{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,11]],"date-time":"2026-09-11T16:05:49Z","timestamp":1789142749005,"version":"build-2803163510"},"reference-count":33,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2025,10,1]],"date-time":"2025-10-01T00:00:00Z","timestamp":1759276800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Cell. Infect. Microbiol."],"abstract":"<jats:sec><jats:title>Background<\/jats:title><jats:p>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.<\/jats:p><\/jats:sec><jats:sec><jats:title>Methods<\/jats:title><jats:p>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.<\/jats:p><\/jats:sec><jats:sec><jats:title>Results<\/jats:title><jats:p>The assay demonstrated high sensitivity (33\u201369 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).<\/jats:p><\/jats:sec><jats:sec><jats:title>Conclusions<\/jats:title><jats:p>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.<\/jats:p><\/jats:sec>","DOI":"10.3389\/fcimb.2025.1611248","type":"journal-article","created":{"date-parts":[[2025,10,1]],"date-time":"2025-10-01T05:52:05Z","timestamp":1759297925000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":3,"title":["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"],"prefix":"10.3389","volume":"15","author":[{"given":"Guohao","family":"Fan","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yuanlong","family":"Lin","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Liuqing","family":"Yang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yun","family":"Peng","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Guanyong","family":"Ou","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Qi","family":"Qian","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Dongmei","family":"Lai","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Fuxiang","family":"Wang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yingxia","family":"Liu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yang","family":"Yang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1965","published-online":{"date-parts":[[2025,10,1]]},"reference":[{"key":"B1","doi-asserted-by":"publisher","DOI":"10.1016\/j.tmaid.2022.102459","article-title":"Diagnosis of monkeypox virus - an overview","volume":"50","author":"Altindis","year":"2022","journal-title":"Travel Med. 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