Triplex visual detection of tobacco potyviruses using reverse transcription recombinase polymerase amplification assay combined with lateral flow dipstick

Currently, several analytical methods have been developed for identifying tobacco viral species. However, conventional methods are based on labor-intensive, time-consuming, and instrument-required approaches. A novel, rapid and visual method based on the combination of reverse transcription recombinase polymerase amplification assay (RT-RPA) and multiplex lateral flow dipstick (LFD) was established specifically for the rapid on-site detection of three tobacco viruses and evaluated. The biosensor used coat protein (CP) genes of potyviruses potato Y virus, chilli veinal mottle virus, and tobacco vein banding mosaic virus as detection markers. A triplex RT-RPA reaction for both targets was constructed and three chemical labels of the amplification products were carefully tested for effective and accurate visualization on the strip. This method demonstrated reasonable specificity and achieved a sensitivity of 103 copies per reaction. Validation with clinical samples showed results consistent with a real-time polymerase chain reaction. The detection process was simple, with little dependence on laboratory settings, and rapid, with a 30-min reaction at 39 degrees C and visualization on the strip within 5 min. This newly developed triplex RT-RPA-LFD assay was sensitive, specific, and suitable for the on-site detection of three tobacco viruses simultaneously.