Tamil Nadu Agricultural University, Coimbatore-641003.
Among the different ELISA methods, triple antibody sandwich (TAS)-ELISA was more sensitive and reliable in detecting BSV antigens in leaves, meristem, suckers and other parts of plants. The plate was coated with polyclonal antiserum (1:5000) produced against Indian BSV and then samples were added. Samples were ground @ 1:2 with extraction buffer (pH 7.4) followed by addition of skimmed milk powder 5% in PBST containing 2% polyvinyl pyrpllidone. After 2 h the solution was discarded and BSV monoclonal antibody was added @ 1:500 μl in PBST. The rabbit antimouse IgG conjugate (Sigma) was used at 1:1000 in PBST, finally PNP 0.6 mg per ml in diethanolamine buffer (10%) used for colour development. Major constraint in BSV detection was periodicity of symptom expression of BSV, which can be over come by PCR method. The PCR reaction mix tube of 50 μl contained 20 milli mol per L Tris – HCI (pH 8.4) 50 milli mol per L KCI, 200 m mol per Leach dATP, dCTP, dGTP, dTTP, 2.5 m mol per L MgCl2, 10 pico mol each forward and reverse primer and 2U Taq polymerase. Forward primer BSV 4673 5’GGA ATG AAA GAG CAG GCC3’. Reverse primer BSV 5317 5’AGT CAT TGG GTY CAA CCT CTG TCC C3’. PCR cycle condition was an initial denaturation at 94°C for 1 min, 33 cycles (94°C for 1 min, annealing at 50°C for 1 min and extension at 72°C for 1 min) and a final extension at 72°C for 5 min. The PGR products amplified at 644bp level product in 1.5 per cent agarose gel electrophoresis showed a positive reaction. Results indicate that meristem was not free of BSV infection.
