Genomic and antibody-based assays for the detection of Indian strains of Macrobrachium rosenbergii nodavirus and extra s
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ORIGINAL ARTICLE
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 Singaiah Naveen Kumar1 Iddya Karunasagar3
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Praveen Rai2
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Indrani Karunasagar2,3
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Received: 1 July 2020 / Accepted: 7 November 2020 Indian Virological Society 2020
Abstract White tail disease (WTD) of cultured Macrobrachium rosenbergii is 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 1 fg (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 10 fg for both MrNV and XSV. Dot-blot hybridization had a detection limit of 10 pg and 0.1 ng for MrNV and XSV respectively when RNA extracted from viral inoculum preparation was used; 0.1 ng and 1 ng when WTD positive whole tissue total RNA was & Indrani Karunasagar [email protected] 1
Fisheries Research Centre, Ministry of Environment, Water and Agriculture, P.O. Box 134, Saihat 31972, Eastern Province, Kingdom of Saudi Arabia
2
Nitte (Deemed to be University), Nitte University Centre for Science Education and Research, Deralakatte, Mangaluru 575018, India
3
Nitte (Deemed to be University), University Enclave, Medical Sciences Complex, Deralakatte, Mangaluru 575018, India
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. Keywords MrNV XSV Molecular detection Indian isolate
Introduction Giant freshwater prawn, Macrobrachium rosenbergii is an important cultured palaemonid, and its culture represents economically important activity in many of the developing Southeast Asian countries. Global production of freshwater prawn has got tremendous scope for the growth due to market demand; however, production could be seriously affected by the occurrence of white tail disease (WTD). WTD affects post larvae (PL) and juveniles of M. rosenbergii in hatcheries and farms, causing m
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