BRIEF RESEARCH REPORT article
Front. Cell. Infect. Microbiol.
Sec. Clinical Infectious Diseases
Volume 15 - 2025 | doi: 10.3389/fcimb.2025.1611248
This article is part of the Research TopicMolecular Tests to Detect Tropical Viruses: Where We Are, and Where We Are GoingView all 3 articles
Development of a two-tube multiplex real-time fluorescent PCR for the simultaneous differentiation of the mpox virus clades and the A.1, B.1 and C.1 lineages within clade IIb
Provisionally accepted
Guohao Fan
Yuanlong LinLiuqing YangYun Peng
Guanyong OuQi QianDongmei Lai
Fuxiang Wang
Yingxia Liu
Yang Yang*- Second Affiliated Hospital of Southern University of Science and Technology, Shenzhen, China
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Background New clades and lineages emerged with the globally prevalent of Mpox virus (MPXV), accompanied by changing clinical symptoms, pathogenesis and transmission dynamics in associated with specific clades and lineages. Methods Here, we developed a two tube multiplex real-time fluorescent quantitative PCR (mrt-qPCR) assay for simultaneous differentiation of MPXV clades Ia, Ib, II, and innovative binding lock nucleic acid (LNA) probes to detect A.1, B.1 and C.1 lineages within the clade IIb. Results The assay demonstrated high sensitivity (33–69 copies/reaction) and specificity with expected linearity and stability. The intra-assay and intre-assay coefficients of variations (CV) were below the acceptable threshold of 5%, and the mrt-qPCR method has good stability and reproducibility. Clinical validation using 109 qPCR positive, 1 clade IIb B.1 virus strain and 15 negative specimens revealed 100% concordance for the differentiation of the three clade II and 97.60% for the differentiation the three lineages. The two tube multi-test system streamlined workflows, enabling efficient screening of diverse clinical samples (swabs from skin lessions, oropharynx and rectum, saliva and plasma). Conclusions We have established a two-tube multiplex qPCR method for detecting different clades and lineages of the MPXV. This method addresses the issue of false-negative detection of MPXV clade Ib caused by gene fragment deletion, and has also enabled the development of a rapid detection approach for the predominantly circulating clade IIb (including lineages A.1, B.1, and C.1). This cost-effective assay provides an important tool for accurate diagnosis, typing and epidemiological surveillance of MPXV.
Keywords: Mpox virus, Multiplex qPCR, Clade Ib, locked nucleic acid (LNA), diagnosis
Received: 17 Apr 2025; Accepted: 17 Sep 2025.
Copyright: © 2025 Fan, Lin, Yang, Peng, Ou, Qian, Lai, Wang, Liu and Yang. 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: Yang Yang, young@mail.sustech.edu.cn
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