Thermal activation of Argonaute nuclease enables one-pot multiplex detection of viruses

Advances in programmable nucleases such as CRISPR-associated proteins (Cas) and Argonaute (Ago), combined with isothermal amplification, have made point-of-care testing more accessible than before. However, the non-specific binding of the nuclease to nucleic acid results in incompatibility issues between the amplification and nuclease systems, substantially limiting the feasibility of one-step workflow. Herein, we propose a temperature control solution based on thermotolerant Pyrococcus furiosus Ago (PfAgo) beads. The use of immobilized PfAgo can effectively prevent interference with loop-mediated isothermal amplification at 65 °C and accurately identifies amplicons when activated at 95 °C. Following optimization, a sensitivity of 0.6 copies/μL was achieved within 45 min, and the system demonstrated high specificity with no cross-reactivity with 21 other pathogens. Additionally, multiplex detection was designed for herpesvirus sensing, with 86.4% and 100% agreement for positive and negative samples, respectively. Our research presents an effective one-pot method for combining amplification and cleavage through the use of thermo-controllable nucleases, significantly improving the clinical applicability of diagnostic techniques dependent on programmable nucleases.