A crude leaf extract-based RT-RPA assay for rapid detection of onion yellow dwarf virus in Allium crops

Garlic and onion, key Allium crops, are cultivated globally for culinary and medicinal purposes. Onion yellow dwarf virus (OYDV) is a major pathogen affecting these crops, causing leaf crinkling, yellow striping, dwarfing, and reduced yield. To facilitate efficient detection, a rapid, reliable, and cost-effective crude leaf extract-based reverse transcription recombinase polymerase amplification (RT-RPA) assay was developed. This method eliminates the need for labour-intensive RNA isolation, cDNA synthesis, and expensive thermal cycler used in PCR/RT-PCR based assay. The optimized RT-RPA assay showed comparable sensitivity to RT-PCR, detecting OYDV up to 0.1 pg/μl, or 10^-6 using RNA or crude leaf extract. It exhibited high specificity, with no cross-reactions with other Allium viruses. This study presents the first crude leaf extract-based RT-RPA test for OYDV detection, offering a valuable tool for routine indexing, biosecurity, and resistance screening.