Development of a real-time RT-PCR assay combined with ethidium monoazide treatment for RNA viruses and its application to detect viral RNA after heat exposure

A method was developed for discriminating damaged viruses or naked viral RNA from intact viruses by ethidium monoazide (EMA) treatment before RT-PCR. The applied EMA treatment consisted of three steps: (1) EMA dose, (2) exposure to light, and (3) additional purification by spin-column gel filtration. Approximately 4-log reduction in viral RNA concentration was observed by adding a dose of 10 mu g/mL-EMA with 300 s of light irradiation. Although residual EMA can be an inhibitor of RT-PCR, its effect was reduced by spin-column gel filtration or a QIAamp (R) Viral RNA Mini Kit. EMA-RT-PCR was applied to the thermally treated PV1. Results of EMA-RT-PCR were similar to the plaque assay when PV1 was thermally inactivated. Although this is a preliminary study investigating applicability of the EmA-RT-PcR method for RNA viruses, the results suggest that the method is potentially applicable for the selective detection of epidemiologically important enteric viruses in water such as enteroviruses and noroviruses.