Amplification-Free Ribonucleic Acid Detection via Cas13−Csm6−Horseradish Peroxidase Cascade with Single-Molecule Sensitivity

Achieving a single-molecule level for RNA detection remains a significant challenge in point-of-care settings. We present CCHP, a Cas13-Csm6-horseradish peroxidase three-step enzymatic cascade assay that enables amplification-free RNA detection with a simple colorimetric readout at 450 nm within 1 h under standard conditions. Across diverse viral and non-coding targets, CCHP spans a dynamic range of 10-10⁷ copies per reaction, showing strong agreement with RT-qPCR on serial dilutions (|<i>r</i>| ≈ 0.95). In a small blinded feasibility comparison using extracted RNA from infected versus uninfected cells, CCHP showed concordance with the reference method (κ = 1.0). In representative matrices spiked with synthetic RNA and without nucleic acid extraction or purification, positive events were observed near the single-molecule input level for H1N1-NP in oropharyngeal swabs and for SARS-CoV-2-N in oropharyngeal swabs and plasma, consistent with stochastic initiation of the cascade by single-molecule inputs under Poisson-limited conditions. Under these conditions, the assay maintains stable low-copy detection performance. A physics-guided minimal model integrating deterministic kinetics and stochastic partition statistics informed assay design and sensitivity projection, which were subsequently validated experimentally. By coupling single-molecule-level initiation with ultralow-copy detection and operational simplicity, CCHP advances point-of-care RNA diagnostics and surveillance.