Development of a Recombinase Polymerase Amplification and CRISPR-Cas12a-Based Assay for Rapid Detection of Rice Bakanae Disease Caused by Fusarium fujikuroi

Fusarium fujikuroi is the primary causal agent of rice bakanae disease, which can lead to substantial yield losses. Developing a rapid, highly specific, and accurate method for detecting F. fujikuroi is crucial for effective surveillance, prevention, and control of rice bakanae disease. In this study, a novel detection assay, RPA-Cas12a-F, was developed by integrating recombinase polymerase amplification (RPA) and Cas12a for the detection of F. fujikuroi. This assay demonstrated a limit of detection (LOD) of 1 copy/μL of reference plasmid or 0.1 fg/μL of F. fujikuroi genomic DNA (gDNA). Furthermore, to enable on-site detection, the RPA-Cas12a technique was combined with a lateral flow strip (LFS) for visual readout, thereby developing the RPA-Cas12a-LFS assay. The LOD of the RPA-Cas12a-LFS assay was 1000 copies/μL of plasmid or 10 fg/μL of F. fujikuroi gDNA. The RPA-Cas12a-based assays developed in this study enable rapid, highly accurate, sensitive, and specific detection of F. fujikuroi, making them a promising tool for on-site detection without the need for expensive equipment and time-consuming methodologies.