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.

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.

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