{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,6]],"date-time":"2026-10-06T13:57:09Z","timestamp":1791295029186,"version":"4.1.0"},"reference-count":46,"publisher":"American Society for Microbiology","issue":"10","license":[{"start":{"date-parts":[[2026,10,6]],"date-time":"2026-10-06T00:00:00Z","timestamp":1791244800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"},{"start":{"date-parts":[[2026,10,6]],"date-time":"2026-10-06T00:00:00Z","timestamp":1791244800000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/journals.asm.org\/non-commercial-tdm-license"}],"funder":[{"DOI":"10.13039\/501100012166","name":"National Key Research and Development Program of China","doi-asserted-by":"publisher","award":["2025YFC2609600"],"award-info":[{"award-number":["2025YFC2609600"]}],"id":[{"id":"10.13039\/501100012166","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100003392","name":"Natural Science Foundation of Fujian Province","doi-asserted-by":"publisher","award":["2024J01167"],"award-info":[{"award-number":["2024J01167"]}],"id":[{"id":"10.13039\/501100003392","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["journals.asm.org"],"crossmark-restriction":true},"short-container-title":["Microbiol Spectr"],"published-print":{"date-parts":[[2026,10,6]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                  <jats:sec>\n                    <jats:title\/>\n                    <jats:p>\n                      Dengue virus (DENV) remains a critical global health threat, necessitating rapid pan-detection and precise serotyping. In this study, we developed a multiplex real-time RT-qPCR assay for simultaneous detection and identification of DENV1\u20134. Guided by a systematic genomic conservation analysis of 4,754 sequences, we targeted the highly conserved 5\u2032 untranslated region (5\u2032 UTR) to design one pan-DENV and four serotype-specific primer-probe sets. The assay demonstrated robust analytical performance, with the limit of detection (LOD) ranging from 15.8 to 1.58 \u00d7 10\n                      <jats:sup>3<\/jats:sup>\n                      copies\/mL and the lowest LOD (15.8 copies\/mL) observed for serotype I with specific primer-probe sets. There was no cross-reactivity with other common pathogens of the\n                      <jats:italic toggle=\"yes\">Orthoflavivirus<\/jats:italic>\n                      or\n                      <jats:italic toggle=\"yes\">Alphavirus<\/jats:italic>\n                      genus pathogens\n                      <jats:italic toggle=\"yes\">,<\/jats:italic>\n                      and clinical specificity was further validated as 100% using a negative cohort of non-dengue febrile samples. Validation using 53 clinical DENV-positive samples showed 100% concordance with a reference diagnostic kit for serotype identification. While the pan-DENV set served as a high-specificity screening tool with broad reactivity, it exhibited a clinical sensitivity of 88.7%, particularly missing low-titer samples (Ct &gt; 30). This discrepancy, coupled with the superior sensitivity of the serotype-specific sets, suggests the presence of incomplete genomic fragments in acute-phase sera, highlighting the advantage of our 5\u2032 UTR-targeted approach in assessing viral genomic integrity. This dual-layered multiplex assay provides a robust, rapid, and cost-effective tool for clinical diagnosis and epidemiological surveillance.\n                    <\/jats:p>\n                    <jats:sec>\n                      <jats:title>IMPORTANCE<\/jats:title>\n                      <jats:p>Dengue fever is a leading cause of systemic viral disease worldwide. Co-circulation of four distinct serotypes complicates clinical management and increases severe disease risk. Here, we presented a novel multiplex RT-qPCR assay enabling simultaneous pan-DENV detection and serotype identification in two tubes, comprising six reactions including an internal control (IC), thereby streamlining dengue diagnosis. A key innovation is the systematic selection of the 5\u2032 UTR as the diagnostic target, guided by big-data genomic analysis. Unlike assays targeting the 3\u2032 UTR, which may overrepresent viral loads by capturing redundant subgenomic orthoflavivirus RNA (sfRNA), our 5\u2032 UTR-targeted design provides a more stringent measure of genomic integrity. Furthermore, the modular performance of our assay combining broad screening via a pan-DENV set with high-sensitivity subtyping which offers a dual-layer diagnostic framework. This work provides a high-performance tool for timely clinical intervention and enhances our molecular understanding of DENV fragments in clinical specimens.<\/jats:p>\n                    <\/jats:sec>\n                  <\/jats:sec>","DOI":"10.1128\/spectrum.00718-26","type":"journal-article","created":{"date-parts":[[2026,9,2]],"date-time":"2026-09-02T16:44:50Z","timestamp":1788367490000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":0,"title":["Comprehensive conservation profiling enables a robust multiplex RT-qPCR assay for simultaneous pan-detection and serotype discrimination of dengue virus"],"prefix":"10.1128","volume":"14","author":[{"ORCID":"https:\/\/orcid.org\/0009-0005-2448-6564","authenticated-orcid":false,"given":"Bohan","family":"Xu","sequence":"first","affiliation":[{"id":[{"id":"https:\/\/ror.org\/034t30j35","id-type":"ROR","asserted-by":"publisher"}],"name":"State Key Laboratory of Virology and Biosafety, Center for Biosafety Mega-Science, Wuhan Institute of Virology, Chinese Academy of Sciences","place":["Wuhan, China"]},{"id":[{"id":"https:\/\/ror.org\/05qbk4x57","id-type":"ROR","asserted-by":"publisher"}],"name":"University of Chinese Academy of Sciences","place":["Beijing, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Dongyan","family":"Xiong","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/034t30j35","id-type":"ROR","asserted-by":"publisher"}],"name":"State Key Laboratory of Virology and Biosafety, Center for Biosafety Mega-Science, Wuhan Institute of Virology, Chinese Academy of Sciences","place":["Wuhan, China"]},{"id":[{"id":"https:\/\/ror.org\/05qbk4x57","id-type":"ROR","asserted-by":"publisher"}],"name":"University of Chinese Academy of Sciences","place":["Beijing, China"]},{"name":"Centre for Novostics","place":["Hong Kong, China"]},{"id":[{"id":"https:\/\/ror.org\/00t33hh48","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Chemical Pathology, Chinese University of Hong Kong, Prince of Wales Hospital","place":["Hong Kong, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jinzhang","family":"Wang","sequence":"additional","affiliation":[{"name":"Fujian Provincial Center for Disease Control and Prevention, Fujian Provincial Key Laboratory of Zoonosis Research","place":["Fuzhou, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Chunjing","family":"Zhao","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/034t30j35","id-type":"ROR","asserted-by":"publisher"}],"name":"State Key Laboratory of Virology and Biosafety, Center for Biosafety Mega-Science, Wuhan Institute of Virology, Chinese Academy of Sciences","place":["Wuhan, China"]},{"id":[{"id":"https:\/\/ror.org\/05qbk4x57","id-type":"ROR","asserted-by":"publisher"}],"name":"University of Chinese Academy of Sciences","place":["Beijing, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Junhua","family":"Li","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/034t30j35","id-type":"ROR","asserted-by":"publisher"}],"name":"State Key Laboratory of Virology and Biosafety, Center for Biosafety Mega-Science, Wuhan Institute of Virology, Chinese Academy of Sciences","place":["Wuhan, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9948-8880","authenticated-orcid":true,"given":"Hongping","family":"Wei","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/034t30j35","id-type":"ROR","asserted-by":"publisher"}],"name":"State Key Laboratory of Virology and Biosafety, Center for Biosafety Mega-Science, Wuhan Institute of Virology, Chinese Academy of Sciences","place":["Wuhan, China"]},{"id":[{"id":"https:\/\/ror.org\/05qbk4x57","id-type":"ROR","asserted-by":"publisher"}],"name":"University of Chinese Academy of Sciences","place":["Beijing, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9024-0793","authenticated-orcid":true,"given":"Junping","family":"Yu","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/034t30j35","id-type":"ROR","asserted-by":"publisher"}],"name":"State Key Laboratory of Virology and Biosafety, Center for Biosafety Mega-Science, Wuhan Institute of Virology, Chinese Academy of Sciences","place":["Wuhan, China"]},{"id":[{"id":"https:\/\/ror.org\/05qbk4x57","id-type":"ROR","asserted-by":"publisher"}],"name":"University of Chinese Academy of Sciences","place":["Beijing, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"235","reference":[{"key":"e_1_3_4_2_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.jviromet.2019.113677"},{"key":"e_1_3_4_3_2","doi-asserted-by":"publisher","DOI":"10.1093\/infdis\/jiw649"},{"key":"e_1_3_4_4_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.ijbiomac.2023.127613"},{"key":"e_1_3_4_5_2","doi-asserted-by":"publisher","DOI":"10.1007\/s13206-020-4409-7"},{"key":"e_1_3_4_6_2","doi-asserted-by":"publisher","DOI":"10.1099\/vir.0.18807-0"},{"key":"e_1_3_4_7_2","doi-asserted-by":"publisher","DOI":"10.1093\/jmedent\/36.4.508"},{"key":"e_1_3_4_8_2","first-page":"243","article-title":"The history of dengue in tropical Asia and its probable relationship to the mosquito Aedes aegypti","volume":"59","author":"Smith CEG","year":"1956","unstructured":"Smith CEG. 1956. The history of dengue in tropical Asia and its probable relationship to the mosquito Aedes aegypti. J Trop Med Hyg 59:243\u2013251.","journal-title":"J Trop Med Hyg"},{"key":"e_1_3_4_9_2","doi-asserted-by":"publisher","DOI":"10.1016\/S0140-6736(07)61687-0"},{"key":"e_1_3_4_10_2","doi-asserted-by":"publisher","DOI":"10.1128\/spectrum.00922-25"},{"key":"e_1_3_4_11_2","doi-asserted-by":"publisher","DOI":"10.1038\/nature12060"},{"key":"e_1_3_4_12_2","doi-asserted-by":"publisher","DOI":"10.1186\/s12916-015-0336-1"},{"key":"e_1_3_4_13_2","doi-asserted-by":"publisher","DOI":"10.1098\/rspb.2024.0724"},{"key":"e_1_3_4_14_2","doi-asserted-by":"publisher","DOI":"10.1186\/s40249-023-01083-2"},{"key":"e_1_3_4_15_2","doi-asserted-by":"publisher","DOI":"10.1038\/s41586-020-2008-3"},{"key":"e_1_3_4_16_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.virusres.2022.199018"},{"key":"e_1_3_4_17_2","doi-asserted-by":"publisher","DOI":"10.1111\/imr.12367"},{"key":"e_1_3_4_18_2","doi-asserted-by":"publisher","DOI":"10.1089\/088282403763635465"},{"key":"e_1_3_4_19_2","doi-asserted-by":"publisher","DOI":"10.1186\/1743-422X-5-20"},{"key":"e_1_3_4_20_2","doi-asserted-by":"publisher","DOI":"10.1128\/CMR.00035-09"},{"key":"e_1_3_4_21_2","doi-asserted-by":"publisher","DOI":"10.1128\/JVI.73.6.4738-4747.1999"},{"key":"e_1_3_4_22_2","doi-asserted-by":"publisher","DOI":"10.1086\/315215"},{"key":"e_1_3_4_23_2","first-page":"51","volume-title":"Methods in molecular biology","author":"Tripathi NK","year":"2011","unstructured":"Tripathi NK, Shrivastava A, Dash PK, Jana AM. 2011. Detection of dengue virus, p 51\u201364. In Methods in molecular biology. Vol. 665. Humana Press, Clifton, N.J."},{"key":"e_1_3_4_24_2","doi-asserted-by":"publisher","DOI":"10.1099\/jmm.0.001833"},{"key":"e_1_3_4_25_2","doi-asserted-by":"publisher","DOI":"10.1007\/s00705-025-06235-3"},{"key":"e_1_3_4_26_2","doi-asserted-by":"publisher","DOI":"10.1080\/17843286.2020.1837576"},{"key":"e_1_3_4_27_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.jcv.2017.05.001"},{"key":"e_1_3_4_28_2","doi-asserted-by":"publisher","DOI":"10.4269\/ajtmh.2012.11-0346"},{"key":"e_1_3_4_29_2","doi-asserted-by":"publisher","DOI":"10.1093\/trstmh\/trz068"},{"key":"e_1_3_4_30_2","doi-asserted-by":"publisher","DOI":"10.1126\/scitranslmed.aan1589"},{"key":"e_1_3_4_31_2","doi-asserted-by":"publisher","DOI":"10.3390\/medicina60091557"},{"key":"e_1_3_4_32_2","doi-asserted-by":"publisher","DOI":"10.15585\/mmwr.rr6801a1"},{"key":"e_1_3_4_33_2","doi-asserted-by":"publisher","DOI":"10.1128\/JCM.00548-13"},{"key":"e_1_3_4_34_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.jcv.2016.01.007"},{"key":"e_1_3_4_35_2","doi-asserted-by":"publisher","DOI":"10.4269\/ajtmh.18-0024"},{"key":"e_1_3_4_36_2","doi-asserted-by":"publisher","DOI":"10.3390\/v14061271"},{"key":"e_1_3_4_37_2","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-021-26616-x"},{"key":"e_1_3_4_38_2","doi-asserted-by":"publisher","DOI":"10.3390\/v9060137"},{"key":"e_1_3_4_39_2","doi-asserted-by":"publisher","DOI":"10.1186\/s12985-025-02800-z"},{"key":"e_1_3_4_40_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.heliyon.2024.e31252"},{"key":"e_1_3_4_41_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.gene.2024.148306"},{"key":"e_1_3_4_42_2","unstructured":"Thermo Fisher Scientific. 2025. Multiple Primer Analyzer. Available from: https:\/\/www.thermofisher.cn\/cn\/zh\/home\/brands\/thermo-scientific\/molecular-biology\/molecular-biology-learning-center\/molecular-biology-resource-library\/thermo-scientific-web-tools\/multiple-primer-analyzer.html"},{"key":"e_1_3_4_43_2","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pntd.0006381"},{"key":"e_1_3_4_44_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.tim.2013.12.011"},{"key":"e_1_3_4_45_2","unstructured":"World Health Organization. 2024. Dengue and Severe Dengue. Available from: https:\/\/www.who.int\/news-room\/fact-sheets\/detail\/dengue-and-severe-dengue"},{"key":"e_1_3_4_46_2","doi-asserted-by":"publisher","DOI":"10.3390\/bios11070206"},{"key":"e_1_3_4_47_2","doi-asserted-by":"publisher","DOI":"10.1186\/s12864-024-10350-x"}],"container-title":["Microbiology Spectrum"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/journals.asm.org\/doi\/pdf\/10.1128\/spectrum.00718-26","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/journals.asm.org\/doi\/pdf\/10.1128\/spectrum.00718-26","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,10,6]],"date-time":"2026-10-06T13:08:31Z","timestamp":1791292111000},"score":1,"resource":{"primary":{"URL":"https:\/\/journals.asm.org\/doi\/10.1128\/spectrum.00718-26"}},"subtitle":[],"editor":[{"given":"Michael G.","family":"Becker","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"editor"}]}],"short-title":[],"issued":{"date-parts":[[2026,10,6]]},"references-count":46,"journal-issue":{"issue":"10","published-print":{"date-parts":[[2026,10,6]]}},"alternative-id":["10.1128\/spectrum.00718-26"],"URL":"https:\/\/doi.org\/10.1128\/spectrum.00718-26","relation":{},"ISSN":["2165-0497"],"issn-type":[{"value":"2165-0497","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,10,6]]},"assertion":[{"value":"2026-02-28","order":0,"name":"received","label":"Received","group":{"name":"publication_history","label":"Publication History"}},{"value":"2026-07-20","order":2,"name":"accepted","label":"Accepted","group":{"name":"publication_history","label":"Publication History"}},{"value":"2026-09-02","order":3,"name":"published","label":"Published","group":{"name":"publication_history","label":"Publication History"}}],"article-number":"e00718-26"}}