{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,11]],"date-time":"2026-07-11T23:41:44Z","timestamp":1783813304252,"version":"3.55.0"},"reference-count":40,"publisher":"MDPI AG","issue":"23","license":[{"start":{"date-parts":[[2025,11,23]],"date-time":"2025-11-23T00:00:00Z","timestamp":1763856000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Ministry of Health & Welfare, Republic of Korea","award":["RS-2025-25459033"],"award-info":[{"award-number":["RS-2025-25459033"]}]},{"name":"Ministry of Health & Welfare, Republic of Korea","award":["RS-2023-00304686"],"award-info":[{"award-number":["RS-2023-00304686"]}]},{"name":"Ministry of Health & Welfare, Republic of Korea","award":["RS-2025-02214700"],"award-info":[{"award-number":["RS-2025-02214700"]}]},{"DOI":"10.13039\/501100001321","name":"National Research Foundation","doi-asserted-by":"crossref","award":["2022M3A9F3016364"],"award-info":[{"award-number":["2022M3A9F3016364"]}],"id":[{"id":"10.13039\/501100001321","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/501100001321","name":"National Research Foundation","doi-asserted-by":"crossref","award":["RS-2023-00261820"],"award-info":[{"award-number":["RS-2023-00261820"]}],"id":[{"id":"10.13039\/501100001321","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/501100008005","name":"Yonsei University College of Medicine","doi-asserted-by":"crossref","award":["6-2023-0172"],"award-info":[{"award-number":["6-2023-0172"]}],"id":[{"id":"10.13039\/501100008005","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/501100008005","name":"Yonsei University College of Medicine","doi-asserted-by":"crossref","award":["6-2025-0007"],"award-info":[{"award-number":["6-2025-0007"]}],"id":[{"id":"10.13039\/501100008005","id-type":"DOI","asserted-by":"crossref"}]},{"name":"CKDBiO-YONSEI Microbiome Research Center"},{"name":"Ministry of Trade Industry & Energy; MOTIE, Korea","award":["20022947"],"award-info":[{"award-number":["20022947"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Fusobacterium nucleatum (Fn) is increasingly recognized as a cancer-associated bacterium, yet reliable quantification in human specimens is challenging due to low bacterial burden and abundant host DNA. We analyzed 145 Fusobacterium genomes to design primers targeting conserved regions of the fadA adhesin gene and developed a duplex quantitative real-time PCR (qPCR) assay for simultaneous detection of fadA and a human PGT as an internal control. Analytical sensitivity, specificity, precision, and reproducibility were evaluated using serially diluted Fn DNA, spike-in experiments with human DNA, and cross-platform\/operator validation. Clinical performance was assessed in colorectal cancer patient tissues, including fresh tissue (n = 24) and formalin-fixed paraffin-embedded (FFPE) samples (n = 22), using 16S rRNA-based methods as references. The assay successfully detected all four major Fn subspecies (nucleatum, animalis, polymorphum, and vincentii). The limit of detection was \u22640.1 pg, with no interference between duplex targets. Spike-in experiments demonstrated consistent target detection in human-DNA-rich samples, with strong linearity (R2 = 0.998) across dilutions. High precision (coefficient of variations &lt; 5%) was observed across intra-day, inter-day, inter-instrument, and inter-operator evaluations. In fresh tissues, the assay yielded 86% sensitivity, 94% specificity, and 92% accuracy. Using the FFPE samples, the assay achieved 91% sensitivity and 100% specificity, confirming robust classification in both clinical samples. This duplex qPCR assay enables broad detection of Fn with high analytical performance in both fresh and FFPE tissues. Its simplicity, reproducibility, and compatibility with pathology workflows support deployment in multi-center studies and downstream applications in diagnostic studies and prognostic modeling.<\/jats:p>","DOI":"10.3390\/ijms262311319","type":"journal-article","created":{"date-parts":[[2025,11,24]],"date-time":"2025-11-24T09:02:07Z","timestamp":1763974927000},"page":"11319","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["A Duplex qPCR Assay Targeting the fadA Gene Enables Robust Detection of Fusobacterium in Clinical Samples"],"prefix":"10.3390","volume":"26","author":[{"given":"Yurin","family":"Seo","sequence":"first","affiliation":[{"name":"Yonsei Cancer Center, Division of Medical Oncology, Department of Internal Medicine, Yonsei University College of Medicine, Seoul 03722, Republic of Korea"},{"name":"Graduate School of Medical Science, Brain Korea 21 FOUR Project, Severance Biomedical Science Institute, Yonsei University College of Medicine, Seoul 03722, Republic of Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2577-1317","authenticated-orcid":false,"given":"Kyung-A","family":"Kim","sequence":"additional","affiliation":[{"name":"Yonsei Cancer Center, Division of Medical Oncology, Department of Internal Medicine, Yonsei University College of Medicine, Seoul 03722, Republic of Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Suho","family":"Lee","sequence":"additional","affiliation":[{"name":"Yonsei Cancer Center, Division of Medical Oncology, Department of Internal Medicine, Yonsei University College of Medicine, Seoul 03722, Republic of Korea"},{"name":"Graduate School of Medical Science, Brain Korea 21 FOUR Project, Severance Biomedical Science Institute, Yonsei University College of Medicine, Seoul 03722, Republic of Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yujin H.","family":"Lim","sequence":"additional","affiliation":[{"name":"Yonsei Cancer Center, Division of Medical Oncology, Department of Internal Medicine, Yonsei University College of Medicine, Seoul 03722, Republic of Korea"},{"name":"Graduate School of Medical Science, Brain Korea 21 FOUR Project, Severance Biomedical Science Institute, Yonsei University College of Medicine, Seoul 03722, Republic of Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0004-8830-0442","authenticated-orcid":false,"given":"Yura","family":"Seo","sequence":"additional","affiliation":[{"name":"Yonsei Cancer Center, Division of Medical Oncology, Department of Internal Medicine, Yonsei University College of Medicine, Seoul 03722, Republic of Korea"},{"name":"Graduate School of Medical Science, Brain Korea 21 FOUR Project, Severance Biomedical Science Institute, Yonsei University College of Medicine, Seoul 03722, Republic of Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Taeyul K.","family":"Kim","sequence":"additional","affiliation":[{"name":"Yonsei Cancer Center, Division of Medical Oncology, Department of Internal Medicine, Yonsei University College of Medicine, Seoul 03722, Republic of Korea"},{"name":"Graduate School of Medical Science, Brain Korea 21 FOUR Project, Severance Biomedical Science Institute, Yonsei University College of Medicine, Seoul 03722, Republic of Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Chae Hyun","family":"Kim","sequence":"additional","affiliation":[{"name":"Yonsei Cancer Center, Division of Medical Oncology, Department of Internal Medicine, Yonsei University College of Medicine, Seoul 03722, Republic of Korea"},{"name":"Graduate School of Medical Science, Brain Korea 21 FOUR Project, Severance Biomedical Science Institute, Yonsei University College of Medicine, Seoul 03722, Republic of Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yeleem","family":"Kim","sequence":"additional","affiliation":[{"name":"Yonsei Cancer Center, Division of Medical Oncology, Department of Internal Medicine, Yonsei University College of Medicine, Seoul 03722, Republic of Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0001-1731-700X","authenticated-orcid":false,"given":"Francesca","family":"Jereis","sequence":"additional","affiliation":[{"name":"School of Medicine, Georgetown University, Washington, DC 20007, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Won Kyu","family":"Kim","sequence":"additional","affiliation":[{"name":"Center for Natural Product Efficacy Optimization, Korea Institute of Science and Technology (KIST), Gangneung 25451, Republic of Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2136-3578","authenticated-orcid":false,"given":"Yoon Dae","family":"Han","sequence":"additional","affiliation":[{"name":"Division of Colon and Rectal Surgery, Department of Surgery, Yonsei University College of Medicine, Seoul 03722, Republic of Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Minsun","family":"Jung","sequence":"additional","affiliation":[{"name":"Department of Pathology, Yonsei University College of Medicine, Seoul 03722, Republic of Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8523-4126","authenticated-orcid":false,"given":"Hyukmin","family":"Lee","sequence":"additional","affiliation":[{"name":"Department of Laboratory Medicine, Yonsei University College of Medicine, Seoul 03722, Republic of Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kyungwon","family":"Lee","sequence":"additional","affiliation":[{"name":"Department of Laboratory Medicine, Yonsei University College of Medicine, Seoul 03722, Republic of Korea"},{"name":"Seoul Clinical Laboratories Academy, Yongin 16954, Republic of Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Joong Bae","family":"Ahn","sequence":"additional","affiliation":[{"name":"Yonsei Cancer Center, Division of Medical Oncology, Department of Internal Medicine, Yonsei University College of Medicine, Seoul 03722, Republic of Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4401-7803","authenticated-orcid":false,"given":"Jihoon G.","family":"Yoon","sequence":"additional","affiliation":[{"name":"Department of Laboratory Medicine, Yonsei University College of Medicine, Seoul 03722, Republic of Korea"},{"name":"Department of Laboratory Medicine, Gangnam Severance Hospital, Seoul 06273, Republic of Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6504-9927","authenticated-orcid":false,"given":"Han Sang","family":"Kim","sequence":"additional","affiliation":[{"name":"Yonsei Cancer Center, Division of Medical Oncology, Department of Internal Medicine, Yonsei University College of Medicine, Seoul 03722, Republic of Korea"},{"name":"Graduate School of Medical Science, Brain Korea 21 FOUR Project, Severance Biomedical Science Institute, Yonsei University College of Medicine, Seoul 03722, Republic of Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2025,11,23]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"Segata, N., Haake, S.K., Mannon, P., Lemon, K.P., Waldron, L., Gevers, D., Huttenhower, C., and Izard, J. 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