Portable Detection of Monkeypox virus Clades using Bridging LAMP

Orthopoxvirus monkeypox (MPXV) is a zoonotic pathogen with two major clades that cause outbreaks in humans. Differentiation between the historically more clinically severe clade I and clade IIb is needed for effective public health response, such as in the ongoing 2022-2025 MPXV multi-country outbreaks. We have developed three loop-mediated isothermal amplification (LAMP) assays that differentiate between clade I, clade IIa/IIb MPXV, and broader Orthopoxvirus species by exploiting viral genomic rearrangements and amplicon length limitations inherent to LAMP. We demonstrate clade specificity between clade I and clade II viral cell culture lysates with LODs of 81-325 genomic copies per reaction and rapid detection of crude lesion specimens isolated from 45 patients during the latest MPXV USA 2022-2025 outbreaks. MPXV genomic DNA can be detected in as little as 10 minutes and clade-specific results detected in under an hour with limited instrumentation. We adapt a portable microfluidic system for point-of-need outbreak responses to zoonotic orthopoxvirus.