{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,2]],"date-time":"2026-09-02T13:59:43Z","timestamp":1788357583929,"version":"build-2803163510"},"reference-count":40,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2026,3,19]],"date-time":"2026-03-19T00:00:00Z","timestamp":1773878400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100004826","name":"Natural Science Foundation of Beijing Municipality","doi-asserted-by":"publisher","id":[{"id":"10.13039\/501100004826","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Cell. Infect. Microbiol."],"abstract":"<jats:sec>\n                    <jats:title>Background<\/jats:title>\n                    <jats:p>Human cytomegalovirus (CMV) and Epstein-Barr virus (EBV) are globally prevalent herpesviruses. While typically self-limiting in immunocompetent individuals, infections can cause severe consequences even in this population. In immunocompromised groups, such as transplant recipients and HIV-infected individuals, viral reactivation or coinfection frequently triggers graft rejection, multi-organ invasion, and malignancies, often exhibiting synergistic pathogenicity. Current serological assays are limited by \u201cwindow periods\u201d and delayed immune responses, while traditional quantitative PCR (qPCR) relies on standard curves for quantification. Consequently, there is an urgent need for precise, interference-resistant methods. This study aimed to develop and validate a duplex droplet digital PCR (ddPCR) assay for the simultaneous, absolute quantification of CMV and EBV in plasma.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Methods<\/jats:title>\n                    <jats:p>Based on the TD-1 platform, a single-tube duplex detection system targeting conserved viral regions was optimized to minimize the \u201crain effect\u201d and maximize signal-to-noise ratios. Leveraging the absolute quantification capability of ddPCR without standard curves, we compared its performance, including dynamic range and limit of detection (LOD), against a homologous qPCR assay. Clinical validation was conducted using 117 plasma samples from suspected cases, utilizing a commercial qPCR kit as the reference standard. Additionally, tolerance to endogenous interfering substances was assessed.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Results<\/jats:title>\n                    <jats:p>\n                      The optimized duplex ddPCR demonstrated high analytical sensitivity, with LODs for CMV and EBV at 7.9 and 6.5 copies\/reaction, respectively, which were approximately 6- to 7-fold lower than homologous qPCR (53.4 and 45.6 copies\/reaction).No competitive inhibition was observed at low concentrations. In clinical validation, the assay achieved 100% sensitivity and specificity compared to the reference kit, with high quantitative correlation (R\n                      <jats:sup>2<\/jats:sup>\n                      \u00a0=\u00a00.80\u20130.87). Notably, ddPCR detected four weak positive samples (1 CMV, 3 EBV) missed by homologous qPCR. Furthermore, the method maintained accurate quantification in plasma containing hyperlipidemia or hyperbilirubinemia.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Conclusion<\/jats:title>\n                    <jats:p>This study successfully established a duplex ddPCR assay characterized by high sensitivity, specificity, and robust interference resistance. By enabling precise absolute quantification, it serves as a powerful complement to existing technologies for the early diagnosis and monitoring of CMV and EBV active infections.<\/jats:p>\n                  <\/jats:sec>","DOI":"10.3389\/fcimb.2026.1798127","type":"journal-article","created":{"date-parts":[[2026,3,19]],"date-time":"2026-03-19T06:48:18Z","timestamp":1773902898000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":2,"title":["Development and validation of a duplex droplet digital PCR assay for the simultaneous detection of cytomegalovirus and Epstein-Barr virus in plasma"],"prefix":"10.3389","volume":"16","author":[{"given":"Zhaoxiang","family":"Du","sequence":"first","affiliation":[{"name":"Department of Clinical Laboratory Center, Beijing Youan Hospital, Capital Medical University","place":["Beijing, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xingxing","family":"Yuan","sequence":"additional","affiliation":[{"name":"Department of Clinical Laboratory Center, Beijing Youan Hospital, Capital Medical University","place":["Beijing, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sihan","family":"Zhou","sequence":"additional","affiliation":[{"name":"Department of Clinical Laboratory Center, Beijing Youan Hospital, Capital Medical University","place":["Beijing, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lili","family":"Zhang","sequence":"additional","affiliation":[{"name":"Department of Clinical Laboratory Center, Beijing Youan Hospital, Capital Medical University","place":["Beijing, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yifeng","family":"Wang","sequence":"additional","affiliation":[{"name":"Department of Clinical Laboratory Center, Beijing Youan Hospital, Capital Medical University","place":["Beijing, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jie","family":"Yi","sequence":"additional","affiliation":[{"name":"Department of Clinical Laboratory, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences","place":["Beijing, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Manyu","family":"Li","sequence":"additional","affiliation":[{"name":"Division I of In Vitro Diagnostics for Infectious Diseases, Institute for In Vitro Diagnostics Control, National Institutes for Food and Drug Control","place":["Beijing, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yan","family":"Dang","sequence":"additional","affiliation":[{"name":"Department of Clinical Laboratory Center, Beijing Youan Hospital, Capital Medical University","place":["Beijing, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ning","family":"Liu","sequence":"additional","affiliation":[{"name":"Department of Clinical Laboratory Center, Beijing Youan Hospital, Capital Medical University","place":["Beijing, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xin","family":"Liu","sequence":"additional","affiliation":[{"name":"Department of Clinical Laboratory Center, Beijing Youan Hospital, Capital Medical University","place":["Beijing, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Fangfang","family":"Dai","sequence":"additional","affiliation":[{"name":"Department of Clinical Laboratory Center, Beijing Youan Hospital, Capital Medical University","place":["Beijing, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Haiqing","family":"Sun","sequence":"additional","affiliation":[{"name":"Department of Clinical Laboratory Center, Beijing Youan Hospital, Capital Medical University","place":["Beijing, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yanhua","family":"Yu","sequence":"additional","affiliation":[{"name":"Department of Clinical Laboratory Center, Beijing Youan Hospital, Capital Medical University","place":["Beijing, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Gengxia","family":"Yang","sequence":"additional","affiliation":[{"name":"Department of General Surgery, Beijing Youan Hospital, Capital Medical University","place":["Beijing, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1965","published-online":{"date-parts":[[2026,3,19]]},"reference":[{"key":"B1","doi-asserted-by":"publisher","first-page":"226","DOI":"10.3390\/pathogens9030226","article-title":"Recent advances in diagnostic approaches for Epstein\u2013Barr virus","volume":"9","author":"Abusalah","year":"2020","journal-title":"Pathogens"},{"key":"B2","doi-asserted-by":"publisher","first-page":"e01089","DOI":"10.1128\/spectrum.01089-24","article-title":"Development of a droplet digital PCR assay for the detection of BK polyomavirus","volume":"12","author":"Ai","year":"2024","journal-title":"Microbiol. 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