Abstract
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.