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Abstract
Prunus necrotic ringspot virus (PNRSV) is one of the most devastating viruses to Prunus spp. In this study, we developed a diagnostic system RT-CPA-NATSC, wherein reverse transcription-cross-priming amplification (RT-CPA) is coupled with nucleic acid test strip cassette (NATSC), a vertical flow (VF) visualization, for PNRSV detection. The RT-CPA-NATSC assay targets the encoding gene of the PNRSV coat protein with a limit of detection of 72 copies per reaction and no cross-reaction with the known Prunus pathogenic viruses and viroids, demonstrating high sensitivity and specificity. The reaction is performed on 60 °C and can be completed less than 90 min with the prepared template RNA. Field sample test confirmed the reliability of RT-CPA-NATSC, indicating the potential application of this simple and rapid detection method in routine test of PNRSV.
Highlights
Cross-priming amplification (CPA) is a recently developed isothermal DNA amplification method with high specificity and sensitivity[18] and has been successfully used to detect number of human, animal and plant pathogens ranging from bacteria to virus[16,19,20,21,22,23]
By using total RNA extracted from the Prunus necrotic ringspot virus (PNRSV)-infected cucumber (Cucumis sativus) leaves as template, a total of seven primer sets (Supplementary Table 1) were evaluated on their reaction efficiency for PNRSV detection by reverse transcription-cross-priming amplification (RT-CPA) coupled with real-time fluorescence at 60 °C
With the selected primer set G7, reverse transcription (RT)-CPA was performed on total RNA of the PNRSV-infected cucumber leaves with 40 min, following visualization of the resulting amplicons with Nucleic acid test strip cassette (NATSC)
Summary
Cross-priming amplification (CPA) is a recently developed isothermal DNA amplification method with high specificity and sensitivity[18] and has been successfully used to detect number of human, animal and plant pathogens ranging from bacteria to virus[16,19,20,21,22,23]. Nucleic acid test strip cassette (NATSC) bases on the vertical flow hybridization and is designed to visually detect double-labeled amplicons in only 5 to 10 min[24]. We established a method termed RT-CPA-NATSC, in which, CPA was combined with reverse transcription (RT) to amplify the RNAs of PNRSV, and the resulting amplicons were subsequently visualized with NATSC. The specificity and sensitivity of RT-CPA-NATSC assay in detecting PNRSV were evaluated, demonstrating that this method has great potential for PNRSV surveillance
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