Development of a One-Step Multiplex Reverse Transcription-Quantitative Polymerase Chain Reaction Assay for the Simultaneous Detection of Four Lily Viruses

Lilium spp. is an important cut flower in international and Korean markets due to its unique characteristics and fragrances. Millions of lily plants are cultivated, imported, and exported globally. Lily plants, propagated vegetatively through bulbs or scales to maintain the same genetic characteristics of lily flowers, require a longer growth period, increasing their risk of contact with infectious pathogens. Viral infections have been reported to cause both quantitative and qualitative damage to lily production yields worldwide. Common lily-infecting viruses include lily mottle virus (LMoV), lily symptomless virus (LSV), cucumber mosaic virus (CMV), and plantago asiatica mosaic virus (PlAMV). While numerous detection techniques have accurately confirmed the incidences of these viruses in lily plants, a one-step multiplex reverse transcription-quantitative polymerase chain reaction (RT-qPCR) has not yet been established. In this study, a one-step multiplex RT-qPCR was optimized to detect these four viruses in lily leaf samples collected in the Republic of Korea. Optimal primer concentrations were 10, 5, 5, and 2 µM for PlAMV, LMoV, CMV, and LSV detection, respectively, with optimal probe concentrations of 2, 1, 1, and 2 µM. A temperature of 57^oC produced the optimal results among six tested annealing temperatures. The developed probe-based multiplex RT-qPCR demonstrated higher sensitivity compared to the conventional multiplex RT-PCR, detecting viral RNAs at as low as 3×10^-4, 3×10^-5, 3×10^-5, and 3×10^-5 ng for LMoV, PlAMV, CMV and LSV, respectively. This optimized assay proved sensitive, specific, and reproducible in distinguishing and simultaneously detecting four lily viruses effectively in 24 lily field samples.