Abstract
The global outbreak of the monkeypox virus (MPXV) highlights the need for rapid and cost-effective MPXV detection tools to effectively monitor and control the monkeypox disease. Herein, we demonstrated a portable CRISPR-Cas-based system for naked-eye detection of MPXV. The system harnesses the high selectivity of CRISPR-Cas12 and the isothermal nucleic acid amplification potential of recombinase polymerase amplification (RPA). It can detect both the current circulating MPXV clade and the original clades. We reached an LoD of 22.4 aM (13.5 copies/µL) using a microtiter plate reader, while the visual LoD of the system is 75 aM (45 copies/µL) in a two-step assay, which is further reduced to 25 aM (15 copies/µL) in a one-pot system. We compared our results with quantitative (q) PCR and obtained satisfactory consistency. For clinical application, we demonstrated a sensitive and precise visual detection method with attomolar sensitivity and a sample-to-answer time of 35 minutes.
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