Dual-Channel Detection System Using Pyrococcus furiosus Argonaute for Single-Nucleotide Variant Quantification in Rice

A dual-channel detection system using Pyrococcus furiosus Argonaute (PfAgo) was developed for the quantification of single-nucleotide variants (SNVs) in genome-edited rice. The proposed approach advanced PfAgo-based detection by introducing universal guide DNA (gDNA) that simplified the system through simultaneous recognition of both wild-type and SNV targets. It achieved precise dual quantification of single-base differences without cross-interference via a rationally designed mismatch in the molecular beacons. Under the optimal conditions, the developed method exhibited exceptional sensitivity with a detection limit of 0.1% for SNV mutant, while maintaining single-nucleotide discrimination capability across various genome-edited rice variants. In blinded sample tests at 5%-10%, the system exhibited robust performance with relative standard deviation below 6% and bias within ± 8%, confirming its quantitative reliability. The proposed approach integrates high sensitivity, excellent specificity, and precise quantification, thereby providing a powerful analytical platform for traceability and quality control of genome-edited products.