CRISPR-Cas12a-based fluorescent and visual assays for universal detection and clade discrimination of mpox virus

The global mpox outbreak has highlighted critical gaps in diagnostic capabilities, particularly the need for methods that can distinguish between the high-fatality Clade I and more transmissible Clade II of the mpox virus (MPXV). Current PCR-based approaches remain reliant on laboratory infrastructure, limiting their use in resource-limited settings. There is an urgent need for a versatile diagnostic platform that can provide both accurate clade discrimination and flexible deployment across diverse healthcare environments. We developed a dual-mode detection platform by integrating recombinase-aided amplification with Clustered Regularly Interspaced Short Palindromic Repeats-Cas12a technology, creating two distinct assays: a universal assay targeting OPG034, and a Clade I-discriminatory assay targeting OPG033. The platform achieved detection sensitivities of 1 copy/reaction for both fluorescence and visual readouts with OPG034, and 10 copies (fluorescence) and 1 copy/reaction (visual) with OPG033. Both assays demonstrated high specificity, successfully distinguishing MPXV from related orthopoxviruses and common viruses. Clinical validation using 23 Clade II samples and 10 healthy controls showed that, relative to quantitative PCR (qPCR) (cycle threshold ≤37), the OPG034 fluorescence assay detected all 13 qPCR-positive samples and 2 additional positives (100% sensitivity, 80.0% specificity), while the visual assay detected 12 of 13 positives (92.3% sensitivity, 100% specificity). For Clade I-specific detection, both OPG033 fluorescence and visual assays showed 100% specificity in Clade II samples (23/23). While the clade-discriminatory capability was established through sequence-specific design, further evaluation with authentic Clade I clinical specimens is warranted to confirm typing performance. The dual-mode design provides flexibility for both laboratory and field use, advancing mpox surveillance and outbreak response.IMPORTANCEMpox is a significant zoonosis. Accurate discrimination between its highly lethal Clade I and more transmissible Clade II is critical for clinical management and outbreak control, yet current methods primarily enable only general detection. To address this, we identified novel genetic markers (OPG034 for universal detection and OPG033 for Clade I specificity) and developed a dual-mode detection platform integrating Clustered Regularly Interspaced Short Palindromic Repeats-Cas12a with recombinase-aided amplification. Its key advantage is providing two result readouts: a sensitive fluorescence mode for laboratories and an instrument-free visual colorimetric mode for field use. The demonstrated excellent performance on clinical samples confirms that this platform meets the precision requirements of clinical laboratories while remaining suitable for resource-limited settings like field clinics. Thus, it offers a flexible and practical tool for enhancing mpox surveillance and control globally, particularly in regions with constrained medical resources.