Development of a one-step RPA–CRISPR/Cas12a point-of-care detection platform based on poloxamer-mediated temporal-spatial separation strategy and its application in monkeypox virus POCT

Real-time quantitative PCR (qPCR) is the gold standard for pathogen detection, but it relies on professional laboratory conditions and is difficult to meet the on-site application requirements of point-of-care testing (POCT). CRISPR/Cas12a technology offers high specificity, yet its sensitivity alone is limited, necessitating preamplification with recombinase polymerase amplification (RPA). The conventional two‑step recombinase polymerase amplification-clustered regularly interspaced short palindromic repeats (RPA‑CRISPR) method is prone to aerosol contamination, whereas one‑step systems (e.g., SHERLOCKv2) suffer from template competition, leading to a 10‑ to 100‑fold reduction in sensitivity. To address these issues, this study constructed the ChronoGate RPA‑CRISPR platform, which employs a thermosensitive poloxamer to achieve temporal separation of amplification and detection, thereby, establishing a rapid and sensitive point‑of‑care testing method. Poloxamer encapsulates the CRISPR-associated protein 12a (Cas12a)/CRISPR RNA (crRNA) complex at temperatures below 25 °C and releases it at 37 °C with a delay of approximately 8 min, enabling an ordered “amplification‑first, detection‑later” reaction. The optimal reaction conditions were determined to be 15% (w/v) poloxamer in a 15 μL volume. A companion portable fluorescence detector was also developed. Using the monkeypox virus (MPXV) F3L gene as the detection target, sensitivity and specificity were validated by testing serial dilutions of plasmid DNA and nontarget pathogens, followed by analysis of clinical samples. The platform exhibited an encapsulation efficiency of 85.7% and a detection sensitivity of 1 copy/μL, comparable to that of the two‑step method. No cross-reactivity was observed with nontarget pathogens, confirming good specificity. Among 14 clinical samples, the results showed 100% concordance with qPCR, with a detection time of only 40 min, representing a 56% improvement in speed compared with qPCR. The ChronoGate RPA‑CRISPR platform enables rapid, sensitive, and specific single‑tube one‑step detection with simple operation and high portability, providing a reliable POCT solution for monkeypox virus and other pathogens in resource‑limited settings.