The kit achieves a 100% consistency with RT-qPCR results in detecting mpox patient samples.
The kit simplifies nucleic acid extraction, eliminating traditional methods without compromising accuracy.
Results can be visually interpreted within 25 minutes, significantly faster than existing diagnostic methods.
The POCT device's enclosed design reduces contamination risks associated with sample transfer.
In clinical validations, the kit demonstrated high specificity and sensitivity, distinguishing MPXV from other pathogens.
It effectively detects MPXV at concentrations as low as 41 copies/mL in clinical samples.
Simplified
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 (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 (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.
Key numbers
41 copies/mL
Lowest Positive Concentration Detected
Minimum concentration detected in clinical samples.
25 min
Time to Result
Time required for visual interpretation of results.
81 of 121
Sample Cohort Size
Number of mpox patient samples used for validation.
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Declarations. Ethics approval and consent to participate: This study complies with all ethical regulations and has been approved by the Ethics Committee of the Guangzhou Center for Disease Control and Prevention (Guangzhou CDC) (Ethical NO. GZCDC-ECHR-2023P0035). According to the “Diagnosis and Treatment Guidelines for Monkeypox (2022 Edition)” released by the National Health Commission of China, the Guangzhou CDC collects and processes clinical samples from patients with mpox with their informed consent. This study does not involve any epidemiological information related to patient privacy, and the testing of clinical samples has no impact on patients. Consent for publication: Not applicable. Competing interests: The authors declare no competing interests.
