Rapid detection of cardamom mosaic virus in crude plant extracts using reverse transcription-recombinase polymerase amplification-lateral flow assay (RT-RPA-LFA)

Cardamom mosaic virus causing mosaic/katte disease is the most destructive virus infecting cardamom. The development of effective diagnostic assays is essential for the production of virus-free plants, as the primary spread of the virus occurs through vegetative propagation. Currently used PCR-based assays are not suitable for Point-of-Care testing, require sophisticated equipment, and are time-consuming. Hence, in the present study, an assay based on reverse transcription-recombinase polymerase amplification (RT-RPA) combined with lateral flow assay (RT-RPA-LFA) was optimized for the specific, and sensitive detection of CdMV. The forward and reverse primers selected for RT-RPA were labeled with 6-carboxyfluorescein (FAM) and biotin respectively at the 5´end. The tedious total RNA preparation was avoided by using the crude extract as a template for the assay. A magnesium acetate concentration of 14 mM, 0.4 M betaine, temperature from 37 to 42 ℃, and 20 min of incubation time were found optimum for the assay. The entire RT-RPA-LFA from sample preparation to visualization of results could be completed within 40–50 min and the assay is suitable for Point-of-Care testing. The assay is specific for CdMV and could detect the virus up to 10^–5 dilutions of the crude extract. The assay was validated using field samples collected from different cardamom-growing regions of Kerala and Karnataka, India.