Recombinase Polymerase Amplification assay for rapid detection of a geminivirus associated with potato apical leaf curl disease

Recombinase Polymerase Amplification (RPA) assay was developed for specific detection of Tomato leaf curl New Delhi virus-potato (ToLCNDV [potato]), causing potato apical leaf curl disease. The RPA assay was optimized with respect to its isothermal temperature, post-amplification treatment and its components of the reaction mix. The assay showed a clear and sharp expected amplicon at 40 °C for 30 min in the thermal cycle followed by post-amplification denaturation, i.e., heat treatment at 65 °C for 10 min. The target specificity of the developed assay was determined by sequencing the amplified product. Pairwise sequence alignment showed 73 numbers of significant hits with ToLCNDV [potato] isolates within the taxonomy of begomovirus with query coverage of 100% with 99% identity indicating that the assay is specific to ToLCNDV [potato]. The comparative sensitivity of the assay was ten times more sensitive than PCR. It was successfully applied for the detection of ToLCNDV [potato] from potato leaf samples, tissue culture raised plants and also from tuber and sprouts. The assay was proved to be rapid, sensitive, highly specific and suitable in diagnostic laboratories for virus indexing and selection of virus-free planting material. To the best of our knowledge, this is the first report of the development and application of RPA assay for the detection of ToLCNDV [potato].