Development and clinical validation of a colorimetric isothermal assay for rapid detection of monkeypox virus

Abstract

Background Monkeypox virus has re-emerged as a global public health concern, highlighting the need for rapid, accurate, and accessible diagnostic tools. Real-time quantitative polymerase chain reaction is the reference standard for MPXV detection; however, its reliance on specialized infrastructure limits use in decentralized and resource-limited settings. This study reports the development and clinical evaluation of a colorimetric isothermal amplification assay for the rapid MPXV detection. Methods A loop-mediated isothermal amplification assay targeting the Orthopoxvirus genus-specific B6R gene and MPXV-specific F3L gene was developed, with human β-actin as an internal control. Reactions were performed at 65 °C for 40 min and interpreted by visual color change. Clinical performance was evaluated using 130 specimens. Analytical specificity was assessed using Vaccinia virus, Buffalopox virus, Herpes simplex virus, and Varicella zoster virus. qPCR served as the reference standard. Results In this retrospective single-centre evaluation, the assay showed complete concordance with qPCR, correctly identifying all 50 qPCR-positive and 80 qPCR-negative specimens. Detection was achieved across the observed Ct-value range; however, a formal analytical limit of detection was not established. No cross-reactivity was observed with non-Orthopox viruses, while expected differentiation from other Orthopoxviruses was achieved. Conclusions The developed colorimetric isothermal assay provides a rapid, accurate, and equipment-minimal approach for MPXV detection. The assay demonstrated performance comparable to qPCR within the evaluated clinical panel, supporting its potential utility for diagnosis and surveillance, particularly in settings with limited molecular diagnostic capacity. Further validation in larger prospective multicentre studies is required to confirm its clinical utility across diverse settings.