A point-of-care testing device for monkeypox virus integrating recombinase polymerase amplification and lateral flow assay.

BACKGROUND: The global spread of monkeypox virus (MPXV) underscores the need for rapid, accessible diagnostic tools to control outbreaks. Current diagnostics, relying on centralized laboratory testing, require specialized personnel and equipment. We aimed to develop a point-of-care testing (POCT) kit that simplifies nucleic acid extraction, minimizes contamination, and enables immediate, accurate MPXV detection. METHODS: We introduced a novel MPXV POCT kit, featuring a separate MPXV DNA rapid release step and a closed integrated device combining recombinase polymerase amplification (RPA) and lateral flow assay (LFA). A fluorescent RPA assay identified F3L-targeting primers and probes with superior amplification efficiency. The RPA system was coupled with LFA to assess the visual diagnostic performance, and then integrated into a POCT device, complemented by a nucleic acid release agent to form the kit. The kit's specificity, sensitivity, and diagnostic performance were validated through clinical body fluid samples. RESULTS: Our nucleic acid release agent streamlines sample processing without compromising detection accuracy, eliminating the need for traditional nucleic acid extraction. The RPA reaction system was formulated into freeze-dried microspheres and integrated directly into the reaction chamber of the POCT device, ensuring the stability and shelf-life of the reagents and simplifying the preparation and configuration process. The POCT device's enclosed design eradicates transfer steps, significantly reducing RPA-induced aerosol contamination. Results can be visually interpreted within 25 min, a substantial improvement over existing methods. In clinical sample validation, the POCT kit showed high specificity and sensitivity, distinguishing other pathogens, with 100% consistency with RT-qPCR results in detecting mpox patient samples. Compared to RT-qPCR and ddPCR, our POCT kit demonstrates stronger diagnostic performance, with the lowest positive concentration we detected being 41 copies/mL (2.1 copies/reaction) in clinical samples and 2.5 copies/μL (2.5 copies/reaction) of DNA plasmid standards, significantly increasing the detection rate of mpox. CONCLUSIONS: This compact, portable POCT kit, requiring no professional operation or precision instruments, offers a promising solution for on-site and home self-testing. Its high sensitivity and specificity, coupled with the capacity to swiftly adapt to new MPXV strains, positions it as an effective tool for early detection, vital for controlling mpox outbreaks.