Development of a Recombinase Polymerase Amplification and CRISPR-Cas12a-Based Assay for Rapid Detection of Rice Bakanae Disease Caused by <i>Fusarium fujikuroi</i>
2025
Hongyu Li | Yue Qiu | Anpeng Zhang | Yingxiong Hu | Can Cheng | Jihua Zhou | Fuan Niu | Bin Sun | Yuting Dai | Kaizhen Xie | Zhizun Feng | Xiaorui Ding | Bilian Hu | Xueqing Zhang | Liming Cao | Huangwei Chu
<i>Fusarium fujikuroi</i> 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 <i>F. fujikuroi</i> 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 <i>F. fujikuroi</i>. This assay demonstrated a limit of detection (LOD) of 1 copy/μL of reference plasmid or 0.1 fg/μL of <i>F. fujikuroi</i> 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 <i>F. fujikuroi</i> gDNA. The RPA-Cas12a-based assays developed in this study enable rapid, highly accurate, sensitive, and specific detection of <i>F. fujikuroi</i>, making them a promising tool for on-site detection without the need for expensive equipment and time-consuming methodologies.
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