Rapid detection assay for infectious tobamovirus in seeds using PMA-RT-LAMP

Overview

The rapid detection assay for infectious Tobamovirus in seeds employs a novel approach using propidium monoazide (PMA) treatment combined with reverse transcription loop-mediated isothermal amplification (RT-LAMP). This solution addresses the challenge of differentiating between infectious and non-infectious virus particles, which traditional methods fail to do, often leading to false positives. This rapid assay provides results within 60 to 90 minutes, making it ideal for seed certification, quarantine screening, and phytosanitary programs.

Technical specifications

This assay utilizes PMA to bind RNA from non-infectious Tobamovirus particles, preventing their amplification during RT-LAMP. By targeting the coat protein gene of the virus, the method ensures specific detection of only viable virus particles. The process involves treating seed extracts with PMA, followed by light exposure to crosslink non-viable RNA, and then lysing the samples for RT-LAMP analysis. Key features include high sensitivity and specificity, compatibility with various seed matrices, and suitability for high-throughput screening without the need for RNA purification or thermocyclers.

Technology readiness level

The technology is at TRL 3, having been validated in controlled laboratory settings. Future validation steps include benchmarking against RT-qPCR and traditional grow-out bioassays using both spiked and naturally infected seed samples. The development of a standard operating procedure will ensure reproducibility and robustness across different operators and technical replicates.