A Rapid On-Site Visualization Detection System for <i>Fusarium oxysporum</i> f. sp. <i>cubense</i> Tropical Race 4 Utilizing RPA-CRISPR/Cas12a

Fusarium wilt of banana, a disease with devastating impacts on banana plants, is primarily caused by a pathogenic fungus called <i>Fusarium oxysporum</i> f. sp. <i>cubense</i> tropical race 4 (<i>Foc</i> TR4). To facilitate control and interrupt the spread of Fusarium wilt of banana, we developed a rapid on-site visualization system for detecting <i>Foc</i> TR4 based on recombinase polymerase amplification (RPA) combined with CRISPR/Cas12a. Based on <i>Foc</i> TR4-specific sequences, primers, crRNA, and ssDNAs were designed. The detection system exhibited high specificity, with amplification signals observed exclusively in samples containing <i>Foc</i> TR4. Additionally, the system showed high sensitivity, with a limit of detection (LOD) of approximately 20 copies, and high efficiency, with detection results generated within 1 h from time of amplification. Notably, this method does not require large-scale instruments, making it a convenient and rapid approach. The detection system represents the first instance of on-site visualization of <i>Foc</i> TR4. This system can be employed for early detection of <i>Foc</i> TR4 and can provide a technical reference for the rapid detection and applications in the field of other pathogens.