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

## TL;DR

Researchers developed a new, cost-effective test for detecting a plant virus using an alternative enzyme, enabling quick on-site diagnosis.

## Contribution

The study introduces an optimized RPA-LFT assay using an alternative endonuclease for on-site detection of CuLCrV.

## Key findings

- The RPA-LFT assay achieves a detection limit of 10 viral copies in plant samples and whiteflies.
- The assay uses a battery-powered mini heat block, making it scalable and cost-effective.
- The alternative endonuclease performs comparably to Nfo and eliminates the need for nucleic acid purification.

## Abstract

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.