CRISPR/Cas12a-RPA integrated assay for potential rapid detection of Fusarium spp.

Timely detection and accurate identification of crop pathogens are considered fundamental components of sustainable agricultural systems alarming prompt and effective crop protection management. The cutting-edge CRISPR/Cas12a technology is emerging as a nucleic-acid-based platform with high potential for rapid, sensitive, and specific phytopathogen detection. In this study, we aimed to develop a CRISPR/Cas12a-based method to detect Fusarium spp., targeting the acl1 gene. A plasmid carrying the target gene fragment was successfully amplified using Recombinase Polymerase Amplification (RPA), confirming the feasibility of integrating this isothermal amplification with the CRISPR/Cas12a detection method. Optimization of the CRISPR-Cas12a reaction revealed optimal conditions at 100 nM MbCas12a, 100 nM crRNA, and 5 µM ssDNA reporter. Sensitivity assays demonstrated reliable detection of the acl1 gene up to 1:1000 dilution. Specificity evaluation involving different plant-pathogenic fungal species confirmed the high specificity of the developed method. Altogether, this study highlights the potential of the CRISPR/Cas12a-based system as a promising diagnostic approach for agricultural applications to detect Fusarium spp.