A multiplex reverse transcription PCR assay for simultaneous detection of four viruses in Cymbidium kanran

Cymbidium, a genus within the Orchidaceae family, is widely distributed across Asia.Cymbidium kanran is classified as an endangered species in Korea, where it is restricted to Jeju Island.Its conservation and cultivation are threatened by viral infection.However, visual diagnosis is unreliable because viral symptoms often resemble those caused by other pathogens or abiotic stress, highlighting the need for accurate molecular diagnostic tools.In this study, we developed a multiplex reverse transcription PCR (RT-PCR) assay for the simultaneous detection of four major viruses that infect C. kanran: odontoglossum ringspot virus (ORSV), orchid fleck virus (OFV), tobacco mosaic virus (TMV), and cymbidium mosaic virus (CymMV).Four virusspecific primer pairs, designed to target conserved genomic regions, generated distinct amplicons of 594, 482, 400, and 220 bp, respectively.The optimized assay successfully identified all four viruses in a single reaction, with a detection limit of 10 5 copies/μL.Field application of this assay to 114 symptomatic C. kanran leaf samples revealed both single and mixed infections, underscoring its reliability and practical applicability.Collectively, these results demonstrate that the developed multiplex RT-PCR assay is a specific, sensitive, and efficient diagnostic tool and thus a valuable resource for monitoring and managing viral diseases in C. kanran.