Point-of-Care Diagnosis of Respiratory Viruses at Single-Nucleotide Resolution with an Autocatalytic Rolling Circle Amplification System.

Amid the continuing epidemic of respiratory viruses (RVs), clinical diagnostics need scalable technologies that enable point-of-care testing of pathogenic viruses with high accuracy and specificity. Here, we propose an autocatalytic rolling circle amplification (A-RCA) system for the precise identification of RV genomic markers through sequence-specific target recognition and synergistic catalytic signal amplification. The system, which we incorporated into lateral-flow strip tests, exploits the cross-stimulation between deoxyribonuclease (DNAzyme) self-cleavage reaction and rolling circle amplification (RCA) to exponentially amplify the recognition of virus RNA sequences for the visually perceptible signal transduction. With superior amplification efficiency, this innovative autocatalysis-driven artificial reaction system facilitates the rapid and robust detection of various viral RNAs, including those of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), influenza A, and influenza B, with single-nucleotide resolution, offering a portable diagnostic platform for unsupervised self-examination of different RVs and their mutants. This modular A-RCA system is adaptable for multiplexed viral RNA profiling in clinical nasopharyngeal swabs and implemented on lateral-flow strip assays for point-of-care (POC) diagnostics. The accessibility and programmability of the A-RCA system make it a promising candidate for epidemiological surveillance in resource-limited settings, facilitating the prompt response to emerging RVs and the prevention of viral pandemics.