{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,5,21]],"date-time":"2026-05-21T13:09:53Z","timestamp":1779368993359,"version":"3.53.0"},"reference-count":36,"publisher":"Wiley","issue":"4","license":[{"start":{"date-parts":[[2026,3,30]],"date-time":"2026-03-30T00:00:00Z","timestamp":1774828800000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"},{"start":{"date-parts":[[2026,3,30]],"date-time":"2026-03-30T00:00:00Z","timestamp":1774828800000},"content-version":"tdm","delay-in-days":0,"URL":"http:\/\/doi.wiley.com\/10.1002\/tdm_license_1.1"}],"funder":[{"DOI":"10.13039\/501100001809","name":"National Natural Science Foundation of China","doi-asserted-by":"publisher","award":["82173396"],"award-info":[{"award-number":["82173396"]}],"id":[{"id":"10.13039\/501100001809","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["onlinelibrary.wiley.com"],"crossmark-restriction":true},"short-container-title":["Journal of Medical Virology"],"published-print":{"date-parts":[[2026,4]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                  <jats:p>Human mpox, formerly known as monkeypox, has twice been declared a Public Health Emergency of International Concern (PHEIC) by the World Health Organization, in 2022 and 2024. Despite this, global access to rapid, reliable diagnostics remains limited, hindering outbreak control and equitable clinical response. To address this gap, we developed and validated a rapid direct real\u2010time PCR assay capable of detecting and differentiating Clade I and Clade II monkeypox virus (MPXV) directly from clinical samples. We designed a new pan\u2010specific B15L assay to complement the published F3L assay and introduced the first clade\u2010specific assay targeting the B1R gene of Clade I MPXV. In silico validation showed that both B15L and F3L assays detected all available MPXV genomes with 100% sensitivity, while B1R selectively detected only Clade I genomes with 100% specificity. Cross\u2010reactivity screening against 40 high\u2010priority non\u2010target organisms revealed minimal risk of false positives. We confirmed analytical sensitivity of 2 copies per reaction for all assays in vitro, with clade\u2010specific B1R showing 100% specificity. When adapted into a lyophilized direct PCR format for near\u2010point\u2010of\u2010care use, the assay demonstrated detection down to 1 copy per reaction for pan\u2010specific targets and 2 copies for clade\u2010specific detection in spiked clinical samples. This direct PCR assay delivers accurate results in under 1\u2009h without requiring nucleic acid extraction. Our combined genomic, laboratory, and clinical validations demonstrate exceptional sensitivity and specificity, positioning this assay as a highly promising tool in emergency mpox diagnostics and a potential model for future outbreak\u2010responsive testing platforms.<\/jats:p>","DOI":"10.1002\/jmv.70886","type":"journal-article","created":{"date-parts":[[2026,3,30]],"date-time":"2026-03-30T13:10:31Z","timestamp":1774876231000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Evaluation and Clinical Validation of Pan\u2010Specific and Clade\u2010Specific Diagnostic Real\u2010Time PCR Assays for Monkeypox Virus"],"prefix":"10.1002","volume":"98","author":[{"given":"Hong","family":"Chang","sequence":"first","affiliation":[{"name":"Oxford Suzhou Centre for Advanced Research (OSCAR) University of Oxford Suzhou Jiangsu China"},{"name":"ZYTCA Limited Centre for Innovation and Enterprise Begbroke Oxfordshire UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9853-2053","authenticated-orcid":false,"given":"Tin Hang","family":"Hung","sequence":"additional","affiliation":[{"name":"Department of Biology University of Oxford Oxford Oxfordshire UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Binbin","family":"Li","sequence":"additional","affiliation":[{"name":"Oxford Suzhou Centre for Advanced Research (OSCAR) University of Oxford Suzhou Jiangsu China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9626-3128","authenticated-orcid":false,"given":"Bobby Lim\u2010Ho","family":"Kong","sequence":"additional","affiliation":[{"name":"ZYTCA Limited Centre for Innovation and Enterprise Begbroke Oxfordshire UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ashwathi Asha","family":"Madhaven","sequence":"additional","affiliation":[{"name":"ZYTCA Limited Centre for Innovation and Enterprise Begbroke Oxfordshire UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yue","family":"Wang","sequence":"additional","affiliation":[{"name":"Oxford Suzhou Centre for Advanced Research (OSCAR) University of Oxford Suzhou Jiangsu China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6804-1317","authenticated-orcid":false,"given":"Ming\u2010shan","family":"Tsai","sequence":"additional","affiliation":[{"name":"ZYTCA Limited Centre for Innovation and Enterprise Begbroke Oxfordshire UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7317-283X","authenticated-orcid":false,"given":"Dan","family":"Deng","sequence":"additional","affiliation":[{"name":"Department of Dermatology, Shanghai Children's Medical Center, School of Medicine Shanghai Jiao Tong University Shanghai China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zhanfeng","family":"Cui","sequence":"additional","affiliation":[{"name":"Oxford Suzhou Centre for Advanced Research (OSCAR) University of Oxford Suzhou Jiangsu China"},{"name":"Institute of Biomedical Engineering, Department of Engineering Science University of Oxford Oxford Oxfordshire UK"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2026,3,30]]},"reference":[{"key":"e_1_2_10_2_1","doi-asserted-by":"publisher","DOI":"10.2217\/fvl.12.130"},{"key":"e_1_2_10_3_1","unstructured":"World Health Organization.2022\u201324 Mpox Outbreak: Global Trends (2024) 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