Development of a Recombinase Polymerase Amplification and CRISPR-Cas12a-Based Assay for Rapid Detection of Rice Bakanae Disease Caused by Fusarium fujikuroi
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
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/&mu:L of reference plasmid or 0.1 fg/&mu:L 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/&mu:L of plasmid or 10 fg/&mu:L 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.
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