Point of care nucleic acid test using Single Stranded DNAs paper microfluidics for differential detection of Mpox Virus Clades under low resource setting: Development of a ssDNAs-based molecular assay for the simultaneous detection and differentiation of Mpox virus Clade I and II

Since 2022, the Mpox outbreak has resulted in multiple fatalities and has spread to 107 countries across all six World Health Organization (WHO) regions. In response, the WHO declared it a Public Health Emergency of International Concern, emphasizing the urgent need for rapid and precise detection of different Mpox virus (Mpox) clades. Early diagnosis, particularly through point-of-care molecular testing (POCT), is crucial for controlling its transmission. Here, we present the development of a colorimetric lateral flow assay detectable by the naked eye, utilizing gold nanoparticles (AuNPs) capped with single-stranded DNAs (ssDNAs) to specifically identify and differentiate Mpox Clades I and II. The ssDNAs, functionalized with thiol groups and conjugated to AuNPs, selectively agglomerate in the presence of target Mpox DNA, inducing a shift in surface plasmon resonance. This optimized assay achieves a detection limit of 100 copies/μl across all clades and exhibits high specificity for Mpox, with no cross-reactivity to other pathogens. These assays hold great potential for clinical applications, enabling rapid and reliable viral detection.