A novel single-cell quantitative real-time RT-PCR method for quantifying foot-and-mouth disease viral RNA

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.