Clade-Specific, Amplification-Free Molecular Lateral Flow Platform for On-Site Detection of Mpox

Abstract

Mpox, an urgent global health threat, requires rapid and accessible Clade-specific diagnostics. Here, a portable amplification-free molecular lateral flow assay was developed to distinguish Mpox Clades Ia, Ib, and II in 30 minutes with sensitivity of 10 2 copies µL -1 . The assay utilizes novel single-stranded DNA probes targeting C3L (Ia), D14L (Ib), and TNF-receptor (II), with gold nanoparticle colorimetric readout. The developed assay strip exhibited high analytical and clinical accuracy, matching RT-PCR results across all tested samples. This approach supports field deployment and rapid global surveillance of emergent Mpox lineages.The rapid global dissemination of Mpox, driven by Clade II since 2022 and exacerbated by emergent virulent Clade Ib outbreaks in Central Africa, has highlighted a critical diagnostic gap for field-ready, Claderesolving surveillance tools 1-8 6, 9 . Although the emergency status was lifted in May 2023, Mpox resurged in 2024 10 , with over 20,000 cases and >1000 deaths, predominantly in the Democratic Republic of Congo, attributed to a more virulent lineage, Clade Ib 11,12 . Mpox is phylogenetically classified into Clade I (Congo Basin) 13,14 and Clade II (West Africa) 11,15,16 . The emergence of Clade Ib, with higher virulence than Clade II, underscores the urgent need for rapid and field-deployable diagnostics capable of differentiating circulating Clades (Fig. 1a). Delayed detection and limited diagnostic access continue to hinder containment and accelerate viral evolution. The WHO's 2024-2027 highlights the necessity of point-of-care (POC) assays for decentralized surveillance, especially in resource-limited settings. Current Mpox confirmation relies on nucleic acid amplification tests (NAATs) 17,18 , or, less commonly, viral culture 18- 23 , which, despite their high accuracy, require complex instrumentation, cold chain logistics, and trained personnel, factors that restricts use in field conditions protein-based lateral flow assays (LFAs) 24 offer a low cost, portable option but lack the sensitivity required for clinical diagnosis. No amplification-free molecular LFA currently exists that can detect and distinguish Mpox Clades, representing a critical diagnostic gap.