First Report of On-Site Detection of Cucurbit Leaf Crumple Virus by an Optimized RPA-Lateral Flow Assay with an Alternative Endonuclease

Rapid, simple, and robust diagnostics are essential for effectively controlling the spread of plant viruses and mitigating their impact. Although recombinase polymerase amplification-lateral flow test (RPA-LFT) diagnostics currently offer high sensitivity and specificity, they rely on the Nfo endonuclease enzyme and require an expensive heat block. In this study, we present the development of a molecular diagnostic test for cucurbit leaf crumple virus (CuLCrV) using an RPA-LFT assay that employs an alternative endonuclease enzyme instead of Nfo. This alternative endonuclease demonstrates comparable functionality to Nfo and achieves a detection limit of 10 viral copies in plant samples and whiteflies. The assay can be performed using a battery-powered mini heat block, ensuring scalability and cost-effectiveness. Notably, the unavailability of commercially accessible Nfo endonuclease enzymes underscores the necessity for an alternative enzyme for RPA-LFT assay development. The RPA-LFT assay eliminates the need for nucleic acid purification and provides results within approximately 30 min from sample collection. The integration of this new endonuclease into the RPA-LFT assay represents an advancement towards on-site detection of plant viruses, enabling early-stage management of viral infections.