Rapid onsite detection of piper yellow mottle virus infecting black pepper by recombinase polymerase amplification-lateral flow assay (RPA-LFA)

Abstract

Piper yellow mottle virus (PYMoV) is a pararetrovirus associated with stunt disease in black pepper. As the primary spread of the virus occurs through vegetative propagation, effective diagnostics are required for the production of virus-free plants. Currently available assays are time-consuming, require expensive equipment, and are not suitable for on-site detection. In the present study, two rapid assays based on the recombinase polymerase amplification (RPA) coupled with lateral flow assay (LFA) using (i) 6-carboxyfluorescein (FAM) labeled nfo probe and biotin-labeled reverse primer and (ii) FAM labeled forward and biotin-labeled reverse primer was developed for the detection of PYMoV. The assays were performed using TwistAmp DNA amplification reagents and crude extract from the infected plant and mealybug as templates. Both assays were optimized for parameters like concentration of magnesium acetate, temperature, and time. The RPA product was then diluted and applied to the sample pad of a lateral flow device for visualizing the results. The formation of a colored line at the test line was considered positive for PYMoV. The entire process from sample preparation to visualization of results could be completed in about 30 min. The developed assays were specific and 10 times more sensitive than PCR. The assays were validated using field samples of black pepper and mealybug vectors.