ORIGINAL RESEARCH article
Front. Public Health
Sec. Infectious Diseases: Epidemiology and Prevention
Volume 13 - 2025 | doi: 10.3389/fpubh.2025.1626030
This article is part of the Research TopicInnovative Approaches for the Early Detection and Rapid Response to Biothreat and Emerging Infectious AgentsView all 3 articles
Clade I or clade II? Targeting essential viral genes to differentiate monkeypox virus clades by multiplex real-time PCR
Provisionally accepted
- California Department of Public Health, Richmond, United States
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The increasing incidence and global spread of mpox have prompted the World Health Organization to twice declare a Public Health Emergency of International Concern. Sustained human-to-human transmission, largely through sexual contact, and waning population immunity to smallpox have accelerated monkeypox virus evolution and driven the emergence of variants that can adversely affect the performance of existing molecular diagnostic tests. To minimize the risk of PCR target drop out and better detect and monitor emerging monkeypox virus variants, we have developed and validated a multiplex real-time PCR (MpoxEG4-plex rPCR) targeting highly conserved and essential orthopoxvirus genes for the detection of four analytes: orthopoxviruses, monkeypox virus, clade I monkeypox virus, and clade II monkeypox virus. The assay limit of detection was ≤ 9 genome copies per reaction and the clinical accuracy, sensitivity, and specificity were > 96% for each analyte. The new assay was implemented to help confirm the first case of clade I mpox in the United States. The MpoxEG4-plex rPCR offers an accurate, informative, and reliable molecular diagnostic test for identifying cases and tracking case contacts in support of public health efforts to prevent and control the spread of mpox.
Keywords: mpox, Orthopoxvirus, Multiplex Polymerase Chain Reaction, Essential genes, Disease Outbreaks, Public Health Surveillance
Received: 09 May 2025; Accepted: 18 Jul 2025.
Copyright: © 2025 Probert, Espinosa and Hacker. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
* Correspondence: William S Probert, California Department of Public Health, Richmond, United States
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