Respiratory virus detection is a prominent area of molecular diagnostics, yet current clinical diagnostic methods lack ultra-rapid and highly sensitive detection capabilities. Here, we reported an ultra-rapid, one-pot isothermal assay called "ADNA-initiated CRISPR-Cas12a-mediated RCA cycle" (ACRE). ACRE was developed through computational studying, engineered design of nucleic acid, and enzyme kinetics analysis, combining rolling circle amplification (RCA) with Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas12a system for respiratory virus detection. The assay involved an engineered assistant DNA (ADNA) initiation reaction, followed by leveraging the cis-cleavage activity of Cas12a coupled with an engineered Padlock, which converted the linear RCA into the RCA cycle. When the RCA cycle is continuously in operation, the trans-cleavage activity of Cas12a facilitates both signal output and amplification. The limit of detection (LOD) for three respiratory viruses (SARS-CoV-2, Inf A, and Inf B) was as low as several hundred attomoles (751 aM, 3.7 fM, and 863 aM), with single-nucleotide specificity. Remarkably, the current assay can detect targets with concentrations above 10 pM within 2.5 min, without the reverse transcription step or specialized instrumentation. Given its exceptional speed, sensitivity, and specificity, ACRE could serve as a robust assay for detecting respiratory virus, enabling molecular diagnostics in clinical settings.
