RT-PCR Assay Standardization for the Diagnosis of Cowpea Infecting Bean Common Mosaic Virus

Bean common mosaic disease (BCMD) caused by Bean common mosaic virus (BCMV) poses a significant threat to legume crops worldwide. BCMV, a monopartite potyvirus, has been a concern since its detection in 1917 due to its seed-borne nature. Symptoms include mosaic patterns, necrosis, stunting and yield reductions, leading to substantial economic losses in affected regions. Reverse transcription-polymerase chain reaction (RT-PCR) offers a precise and efficient method for BCMV detection, necessitating the development of specific primers for accurate diagnosis.

Specific primers (BCMV-polyp1, BCMV-coatp2 and BCMV-polyp3) were designed by aligning BCMV genome sequences and employing Primer3 software. RNA isolation from infected samples and cDNA synthesis were performed using standard protocols. RT-PCR was conducted with various annealing temperatures to optimize conditions. Primer specificity of BCMV-coatp2 primer set was confirmed via BLAST analysis and in vivo testing against closely related potyviruses. Sensitivity assays were carried out using serially diluted cDNA samples from infected tissue.

The designed primers demonstrated high specificity to BCMV, with no cross-reactivity observed against closely related potyviruses. Optimization of RT-PCR conditions revealed distinct bands at an annealing temperature of 51.9°C for all primer sets. Sequencing confirmed amplicon sizes (175 bp, 157 bp and 189 bp) and alignment with BCMV sequences. Sensitivity analysis showed the assay capable of detecting BCMV at concentrations as low as 0.14 ng/μgl. Validation with field samples confirmed the effectiveness of the primers in identifying BCMV-infected plants.