Simultaneous detection of Macrobrachium rosenbergii nodavirus and extra small virus by a single tube, one-step multiplex RT-PCR assay

Post- larvae of Macrobrachium rosenbergii infected with white tail disease were collected from hatcheries and nursery ponds in India. The causative organisms have been identified as Macrobrachium rosenbergii nodavirus (MrNV) and extra small virus ( XSV). A one-step multiplex reverse transcriptase-polymerase chain reaction ( RT-PCR) has been developed to detect these viruses simultaneously in naturally and experimentally infected prawns. Several parameters were assayed in order to optimize the protocol for simultaneous detection. Naturally and experimentally infected prawns showed two prominent bands of 681 and 500 bp for MrNV and XSV, respectively, as in separate RT-PCR assays. Experimentally infected adult prawns showed two bands for these two viruses in all the organs, except hepatopancreas and eyestalk, as seen in normal RT-PCR. The sensitivity test carried out on the primer sets of MrNV and XSV revealed that these primers could simultaneously detect the two viruses at a level of 25 fg of total RNA prepared from infected samples using this multiplex RT-PCR protocol.