Evaluation of immunological and molecular techniques for the detection of different isolates of banana bunchy top virus in India.

Abstract

In this study, Banana bunchy top virus (BBTV) infecting hill banana cv. Virupakshi (AAB) was purified and raised polyclonal antiserum which had a titre of 1:500. Direct Antigen Coating (DAC)-ELISA, PCR and Nucleic Acid Spot Hybridization (NASH) with non-radioactive probe techniques were optimized and compared for detection of BBTV. In PCR, the virus could be detected at a dilution of 1:1000 whereas DAC-ELISA and NASH detected the virus at 50 and 500 dilutions respectively. Five BBTV isolates collected from various parts of India were tested by three methods, only PCR detected all the isolates. Primers specific to coat protein gene of BBTV was used for PCR and NASH tests. As part of validation, 417 samples collected from different banana growing regions of Tamil Nadu tested, DAC-ELISA detected the virus from 336 samples whereas 364 (87.29%) and 404 (96.8%) samples were positive in NASH and PCR respectively. When NASH test was performed for PCR products, three PCR negative samples tested positive. This is the first report that the coat protein gene specific primers have been used for detection of BBTV. The PCR and PCR-NASH combined techniques can be used for identifying virus-free plants in germplasm and routine indexing of BBTV in certification of tissue culture plants in India.