A highly sensitive on-site duplex genotyping method dRPG for simultaneous detection of SARS-CoV‑2 key mutations with single nucleotide resolution

Genotyping is an important part in nucleic acid detections, showing great application values in precise medicine, molecular breeding, pathogen detection, and food safety control. Multiplexing is an essential requirement by genotyping, but poses challenges on developing on-site genotyping methods. By combining the highly specific recognition and the multi-target distinguishing ability of the Argonaute protein from Pyrococcus furiosus (PfAgo) with the exponential amplification capacity of recombinase polymerase amplification (RPA), this study has established a novel duplex genotyping method suitable for on-site detection, the duplex RPA-PfAgo Genotyping (dRPG). The method can simultaneously detect two key mutations of SARS-CoV-2, L452R and E484Q, with single nucleotide resolution. The entire detection procedure can be completed within 1.5 hours, and the sensitivity is one copy per reaction for both mutations. This study reports the first multiplex genotyping that combines PfAgo with RPA, and meets the accuracy, sensitivity, and simplicity requirements of on-site genotyping. The dRPG method is expected to serve as an effective tool for long-term tracking of SARS-CoV‑2 mutations, and provides a technological repository for on-site multiplex genotyping of important nucleic acid targets.