{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,11]],"date-time":"2026-09-11T06:07:27Z","timestamp":1789106847229,"version":"build-2803163510"},"reference-count":39,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2026,9,11]],"date-time":"2026-09-11T00:00:00Z","timestamp":1789084800000},"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. Microbiol."],"abstract":"<jats:p>Monkeypox virus (MPXV) has caused two Public Health Emergencies of International Concern since 2022, with co-circulation of distinct clades (Ia, Ib, IIa, IIb) that differ in virulence and transmission patterns. Rapid discrimination of these clades is critical for outbreak control and clinical management. Here, we developed and validated a two-tube multiplex real-time quantitative PCR (qPCR) assay targeting the H3L gene, designed to first distinguish clade I from clade II, followed by subtyping into clades Ia, Ib, IIa, and IIb in separate reactions. The assay demonstrated high sensitivity, with detection limits of 100 copies\/reaction for clade Ia, 10 copies\/reaction for clade Ib, 10 copies\/reaction for clade IIa, and 1 copy\/reaction for clade IIb using standard plasmids. Excellent specificity was confirmed by the absence of cross-reactivity with nucleic acids from other human pathogens, including vaccinia virus Tiantan strain (VTT), human adenovirus (HAdV), getah virus (GETV), chikungunya virus (CHIKV), Japanese encephalitis virus (JEV), dengue virus (DENV), influenza A virus (IAV), influenza B virus (IBV), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The assay exhibited high repeatability, with intra-assay coefficients of variation (CVs) ranging from 0.09 to 1.88% and inter-assay CVs from 0.23 to 2.68%. Clinical evaluation using 48 skin samples from infected crab-eating monkeys or SCID mice showed 100% concordance with singleplex qPCR for clade IIb detection. For clades Ia, Ib, and IIa, clinical validation was not feasible due to sample unavailability, and their performance was analytically confirmed using recombinant plasmids. Importantly, the clade assignment of the 48 animal specimens was not confirmed by sequencing, which represents a limitation of the current study. Furthermore, these specimens were derived from animal models rather than human patients; thus, the results represent preclinical validation, and formal clinical validation in human specimens from endemic regions is warranted. This two-tube multiplex qPCR assay provides a rapid, sensitive, and reliable tool for the analytical discrimination of all four major MPXV clades, with preliminary clinical validation for clade IIb. Its ability to distinguish emerging clades makes it a valuable asset for clinical diagnosis, epidemiological surveillance, and future outbreak preparedness, although further clinical validation in endemic regions is required.<\/jats:p>","DOI":"10.3389\/fmicb.2026.1907267","type":"journal-article","created":{"date-parts":[[2026,9,11]],"date-time":"2026-09-11T05:29:31Z","timestamp":1789104571000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":0,"title":["Development and evaluation of a two-tube multiplex real-time PCR assay for differential detection of Monkeypox virus clades Ia, Ib, IIa, and IIb"],"prefix":"10.3389","volume":"17","author":[{"given":"Xiangyu","family":"Zhu","sequence":"first","affiliation":[{"name":"Department of Clinical Laboratory, The Second Hospital of Jilin University, Changchun","place":["Jilin, China"]},{"name":"The Second Department of Infectious Disease Prevention and Control, Center for Disease Control and Prevention in Northern Theater Command, Shenyang","place":["Liaoning, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ning","family":"Shi","sequence":"additional","affiliation":[{"name":"State Key Laboratory for Diagnosis and Treatment of Severe Zoonotic Infectious Diseases, Key Laboratory for Zoonosis Research of the Ministry of Education, Institute of Zoonosis, and College of Veterinary Medicine, Jilin University, Changchun","place":["Jilin, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yong","family":"Liu","sequence":"additional","affiliation":[{"name":"The Second Department of Infectious Disease Prevention and Control, Center for Disease Control and Prevention in Northern Theater Command, Shenyang","place":["Liaoning, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jiachen","family":"Li","sequence":"additional","affiliation":[{"name":"The Second Department of Infectious Disease Prevention and Control, Center for Disease Control and Prevention in Northern Theater Command, Shenyang","place":["Liaoning, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jiaxin","family":"Tian","sequence":"additional","affiliation":[{"name":"State Key Laboratory for Diagnosis and Treatment of Severe Zoonotic Infectious Diseases, Key Laboratory for Zoonosis Research of the Ministry of Education, Institute of Zoonosis, and College of Veterinary Medicine, Jilin University, Changchun","place":["Jilin, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Bocheng","family":"Liu","sequence":"additional","affiliation":[{"name":"State Key Laboratory for Diagnosis and Treatment of Severe Zoonotic Infectious Diseases, Key Laboratory for Zoonosis Research of the Ministry of Education, Institute of Zoonosis, and College of Veterinary Medicine, Jilin University, Changchun","place":["Jilin, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xuerui","family":"Wang","sequence":"additional","affiliation":[{"name":"Department of Clinical Laboratory, The Second Hospital of Jilin University, Changchun","place":["Jilin, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1965","published-online":{"date-parts":[[2026,9,11]]},"reference":[{"key":"B1","doi-asserted-by":"publisher","first-page":"109108","DOI":"10.1016\/j.clim.2022.109108","article-title":"The resurgence of a neglected orthopoxvirus: Immunologic and clinical aspects of monkeypox virus infections over the past six decades.","volume":"243","author":"Al-Musa","year":"2022","journal-title":"Clin. Immunol"},{"key":"B2","doi-asserted-by":"publisher","first-page":"719","DOI":"10.1016\/j.tips.2023.08.003","article-title":"Differences in pathogenicity among the mpox virus clades: Impact on drug discovery and vaccine development.","volume":"44","author":"Andrei","year":"2023","journal-title":"Trends Pharmacol. Sci"},{"key":"B3","doi-asserted-by":"publisher","first-page":"455","DOI":"10.1128\/mr.47.4.455-509.1983","article-title":"The smallpox story: Life and death of an old disease.","volume":"47","author":"Behbehani","year":"1983","journal-title":"Microbiol. Rev"},{"key":"B4","unstructured":"The Mpox Reagent (RT-qPCR).\n          \n          2024"},{"key":"B5","doi-asserted-by":"publisher","first-page":"e0010141","DOI":"10.1371\/journal.pntd.0010141","article-title":"The changing epidemiology of human monkeypox-a potential threat? A systematic review.","volume":"16","author":"Bunge","year":"2022","journal-title":"PLoS Negl. Trop. 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Microbiol"},{"key":"B11","unstructured":"D The Mpox Reagent (RT-qPCR).\n          \n          2024"},{"key":"B12","doi-asserted-by":"publisher","first-page":"708","DOI":"10.3390\/v11080708","article-title":"Genome of alaskapox virus, a novel orthopoxvirus isolated from alaska.","volume":"11","author":"Gigante","year":"2019","journal-title":"Viruses"},{"key":"B13","doi-asserted-by":"publisher","first-page":"e3001769","DOI":"10.1371\/journal.pbio.3001769","article-title":"Urgent need for a non-discriminatory and non-stigmatizing nomenclature for monkeypox virus.","volume":"20","author":"Happi","year":"2022","journal-title":"PLoS Biol"},{"key":"B14","doi-asserted-by":"publisher","first-page":"2200497","DOI":"10.2807\/1560-7917.ES.2022.27.32.2200497","article-title":"Monkeypox: Another test for PCR.","volume":"27","author":"Huggett","year":"2022","journal-title":"Euro. Surveill"},{"key":"B15","unstructured":"Virus Taxonomy.\n          \n          2024"},{"key":"B16","doi-asserted-by":"publisher","first-page":"1200","DOI":"10.1038\/labinvest.3700143","article-title":"Monkeypox virus detection in rodents using real-time 3\u2019-minor groove binder TaqMan assays on the roche lightcycler.","volume":"84","author":"Kulesh","year":"2004","journal-title":"Lab. Invest"},{"key":"B17","doi-asserted-by":"publisher","first-page":"115164","DOI":"10.1016\/j.jviromet.2025.115164","article-title":"Development and clinical evaluation of a MPXV antigen-detecting rapid diagnostic test.","volume":"337","author":"Kurosawa","year":"2025","journal-title":"J. Virol. Methods"},{"key":"B18","doi-asserted-by":"publisher","first-page":"194","DOI":"10.1016\/j.jcv.2006.03.012","article-title":"Detection of monkeypox virus with real-time PCR assays.","volume":"36","author":"Li","year":"2006","journal-title":"J. Clin. 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Med"},{"key":"B33","unstructured":"Emergency Committee Regarding the Upsurge of Mpox 2024 2025.\n          \n          2005"},{"key":"B34","unstructured":"Monkeypox: Experts Give Virus Variants New Names.\n          \n          2022"},{"key":"B35","unstructured":"Mpox Outbreak."},{"key":"B36","unstructured":"Mpox."},{"key":"B37","unstructured":"WHO Director-General Declares Mpox Outbreak a Public Health Emergency Of International Concern."},{"key":"B38","unstructured":"Global Mpox Trends.\n          \n          2026"},{"key":"B39","doi-asserted-by":"publisher","first-page":"2427","DOI":"10.1016\/j.tibtech.2025.04.015","article-title":"Mpox diagnosis at POC.","volume":"43","author":"Yigci","year":"2025","journal-title":"Trends Biotechnol."}],"container-title":["Frontiers in Microbiology"],"original-title":[],"link":[{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fmicb.2026.1907267\/full","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,9,11]],"date-time":"2026-09-11T05:29:34Z","timestamp":1789104574000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fmicb.2026.1907267\/full"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,9,11]]},"references-count":39,"alternative-id":["10.3389\/fmicb.2026.1907267"],"URL":"https:\/\/doi.org\/10.3389\/fmicb.2026.1907267","relation":{},"ISSN":["1664-302X"],"issn-type":[{"value":"1664-302X","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,9,11]]},"article-number":"1907267"}}