Development of a method to detect infectious rotavirus and astrovirus by reverse transcriptase polymerase chain reaction (RT-PCR) with propidium monoazide (PMA)

Rotavirus and astrovirus are two kinds of water-borne pathogens that can cause severe diarrhea in children, infants and immunocompromised humans. Both can be present in untreated and inadequately treated water. Molecular methods, such as the reverse transcriptase polymerase chain reaction (RT-PCR), have been used to detect two viral genomes in a few hours, but they cannot distinguish between infectious and noninfectious rotavirus and astrovirus. In our study, we developed a propidium monoazide-reverse transcriptase polymerase-chain reaction (PMA-RT-PCR) assay to determine the infectivity of these two RNA viruses. Rotavirus and astrovirus stool samples were detected by RT-PCR in conjunction with PMA, respectively. Stool samples inactivated with heat treatment (95°C) were processed at the meantime as controls. The result showed that infectious virus samples gave positive results, while noninfectious virus samples presented negative results. To determine the viability of rotavirus and astrovirus in source water, a total of five source water samples were also collected from different reservoirs in July, 2012. One sample showed a positive result, which meant that PMA-RT-PCR method can be successfully applied to the viral detection in water samples. Data obtained in this study suggest that pretreatment of viruses with PMA prior to RT-PCR is a reliable method for distinguishing between infectious and noninfectious rotavirus and astrovirus. To our knowledge, this is the first report of application of this technique to rotavirus and astrovirus.   Key words:  Rotavirus, astrovirus, propidium monoazide (PMA), source water.