Development of Duplex RT-qPCR Assay for the Simultaneous Detection of Cycas Necrotic Stunt Virus and Lychnis Mottle Virus in Paeonia lactiflora

Background: In this study, a duplex probe-based reverse transcription quantitative polymerase chain reaction (RT-qPCR) assay was used to simultaneously detect Cycas necrotic stunt virus (CNSV) and Lychnis mottle virus (LycMoV) in Paeonia lactiflora (peony) collected from various locations in South Korea. Methods and Results: CNSV and LycMoV infections were verified by using conventional RTPCR using gene-specific primers. A high-precision quantification duplex probe-based qPCR assay was conducted on plasmid DNAs of target viruses and optimized to select the best-performing primers characterized by their ability to yield high relative fluorescence units and low cycle threshold values. Stable and consistent amplification plots and standard curves were achieved with primer and probe concentration of 0.25 µM and 0.2 µM, respectively, and an annealing temperature of 57℃. RT-qPCR was used with the selected primer sets and optimum conditions to detect the total RNA of peony leaves co-infected with CNSV and LycMoV. Successful detection occurred with a slightly weak sensitivity, having a detection limit of 10^-3 ng/㎕. Conclusions: The duplex probe-based RT-qPCR assay demonstrated in this study can improve the screening process for CNSV and LycMoV, leading to a reduction in the spread of these two plant viruses.