A cleavage-gated terminal exposure-driven CRISPR-RCA self-amplifying system for ultra-fast DNA detection.

The one-pot detection technology based on nucleic acid isothermal amplification combined with CRISPR has a significant advantage in on-site detection of infectious diseases. It is superior to quantitative polymerase chain reaction (qPCR) due to its lack of temperature variation and significantly faster reaction speed. Nevertheless, Cas proteins compete with amplification enzymes for reaction substrates, which results in the signal amplification effect being less than expected. To overcome this limitation, we have developed a single-tube self-amplifying system driven by cleavage-gated terminal exposure based on CRISPR and rolling circle amplification (AURORA), enabling ultra-fast and sensitive monkeypox virus (MPXV) detection. This method innovatively designs a dual-function probe (DF probe). In the presence of the target, the trans-cleavage activity of Cas12a is activated, only cleaving the single-stranded DNA to expose the 3' terminal of the DF probe. The cleaved DF probe hybridizes with the circular DNA template and is bound by phi29 DNA polymerase to initiate RCA. Here, we utilize the characteristics of Cas12a and phi29 DNA polymerase acting on substrates in different strand states to avoid substrate competition between the two enzymes in a single-tube reaction. This assay can achieve ultra-fast signal amplification of MPXV DNA within 8 min, with a limit of detection (LOD) of 88 aM (53 copies/μL). Combining the viral nucleic acid thermal lysis method, it is possible to achieve results from sample input to output in 10 min. The AURORA detection strategy was further used to detect MPXV in clinical samples (36 MPXV samples), and the results were completely consistent with qPCR. The AURORA system features ultra-fast and precise detection, providing a more efficient tool for the prevention and control of severe infectious diseases.