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Highly Efficient Immunodiagnosis of Episomal B anana streak MY virus Using Polyclonal Antibodies Raised Against Recombinant Viral‐Associated Protein

2016; Wiley; Volume: 164; Issue: 7-8 Linguagem: Inglês

10.1111/jph.12475

1439-0434

R. Selvarajan, V. Balasubramanian, Thirugnanasambantham Gayathrie,

Plant and Fungal Interactions Research

Abstract B anana streak MY virus ( BSMYV ) is the causal agent of viral leaf streak disease of banana, which leads to considerable losses in banana production in most of the banana‐growing regions worldwide. Developing high‐throughput virus detection system is essential for managing viral diseases especially in vegetatively propagated crops like banana. In this study, viral‐associated protein ( VAP ) coded by ORF II of BSMYV was expressed in E scherichia coli , and polyclonal antibodies were raised against purified recombinant VAP ( rVAP ) fusion protein in rabbits. Specificity and sensitivity of resulting antibodies were tested in Western blot, immunosorbent electron microscopy ( ISEM ) and enzyme‐linked immunosorbent assays ( ELISA s). In direct antigen‐coated ( DAC )‐ ELISA , antibodies reacted specifically to BSMYV in crude sap, up to 1 : 8000 dilutions, but not to healthy leaf extracts. Using this antiserum, an immunocapture polymerase chain reaction ( IC ‐ PCR ) assay was developed and compared with DAC ‐ ELISA . VAP antibody‐based IC ‐ PCR is highly specific and could differentiate episomal virus infection from the integrated endogenous BSV ( eBSV ) sequences. The recombinant antibodies were validated by testing with a large number of banana germplasm conserved in the field gene bank. Field samples collected during surveys and mother cultures used in tissue culture propagation suggest that antibodies generated against rVAP are sensitive and useful for large‐scale detection of BSMYV . To the best of our knowledge, this is the first report on the production of polyclonal antiserum against recombinant VAP of BSMYV and its suitability for serology‐based testing by ELISA and IC ‐ PCR . This VAP ‐based immunodiagnosis can be applied in quarantine, germplasm exchange and certification programmes.