Reverse Transcription-polymerase Chain Reaction (RT-PCR) Based Detection and Identification of Monopartite Bean Common Mosaic Virus in Mungbean

Abstract

Background: Bean common mosaic virus (BCMV) which belongs to the genus Potyvirus and the family Potyviridae, causes significant yield losses in legumes across the globe. Traditional methods for BCMV detection and diagnosis, such as visual inspection and serological assays, are often time-consuming, labour-intensive and may lack the sensitivity required for early detection. Advances in molecular techniques have made RT-PCR a powerful tool for the accurate detection of BCMV-infected plants. Methods: Three pairs of specific primers that flank the BCMV coat protein and polyprotein gene were designed using the Primer 3web tool to amplify fragments approximately ranging from 191 to 205 bp. The RT-PCR protocol was then standardized, including the optimization of annealing temperatures for all three primers, followed by the sequencing of the amplified PCR products. The, specificity and sensitivity of primer pair BCMV2 were then established. Result: Three pairs of specific primers were designed and RT-PCR protocol was standardized. The sequenced PCR products showed 98.03% to 99.50 % nucleotide similarity with the corresponding sequences of BCMV isolates used in primer designing. Further, the BCMV2 primer pair specifically amplified the target amplicon (200 bp) from the suspected leaf sample and no amplicon was observed when tested against other pathotypes of potyvirus such as BCMNV, BYMV, CABMV, SMV and PeMoV. The designed primer was sensitive enough to detect 0.05% of target cDNA. Additionally, the RT-PCR protocol was validated using field samples collected from Hyderabad, India. ELISA in combination with RT-PCR using the developed specific BCMV primer pair will ensure a foolproof, sensitive and rapid, procedure for diagnosis of BCMV in the quarantine processing of imported germplasm.