A field‐deployable <scp>RPA</scp> ‐ <scp>CRISPR</scp> /Cas12a dual‐mode assay for rapid detection of <i>Fusarium oxysporum</i> in <i>Nicotiana tabacum</i>

BACKGROUND: Fusarium oxysporum is a devastating soil-borne pathogen that causes severe economic losses in tobacco and other crops, necessitating rapid and accurate detection methods for effective disease management. Herein, we developed a dual-mode RPA-Cas12a platform incorporating both lateral flow dipstick (LFD) and fluorescence-based detection (FBD), targeting the CYP51C gene to enable rapid and equipment-facilitated detection of F. oxysporum under isothermal conditions. RESULTS: The entire assay from sample to result can be completed within 68 min. The platform offers two complementary detection formats. The LFD mode provides visual qualitative results with a detection limit of 360 copies, making it ideally suited for rapid on-site screening. In contrast, the FBD mode achieves a detection limit of 3.6 copies, demonstrating 100-fold higher sensitivity and enabling precise quantitative analysis. CONCLUSION: The assay successfully identified F. oxysporum in both artificially inoculated and field-collected tobacco samples, showing high concordance with fluorescence intensity. This work provides a sensitive, rapid, and practical diagnostic tool for the on-site detection and monitoring of tobacco root rot caused by F. oxysporum. © 2026 Society of Chemical Industry.