# Simplified Sample Preparation and Lateral Flow Immunoassay for the Detection of Plant Viruses

## TL;DR

A simplified lateral flow immunoassay with an easy sample preparation method enables rapid and sensitive detection of plant viruses like cowpea chlorotic mottle virus in the field.

## Contribution

The paper introduces a simplified dipstick LFA format and an on-site sampling method for plant virus detection without laboratory equipment.

## Key findings

- The assay can detect CCMV at concentrations as low as 3.5 µg/L in 15 minutes.
- The new sampling method combines grinding, extraction, filtration, and reconstitution using a manual punch and syringe.
- The assay detected systemic CCMV infection in cowpea plants before visual symptoms appeared.

## Abstract

Lateral flow immunoassays (LFAs) are widely used for on-site testing; however, their use for the rapid detection of plant viruses in the field is often limited by inconvenient sample preparation. Here, we present a new sampling method and a simplified dipstick LFA format for the detection and monitoring of cowpea chlorotic mottle virus (CCMV) as a model plant pathogen. The assay employs a monoclonal mouse antibody for capture and a poly-clonal rabbit antibody conjugated to 80 nm gold nanoparticles for detection. Conventional sample and conjugate pads are omitted, allowing the test strips to be dipped directly into wells containing plant extract and antibody–gold conjugate. No plastic casing was required, which could lead to a reduction in waste. It was shown that CCMV concentrations as low as 3.5 µg/L or 350 pg per sample could be reliably detected in 15 min. Specificity tests confirmed that other plant viruses, cowpea mosaic virus (CPMV) and tobacco mosaic virus (TMV), did not produce false-positive results. In addition, we describe a new method for on-site sampling using a manual punch and a syringe equipped with a frit. This step combines grinding the sample, extraction, filtration, and reconstitution and mixing of the antibody-gold conjugate, enabling the analysis of punched leaf disks without laboratory equipment. When applied to CCMV-infected cowpea plants, the assay revealed systemic infection before visual symptoms became apparent. This work demonstrates that simplified LFAs combined with innovative sampling techniques can provide sensitive, specific, and rapid diagnostics for crop monitoring and support early intervention strategies in agriculture.