Key result
Ethidium monoazide (EMA) treatment before RT-PCR achieved an approximately 4-log reduction in viral RNA concentration, enabling discrimination of damaged or naked viral RNA from intact viruses.
EMA-RT-PCR is a potentially applicable method for the selective detection of intact, epidemiologically important enteric viruses in water.
EMA pretreatment may refine intact virus detection in water; leaves open standardization and field validation before surveillance use.
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 μg/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® 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.
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Kim et al. (2011) studied RNA viruses. Ethidium monoazide (EMA) treatment before RT-PCR was evaluated on Reduction in viral RNA concentration. Ethidium monoazide (EMA) treatment before RT-PCR achieved an approximately 4-log reduction in viral RNA concentration, enabling discrimination of damaged or naked viral RNA from intact viruses.
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