Visual Detection of Multiple Fusarium Species via RAA-CRISPR/Cas12a Dual Fluorescence–Lateral Flow Assay

Fusarium verticillioides, F. graminearum , and F. oxysporum are plant pathogens that produce harmful mycotoxins and severely impact crop yields. Given their widespread distribution and threat to agricultural production, there is an urgent need for rapid and accurate detection methods to improve monitoring and mitigation strategies. This study presents a highly sensitive system for detecting Fusarium species that integrates CRISPR/Cas12a-based nucleic acid diagnostics and recombinase-aided amplification (RAA). A triple-labelled single-stranded DNA reporter (6-carboxyfluorescein, black hole quencher, and biotin) enables real-time naked-eye observation of fluorescence during the Cas12a reaction, followed by visual confirmation via lateral flow strip (LFS). The optimised dual-mode system achieves a genus-level detection limit of 0.3 pg genomic DNA within 1 hour, with consistent results in both fluorescence and LFS modes. Species-specific detection for F. verticillioides, F. graminearum , and F. oxysporum reached limits of 3 pg per reaction. These sensitivities surpass conventional PCR (10–100 pg in 2–3 hours) and are comparable to TaqMan qPCR (1–5 pg in 2–3 hours), without requiring sophisticated instrumentation. The specificity, sensitivity, and speed of this dual-mode detection system were validated using simulated field samples, demonstrating its applicability to Fusarium screening and surveillance in resource-limited settings.