Rapid detection of Broad bean wilt virus 2 and Turnip mosaic virus in Pseudostellaria heterophylla by reverse transcription loop-mediated isothermal amplification assay

Pseudostellaria heterophylla (P. heterophylla) is a widespread Chinese herb that is widely used as medicine, especially for the treatment of lung disease and nourishing the spleen. Broad bean wilt virus 2 (BBWV2) and Turnip mosaic virus (TuMV) threaten the quality and yield of P. heterophylla. To improve the detection efficiency of P. heterophylla viral disease pathogens, a rapid, simple, intuitive, accurate, sensitive and economical reverse transcription loop-mediated isothermal amplification (RT-LAMP) method was established. Primers were designed based on the conserved sequences of BBWV2 and TuMV coat protein nucleotide sequences. The specificity of the primers and sensitivity of the LAMP method to the cDNA template were analysed by real-time fluorescence-based quantitative PCR, SYBR Green I staining, and agarose gel electrophoresis. No cross-reactions occurred with other viruses tested in P. heterophylla. The detection limit of RT-LAMP was 10 pg/mu l and was found to be 10 times more sensitive on comparing with RT-LAMP and reverse transcription-PCR (RT-PCR). Furthermore, the tube-closed dye method facilitated the inspection of the LAMP reaction while avoiding cross-contamination. This detection assay could serve as an important tool for field samples diagnoses and forecasting P. heterophylla viral disease intensities.