Huang Xuan, Li Yong, Zheng Cong-yi
State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan 430072, China.
J Virol Methods. 2009 Feb;155(2):150-6. doi: 10.1016/j.jviromet.2008.10.007. Epub 2008 Nov 30.
Foot-and-mouth disease virus is a positive-sense, single-stranded RNA virus with a negative strand as its replication intermediate, which can cause severe acute infection in sensitive cell lines. To investigate better the actual state of virus infection, there is a need to measure the amount of FMDV RNA in a single acutely infected cell rather than in a large number of cells. Therefore, in the present study, a strand-specific single-cell quantitative real-time RT-PCR was developed to analyze the RNA or FMDV. This new method uses two techniques in concert with each other: a technique for isolating single cells with micromanipulators, which is coupled to an assay for detecting viral RNA by real-time RT-PCR. In the assay of acute infection, 185 of 224 (82.6%) single-cell samples were positive and contained viral genome copies ranging from several to thousands, and up to 1,000,000 copies. However, not all cells were infected and there were differences in the number of viral RNA copies between cells. A single-cell quantitative RT-PCR was validated to be feasible and effective.
口蹄疫病毒是一种正链单链RNA病毒，其复制中间体为负链，可在敏感细胞系中引起严重的急性感染。为了更好地研究病毒感染的实际状态，需要测量单个急性感染细胞中的口蹄疫病毒RNA量，而不是大量细胞中的量。因此，在本研究中，开发了一种链特异性单细胞定量实时RT-PCR来分析RNA或口蹄疫病毒。这种新方法将两种技术协同使用：一种是用显微操作器分离单细胞的技术，该技术与通过实时RT-PCR检测病毒RNA的检测方法相结合。在急性感染检测中，224个单细胞样本中有185个（82.6%）呈阳性，含有从几个到数千个，最多可达1,000,000个拷贝的病毒基因组。然而，并非所有细胞都被感染，细胞之间的病毒RNA拷贝数存在差异。单细胞定量RT-PCR被验证是可行和有效的。
