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

UNLABELLED

Cardamom mosaic virus causing mosaic/ 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 dilutions of the crude extract. The assay was validated using field samples collected from different cardamom-growing regions of Kerala and Karnataka, India.

SUPPLEMENTARY INFORMATION

The online version contains supplementary material available at 10.1007/s13205-024-04191-4.