One-Pot RPA-CRISPR/Cas12a Assay With Visual Readout for the Ultra-Specific Detection of Monkeypox Virus Clade I

In 2024, Monkeypox virus (MPXV) clade I has triggered outbreaks in several countries world-wide. MPXV clade I demonstrates enhanced virulence and transmissibility, with a case fatality rate reaching 10%. In response, we have developed a one-pot detection assay specifically targeting MPXV clade I, combining recombinase polymerase amplification (RPA) and the CRISPR/Cas12a system. The assay can be completed within 40 min and achieved a 95% limit of detection (LOD95) of 27.16 copies/μL. No cross-reactivity was observed with MPXV clade II or other tested viral templates, including Vaccinia virus (Tiantan strain). A preliminary room-temperature evaluation showed that the assay retained detectable performance at 25°C, supporting its potential use in equipment-limited settings. The assay also showed good intra-assay and inter-assay repeatability for recombinant plasmid templates, with all coefficient of variation (CV) values below 10%. In simulated clinical samples, the RPA-CRISPR/Cas12a assay detected more low-concentration plasmid-spiked samples than quantitative polymerase chain reaction (qPCR). These results indicate that the established assay is specific, sensitive, repeatable, and easy to perform, providing a practical tool for field-based screening and decentralized detection of MPXV clade I.