Development of an isothermal reverse transcriptase-recombinase polymerase amplification assay for the rapid diagnosis of four viroids in different grape cultivars

Rapid, isothermal detection is possible using recombinase polymerase amplification assay (RPA) offering great specificity plus sensitivity. An RPA assay was standardized and evaluated to detect four viroids using RNA from infected grapevine plants. From the sequences of the whole genome of four viroids, four pairs of oligonucleotide primers were created. The four viroids employed in this study included Grapevine yellow speckle viroid 1 and 2 (GYSVd-1; GYSVd-2), Australian grapevine viroid (AGVd) and Hop stunt viroid (HSVd). The primers were tested to function both in RPA in addition to polymerase chain reaction (PCR). Applying the RT-RPA assay, the presence of four viroids was determined in a total of 119 symptomatic and asymptomatic grapevine leaf samples from several 22 genotypes obtained from the various regions of India. The results of RT-RPA were compatible with RT-PCR-based detection utilising pure RNA generated cDNA as a template. GYSVd-2 was effectively detected using crude leaf sap in RPA as well. To our awareness, this is the earliest report of consistent diagnosis of most grapevine viroids infection by RT-RPA using RNA as well as crude leaf sap as a template.