Development of a Reverse-Transcription Loop-Mediated Isothermal Amplification Assay for Detecting Brassica Yellows Virus in China

Le résumé fourni par la source

Brassica yellows virus (BrYV) mainly infects cruciferous crops and has been widely prevalent across China. To develop a rapid and highly sensitive method for detecting BrYV in oilseed rape, a reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay was established. Four specific primers were designed to target the conserved gene of BrYV, with total RNA extracted from BrYV-infected oilseed rape leaves used as the template for the RT-LAMP assay. The optimal reaction conditions were determined, including a primer concentration ratio of 1:8, MgSO4 concentration of 4 mM, reaction temperature of 64 °C, and a suitable reaction time of 60 min. Sensitivity analysis demonstrated that the RT-LAMP assay could detect total RNA at a concentration of 0.091 × 10−3 μg/μL, which was 100-fold more sensitive than conventional RT-PCR for BrYV detection. In addition to visualizing results by electrophoresis, the RT-LAMP assay could also be easily visualized using calcein-MnCl2. These results indicate the potential of the established RT-LAMP assay for rapid BrYV detection in oilseed rape plants, which can provide better technical support for field diagnosis, disease forecasting, and the implementation of effective control strategies against the virus.

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