Refinement of RNA purification significantly enhances detection of potato viruses from tuber extracts

Potato viruses substantially reduce seed quality and crop yield, with six principal viruses posing the greatest threats to global potato production. Conventionally, the procedure consists of a grow-out test followed by DAS‑ELISA, which facilitates reliable detection of viruses. However, it is time-consuming and costly. This study attempted to optimize RNA purification protocols for consistent molecular detection of potato viruses in tuber tissue. Five commercial RNA isolation kits representing six extraction workflows, together with two non‑column procedures, were evaluated. RNA yield, purity, and integrity were measured using spectrophotometry, fluorimetry, and capillary electrophoresis, while functional RNA quality was assessed by RT‑qPCR targeting viral and potato cytochrome oxidase ( StCOX ) mRNAs. Selected protocols were further evaluated for reproducibility, compatibility with automation, and RNA stability during refrigerated storage. Automated magnetic particle‑based isolation demonstrated high repeatability and throughput, indicating suitability for routine diagnostic use. Analysis of refrigerated RNA samples showed a progressive, transcript‑dependent decline in RT‑qPCR detectability over 14 days, yet remaining reliably detectable by RT‑qPCR during the first 48 h, particularly in samples with sufficient initial viral load. To summarize, optimized RNA extraction workflows allow robust, high‑throughput molecular detection of potato viruses directly from tubers. These protocols offer a feasible alternative to labor‑intensive grow‑out assays and facilitate the adoption of molecular testing in potato seed certification and phytosanitary monitoring programs.