Rapid and visual field diagnosis of tomato brown rugose fruit virus using reverse transcription recombinase aided amplification (RT RAA) combined with lateral flow strips (LFS)

Tomato brown rugose fruit virus (ToBRFV) causes significant disease outbreaks in tomato and pepper production areas worldwide. A simple, fast, and visual detection method for ToBRFV is critical for controlling the viral disease. Here, a method was constructed to detect ToBRFV by combining reverse transcription recombinase-assisted amplification (RT-RAA) and lateral flow test strips (LFS). After selecting the primers and probe, the concentration of the modified reverse primer and magnesium acetate in the RT RAA-LFS assay were optimized. The RT RAA could amplify the target sequence in 16 minutes at a constant temperature of 39 °C, and the amplification products could be visualized by LFS within five minutes. Importantly, there was no cross-reaction with other viruses that can infect tomatoes, such as tobacco mosaic virus, tomato mild mottle virus, pepper mild mottle virus, tobacco mild green mosaic virus, broad bean wilt virus, pepper vein yellows virus, and chilli veinal mottle virus. Furthermore, the RT RAA-LFS assay was highly sensitive, with a detection limit of 2.1×101 copies/50-μL reaction. Using 138 field samples and 6 commercial seeds with suspected infection of ToBRFV, the RT RAA-LFS and RT-PCR methods gave identical results, indicating that the RT RAA-LFS method is as reliable as the standard RT-PCR method for detecting ToBRFV. Our RT RAA-LFS assay could be a valuable tool for the rapid and accurate detection of ToBRFV.