9):37-42. |
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| 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。 |
| 关键词: TSV Taqman-MGB 探针 一步法荧光定量RT-PCR |
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| 基金项目: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(2060302 GXIF-2009-05) |
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| TaqMan-MGB probe real-time fluorescence quantitative RT-PCR for rapid detection of taura syndrome virus in shrimps |
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| Abstract: |
| 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 (㏒ X) and cycle threshold (Ct). The real-time RT-PCR assay can exactly detect and quantify TSV from 2×101copies/μL to 2×107copies/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. |
| Key words: TSV TaqMan-MGB probe one step real-time RT-PCR |
