A rapid and sensitive detection assay of grapevine virus L using real-time fluorescence recombinase polymerase amplification based on RNA and crude extracts.

Grapevine virus L (GVL) is an emerging vitivirus with global distribution and potential economic impact due to frequent co-infections. In this study, a highly sensitive and specific one-step reverse transcription-recombinase polymerase amplification coupled with an exo probe (RT-Exo-RPA) assay was developed for the rapid detection of GVL. The optimized assay achieved amplification at 41 °C within 15 min, with a sensitivity 10,000-fold higher than conventional RT-PCR and no cross-reactivity with other grapevine viruses. The method demonstrated superior performance in detecting GVL across field grapevine samples from different seasons and positions, yielding a 96.6% (112/116) positive detection rate compared to 68.9% (80/116) by RT-PCR. Additionally, RT-Exo-RPA exhibited a slightly higher detection rate than conventional RT-PCR across 60 samples from major viticultural areas in Liaoning Province. Importantly, all samples that tested positive with RT-PCR also yielded positive results with this method, confirming its accuracy. Furthermore, we established a simplified protocol using a NaOH-EDTA buffer for the preparation of plant crude extract, enabling direct detection within 15 min, including sample processing. This field-deployable approach was achieved with an inexpensive and simple portable device, showing promise for Point-of-Care testing. Overall, the developed RT-Exo-RPA assay offers a robust, rapid, and cost-effective solution for early diagnosis and certification of GVL in vineyards.