The development of a duplex RT-qPCR assay for the simultaneous detection of cycas necrotic stunt virus and lychnis mottle virus in Paeonia lactiflora

Abstract In this study, a duplex probe based reverse transcription quantitative polymerase chain reaction (RT-qPCR) assay was utilized to simultaneously detect cycas necrotic stunt virus (CNSV) and lychnis mottle virus (LycMoV) in Paeonia lactiflora collected from various locations in South Korea. CNSV and LycMOV infections were verified by using conventional reverse transcription polymerase chain reaction (RT-PCR) using gene-specific primers. Due to peony’s high secondary metabolites, multiple standard templates in the form of both DNA and RNA were evaluated. The circular plasmid was observed to produce the finest results and was used in dye-based qPCR to select the best-performing primers characterized by their ability to yield a low threshold cycle (Ct) and high fluorescence. The high precision quantification duplex probe-based qPCR assay was conducted and then optimized. The combination of primer concentration of 5 pmol/µl coupled with probe concentration of 4 pmol/µl at the annealing temperature of 57 ℃ produced stable and consistent amplification plots and standard curves. This combination demonstrated the capability to simultaneously detect plasmid DNA of both CNSV and LycMoV at concentrations as low as 10-6 ng/µl. These primer sets and optimum conditions were applied in RT-qPCR to detect total RNA of peony leaves co-infected with CNSV and LycMoV. Successful detection occurred with a slightly weaker sensitivity, having a detection limit of 10-3 ng/µl. The use of duplex probe-based RT-qPCR assay demonstrated in this study should improve the virus screening process of CNSV and LycMoV, leading to a reduction of the spread of these two plant viruses.