Polymerase chain reaction based detection of banana bunchy top virus(BBTV) and banana streak virus(BSV)

Abstract

Banana is an important fruit crop of Kerala. It is affected by various diseases including viral diseases, of which Banana Bunchy Top Virus (BBTV) and Banana Streak Virus (BSV) assume economic importance. Detection of these viruses in early stage warrants great significance. The present study entitled “Polymerase Chain Reaction based detection of Banana Bunchy top virus (BBTV) and Banana streak virus (BSV)’’ was taken up in this context. It was carried out at the Department of Plant Biotechnology, College of Agriculture, Vellayani, Thiruvananthapuram during October 2006 to July 2008 with the objective of developing a protocol for the detection of these two DNA viruses of banana using polymerase chain reaction (PCR) technique.

DNA from infected samples of BBTV and BSV as well as healthy plants was isolated using CTAB and modified CTAB methods. In terms of quality and quantity of DNA isolated, both the methods were good. The isolation of the genomic DNA using the CTAB and modified CTAB method was also standardized.

Polymerase Chain Reaction was carried out using designed as well as reported primers. DNA sequence used for the primer designing was taken from National Centre for Biotechnology Information. The primers were designed for the coat protein gene of the two viruses viz., BBTV and BSV. The primer designing was done manually using certain fixed parameters such as primer length, GC content, melting temperature etc. The length of the designed primers ranged from 16nt to 24nt and Tm from 51.7oC to 62.9oC. Designed primers viz., Af and Ar in the case of BBTV and Bf and Br in the case of BSV were analysed using the oligocalc-oligonucleotide calculation program and the similarity search was done using the BLASTN. The result of the BLASTN showed that the primer sequences were similar not only to banana but also to other organism like Arabidopsis thaliana, Drosophila melanogaster etc. The reported primers were obtained from Su et al. (2003) in the case of BBTV and Braithwaite et al. (1995) in the case of BSV.

The amplification gave a band of about 500bp in the case of BBTV with the primer Af and Ar and 1000bp in the case of BSV with the primers Bf and Br and 1Kb in the case of the reported primers of BBTV. There was no amplification in the BSV samples using reported primer as well as using IC-PCR technique.

The present study made it possible to detect the presence or absence of the viruses through PCR. More number of samples and primers can be used in the future to detect the virus. This technique can also be extended to other viruses also.