Genomic and antibody-based assays for the detection of Indian strains of Macrobrachium rosenbergii nodavirus and extra small virus associated with white tail disease of Macrobrachium rosenbergii.

Abstract

White tail disease (WTD) of cultured Macrobrachium rosenbergiiis caused by Macrobrachium rosenbergii nodavirus (MrNV) and extra small virus (XSV). Since both the viruses have small single strand RNA as genetic material with short generation time, they are more prone to mutations. Hence detection methods developed for one strain may be suboptimal for the detection of isolates from the different geographical locations. In the present study two new genomic based methods (RT-PCR and dot-blot hybridization) along with one immunological method (polyclonal antibodies based detection) were developed for the detection of Indian isolates of MrNV and XSV. Among genomic based methods, RT-PCR assay developed was most sensitive. Sensitivity of detection of RT-PCR was 1fg (both MrNV and XSV) of total RNA extracted from purified viral inoculum preparation. In case of WTD positive whole tissue total RNA, the limit of detection was 10fg for both MrNV and XSV. Dot-blot hybridization had a detection limit of 10pg and 0.1ng for MrNV and XSV respectively when RNA extracted from viral inoculum preparation was used; 0.1ng and 1ng when WTD positive whole tissue total RNA was used. Polyclonal antibodies against recombinant proteins (MrNV and XSV capsid) were synthesised. Western blotting and indirect ELISA revealed that the antibodies produced to be specific and highly sensitive. Recombinant protein (antigen) of MrNV and XSV capsid were detected at the dilution of 1:8000. However in case of infected prawn tissue sample, MrNV and XSV were detected at the dilution of 1:32,000 and 1:64,000 respectively. All methods developed are field applicable.