Development of Duplex RT-qPCR Assay for the Simultaneous Detection of Cycas Necrotic Stunt Virus and Lychnis Mottle Virus in Paeonia lactiflora
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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 RT- PCR 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 thresh- old 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 tempera- ture 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.
Abstract
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 RT- PCR 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 thresh- old 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 tempera- ture 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.
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