| Sumario: | Alfalfa mosaic virus (AMV) and Southern bean mosaic virus (SBMV) are major seedborne pathogens affecting legumes and forage crops, causing substantial reductions in grain and biomass yield, and feed quality. Accurate and timely detection of these viruses is critical for effective disease management and seed certification. Conventional diagnostic methods, including Enzyme-Linked Immunosorbent Assay (ELISA) and single-target Reverse Transcription Polymerase Chain Reaction (RT-PCR), are limited by their inability to detect multiple viruses simultaneously, which is particularly challenging in crops prone to mixed infections. This study reports the optimization of a multiplex polymerase chain reaction (m-PCR) assay for the simultaneous detection of AMV and SBMV in forage species. Known positive and negative reference samples, as well as field and greenhouse leaf samples, were used to optimize the assay. The optimized m-PCR conditions:0.5 μL cDNA template, 54°C annealing temperature, and 0.5μL of each virus-specific primer, produced clear and distinct amplicons of 632bp for AMV and 211bp for SBMV without nonspecific amplification. Specificity tests using non-target viruses, including potyvirus, bean yellow mosaic virus (BYMV), bean common mosaic virus (BCMV), cowpea mosaic virus (CPMV), peanut mottle virus (PeMoV), and Johnson grass mosaic virus (JGMV), confirmed absence of cross amplification of non-target viruses, demonstrating the specificity of the assay. Validation with 30 field and greenhouse samples showed that the multiplex PCR results were largely consistent with conventional RT-PCR, with 90% and 93% agreement for AMV and SBMV, respectively. The optimized multiplex PCR provides a rapid, sensitive, and cost-effective diagnostic tool suitable for routine virus indexing, seed health testing, and phytosanitary monitoring in legume and forage crops.
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