Pyrococcus furiosus Argonaute-Mediated Dual-Recognition Enables the Detection of Trace Single-nucleotide-Mutated Fungicide-resistant Fungal Pathogens

The emergence of fungicide resistance in fungal pathogens, often caused by single-nucleotide mutations (SNMs) in target genes, poses a significant threat to global food security. Current detection methods for these mutations face challenges in terms of sensitivity, specificity, and simplicity. Herein, we developed a Pyrococcus furiosus Argonaute (PfAgo)-mediated dual-recognition biosensor for the accurate and sensitive detection of SNM-containing DNA fragments. This biosensor utilizes the unique ability of PfAgo to recognize both the DNA guide strand and the target DNA sequence, enabling a dual-recognition mechanism that significantly enhances specificity. By integrating this with a simple colorimetric assay, we achieved a limit of detection (LOD) of 0.1 fM for SNM-containing DNA. The method was successfully applied to detect fungicide-resistant genes in real samples of Botrytis cinerea, demonstrating its potential for practical applications. This PfAgo-based biosensor provides a promising tool for the early diagnosis and monitoring of fungicide-resistant fungal pathogens, contributing to sustainable agriculture.