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

Abstract 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.