One-pot virus detection based on isothermal amplification coupled with temperature-activated argonaute

Abstract

Advances in programmable nucleases like CRISPR-associated protein (Cas) and Argonaute (Ago), combined with isothermal amplification, have made point-of-care testing (POCT) more accessible. However, the specific binding of the nuclease resulted in compatibility issues between the amplification and nuclease systems, substantially limiting the feasibility of a one-step workflow. Here, a temperature control solution based on immobilized thermotolerant Pyrococcus furiosus Ago (PfAgo) has been proposed. The use of immobilized PfAgo can effectively prevent interference with loop-mediated isothermal amplification (LAMP) at 65 degree and accurately identifies amplicons when activated at 95 degree. Following optimization, a sensitivity of 0.6 copies/μL was achieved within 45 minutes, and high specificity was verified with no cross-reactivity among 22 other viruses. Additionally, the multiplex detection was designed for herpes virus sensing, with agreements of 86.4% for positive and 100% for negative samples. Our research presents an effective method for combining amplification and cleavage through the use of controllable nucleases, significantly improving the clinical applicability of diagnostic techniques dependent on programmable nucleases.