Field-deployable seed health diagnostic assay: RT-RPA-based detection of turnip mosaic virus in mustard leaf and seed lots

Abstract

Turnip mosaic virus (TuMV) is a major constraint in mustard production, with emerging evidence of its seed-borne nature posing significant risks for long-distance dissemination and disease outbreaks. Rapid and sensitive detection methods tailored for seed health diagnostics are therefore critically needed. In the present study, a reverse transcription recombinase polymerase amplification (RT-RPA) assay targeting the coat protein (CP) gene of TuMV was optimized across multiple mustard tissues, including mature seeds. The assay was standardized at 42 °C for 25 min, and it demonstrated high analytical sensitivity, detecting viral RNA up to 10 fg dilution, particularly from crude extracts, outperforming conventional RT-PCR by 2–3 orders of magnitude. A simplified crude sap extraction using 0.1 M phosphate buffer enabled direct amplification without RNA purification, and assay compatibility with hydroxynaphthol blue (HNB) allowed visual, colorimetric detection suitable for field applications. Validation across diverse genotypes and field samples showed complete concordance of RT-RPA with RT-PCR. To establish practical seed health testing thresholds, seed pool optimization was performed using two mustard varieties differing in seed size. RT-RPA results were quantitatively validated using RT-qPCR, revealing strong agreement between qualitative and quantitative detection. Reliable detection was achieved with crude seed extracts of as few as 2–6 seeds in large-seeded genotypes and 15–25 seeds in small-seeded genotypes, highlighting the influence of seed size on detection sensitivity. This study developed and validated the first crude extract-based RT-RPA assay for detecting TuMV infection in mustard plant parts, including seed samples. It provides an ISTA (International Seed Testing Association)-compatible framework for rapid, sensitive, and field-deployable seed health diagnostics. This study delivers a crude extract based RT-RPA, with RT-qPCR validation, providing a rapid, field-deployable framework for the sensitive detection of turnip mosaic virus in seed-lot testing of mustard.