Validation of DAC-ELISA and RT-PCR for diagnosing peanut bud necrosis virus in groundnut

Abstract

Peanut Bud Necrosis Disease (PBND), caused by the Peanut Bud Necrosis Virus (PBNV), poses a significant threat to groundnut cultivation, particularly in Northeastern Karnataka. Visual symptoms can aid in preliminary diagnosis, however, distinguishing PBND from infections caused by closely related tospoviruses like Tomato spotted wilt virus (TSWV) and Tobacco streak virus (TSV) remains challenging due to overlapping symptom expressions. In this study, symptomatic groundnut samples collected during a regional survey were subjected to serological analysis using Direct Antigen Coating Enzyme-Linked Immunosorbent Assay (DAC-ELISA) and molecular confirmation through Reverse Transcription Polymerase Chain Reaction (RT-PCR). Among the 30 symptomatic samples tested, 19 samples showed positive reaction in DAC-ELISA using PBNV-specific polyclonal antiserum, with optical density (OD) values ranging from 0.31 to 0.92. RT-PCR amplification targeting the coat protein gene (PBNV-Nc) yielded an ~830 bp amplicon in all DAC-ELISA-positive samples and an additional ELISA-negative sample, demonstrating RT-PCR's superior sensitivity. The integration of serological and molecular diagnostic techniques ensured high specificity and reliability in detecting PBNV, overcoming the limitations of symptom-based diagnosis. These findings emphasize the importance of combining DAC-ELISA and RT-PCR for effective surveillance and accurate identification of PBND in groundnut, which is critical for timely disease management.