Comparative detection of banana bunchy top virus in banana cv. Safed velchi utilizing DNA/RNA by PCR technique

Abstract

The present study was carried out to describe the ideal PCR protocol to find viral infection in plantlets of banana. 50 plantlets selected from Plant Biotechnology Center, Dapoli from which DNA (50 samples bulked into 10) as well as RNA (20 samples) were extracted along with BBTV infected samples. The priviously standard rapid DNA isolation protocol was used. A protocol using 1.80 g ml-1 Tris-base, 3.75 g ml-1 EDTA, 1.5 g ml-1 SDS and 100µl ml-1 βMercaptoethanol produced the highest yields and best quantity RNA. cDNA synthesis (M-MuLV kit) containing Oligo-dT, dNTP’s, Molecular Biology Grade Water and isolated RNA template were used to prepare reaction mixture. The optimum PCR procedure utilizes to synthesized cDNA from template RNA. 21 samples get assayed at RNA level by preparing cDNA. Strong reactions were obtained from the sample no 10, 11, 22 and 23 of BBTV infected samples and a positive control plant at no 12 and 24 confirms the presence of BBTV with amplicon size of 389 bp with primer BBTV F & R. All 50 DNA samples were tested for BBTV gene specific primers (CP, NSP and FPCR’4+30MER gene) all the tested regenerated plantlets were found to be negative and the lot no 11 of BBTV infected samples and a positive control plant at no 12 confirms the presence of BBTV with amplicon size of 511bp, 349 bp and 1,349 bp respectively. But, RNA level mechanism was effective in which the actual coding genome was transcribed. So it is easy and useful to detect the genome at RNA level than DNA which contains large genome size.