Department of Microbiology, College of Veterinary Science, Acharya N.G. Ranga Agricultural University, Tirupati - 517 502, (AP).
*Corresponding author: Assoc. Professor.
1Part of M.V.Sc. thesis, ANGRAU, Department of Microbiology, College of Vety. Sci., Tirupati - 517 502, (AP).
NS3 specific RT-PCR for detection of bluetongue virus (BTV) in clinical samples has been standardized using NS3 primers. Trizol® method of RNA extraction was used to isolate RNA from infected cell culture fluids, embryonated chicken eggs and blood samples. Denaturation of dsRNA by heating at 95°C for 5 minutes in the pesence of 5% DMSO and a 35 amplification cycle PCR with annealing temperature of 59°C for 30 seconds at 1.5 mM concentration of MgCl2 and primer extension at 72°C for 1 minute were found optimum to obtain 780 bp PCR product. The RT- PCR thus standardized has detected BTV RNA in blood samples collected between days 5 to 11 post inoculation from experimentally infected sheep. Out of 32 field blood samples collected from bluetongue suspected outbreaks of A.P. during the months of August and September, 2005, ten were found positive for the presence of BTV RNA by NS3 RT-PCR. Further, BTV was isolated from PCR positive blood samples by intravenous inoculation into embryonated chicken eggs and subsequent passages on to BHK21 cell line and identified as BTV by nucleic acid migration pattern in 1% agarose gel electrophoresis. This confirmed the utility of NS3 RT-PCR as a test for rapid and confirmatory diagnosis of bluetongue infection in sheep.
