{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,5]],"date-time":"2026-08-05T03:27:26Z","timestamp":1785900446910,"version":"3.56.0"},"reference-count":31,"publisher":"Public Library of Science (PLoS)","issue":"8","license":[{"start":{"date-parts":[[2025,8,18]],"date-time":"2025-08-18T00:00:00Z","timestamp":1755475200000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100010269","name":"Wellcome Trust","doi-asserted-by":"publisher","award":["220211\/Z\/20\/Z"],"award-info":[{"award-number":["220211\/Z\/20\/Z"]}],"id":[{"id":"10.13039\/100010269","id-type":"DOI","asserted-by":"publisher"}]},{"name":"Institut de Recherche pour le D\u00c3\u00a9veloppement"},{"DOI":"10.13039\/100007586","name":"Aix-Marseille Universit\u00c3\u00a9","doi-asserted-by":"publisher","id":[{"id":"10.13039\/100007586","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["www.plosone.org"],"crossmark-restriction":false},"short-container-title":["PLoS One"],"abstract":"<jats:sec id=\"sec001\">\n<jats:title>Introduction<\/jats:title>\n<jats:p>Dengue fever is a growing global concern with an estimated 100\u2013400 million infections every year and rising mortality over the past decade. In 2017, 40,000 deaths were attributed to dengue. Real-time reverse transcription PCR (RT-qPCR) is the gold standard technique to detect dengue virus (DENV) during the acute phase of the infection. However, it requires prior RNA purification which is costly and time consuming. We evaluated direct RT-qPCR using Luna Universal Probe One-Step RT-qPCR kit (Luna RT-qPCR) for the detection of DENV in sera.<\/jats:p>\n<\/jats:sec>\n<jats:sec id=\"sec002\">\n<jats:title>Methods<\/jats:title>\n<jats:p>Luna RT-qPCR conditions were optimized using DENV2 isolates. The efficiency of direct Luna RT-qPCR was evaluated on a panel of 132 patient sera using RNA purification (EZ1&amp;2 Virus Mini Kit) followed by RT-qPCR (SuperScript III Platinum One-Step qRT-PCR system) as reference standard.<\/jats:p>\n<\/jats:sec>\n<jats:sec id=\"sec003\">\n<jats:title>Results<\/jats:title>\n<jats:p>The sensitivity (95% CI) of direct Luna RT-qPCR using neat patient sera was 34% (25\u201345). By reducing PCR inhibitors through a 1\/10 dilution of the sera, the sensitivity improved to 86% (95% CI: 77\u201392). Comparable results were obtained between direct Luna RT-qPCR and reference standard process for samples with Cq\u2009&lt;\u200935.<\/jats:p>\n<\/jats:sec>\n<jats:sec id=\"sec004\">\n<jats:title>Conclusion<\/jats:title>\n<jats:p>The results obtained in this study are promising. Direct RT-qPCR for DENV detection in patient sera, could make PCR-based dengue detection and typing, and potentially other target detections, more affordable for reference laboratories in LMICs by reducing reagent cost by approximately two-thirds. Further studies are needed to evaluate DENV direct RT-qPCR on prospective samples in diagnosis context and to improve the sensitivity by minimizing the impact of inhibitors.<\/jats:p>\n<\/jats:sec>","DOI":"10.1371\/journal.pone.0330459","type":"journal-article","created":{"date-parts":[[2025,8,18]],"date-time":"2025-08-18T17:39:34Z","timestamp":1755538774000},"page":"e0330459","update-policy":"https:\/\/doi.org\/10.1371\/journal.pone.corrections_policy","source":"Crossref","is-referenced-by-count":2,"title":["Direct real-time RT-PCR for the detection of dengue virus from patient serum in Lao PDR"],"prefix":"10.1371","volume":"20","author":[{"given":"Vilayouth","family":"Phimolsannousith","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Malavanh","family":"Vongsouvath","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Padthana","family":"Kiedsathid","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Manivanh","family":"Vongsouvath","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7620-4822","authenticated-orcid":true,"given":"Elizabeth A.","family":"Ashley","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1267-0725","authenticated-orcid":true,"given":"Audrey","family":"Dubot-P\u00e9r\u00e8s","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"340","published-online":{"date-parts":[[2025,8,18]]},"reference":[{"issue":"10427","key":"pone.0330459.ref001","doi-asserted-by":"crossref","first-page":"667","DOI":"10.1016\/S0140-6736(23)02576-X","article-title":"Dengue","volume":"403","author":"G Paz-Bailey","year":"2024","journal-title":"Lancet Lond Engl"},{"key":"pone.0330459.ref002","unstructured":"WHO. Dengue and severe dengue. https:\/\/www.who.int\/news-room\/fact-sheets\/detail\/dengue-and-severe-dengue. 2024. 2024."},{"issue":"12","key":"pone.0330459.ref003","doi-asserted-by":"crossref","DOI":"10.1371\/journal.pgph.0002598","article-title":"Facing the escalating burden of dengue: Challenges and perspectives","volume":"3","author":"GN Malavige","year":"2023","journal-title":"PLOS Glob Public Health"},{"key":"pone.0330459.ref004","doi-asserted-by":"crossref","first-page":"3","DOI":"10.2149\/tmh.2011-S05","article-title":"Dengue, Urbanization and Globalization: The Unholy Trinity of the 21(st) Century","volume":"39","author":"DJ Gubler","year":"2011","journal-title":"Trop Med Health"},{"issue":"8","key":"pone.0330459.ref005","doi-asserted-by":"crossref","DOI":"10.1371\/journal.pntd.0002360","article-title":"An epidemic of dengue-1 in a remote village in rural Laos","volume":"7","author":"A Dubot-P\u00e9r\u00e8s","year":"2013","journal-title":"PLoS Negl Trop Dis"},{"issue":"6","key":"pone.0330459.ref006","article-title":"Characterizing dengue transmission in rural areas: A systematic review","volume":"17","author":"O Man","year":"2023","journal-title":"PLoS Negl Trop Dis"},{"issue":"1","key":"pone.0330459.ref007","doi-asserted-by":"crossref","DOI":"10.1016\/S2214-109X(13)70008-1","article-title":"Causes of non-malarial fever in Laos: a prospective study","volume":"1","author":"M Mayxay","year":"2013","journal-title":"Lancet Glob Health"},{"issue":"3","key":"pone.0330459.ref008","doi-asserted-by":"crossref","first-page":"517","DOI":"10.4269\/ajtmh.14-0772","article-title":"Causes of Fever in Rural Southern Laos","volume":"93","author":"M Mayxay","year":"2015","journal-title":"Am J Trop Med Hyg"},{"issue":"1","key":"pone.0330459.ref009","first-page":"7","article-title":"National dengue surveillance in the Lao People\u2019s Democratic Republic, 2006-2012: epidemiological and laboratory findings","volume":"5","author":"B Khampapongpane","year":"2014","journal-title":"WPSAR"},{"issue":"9","key":"pone.0330459.ref010","doi-asserted-by":"crossref","first-page":"728","DOI":"10.3390\/pathogens9090728","article-title":"Trends of the Dengue Serotype-4 Circulation with Epidemiological, Phylogenetic, and Entomological Insights in Lao PDR between 2015 and 2019","volume":"9","author":"E Calvez","year":"2020","journal-title":"Pathog"},{"issue":"2","key":"pone.0330459.ref011","doi-asserted-by":"crossref","first-page":"243","DOI":"10.3390\/microorganisms11020243","article-title":"A six years (2010-2016) longitudinal survey of the four serotypes of dengue viruses in Lao PDR","volume":"11","author":"C Bali\u00e8re","year":"2023","journal-title":"Microorganisms"},{"issue":"12","key":"pone.0330459.ref012","article-title":"Co-circulation of dengue virus type 3 genotypes in Vientiane capital, Lao PDR","volume":"9","author":"M Lao","year":"2014","journal-title":"PLoS One"},{"issue":"1","key":"pone.0330459.ref013","doi-asserted-by":"crossref","DOI":"10.1371\/journal.pntd.0006203","article-title":"Molecular epidemiology of dengue viruses in three provinces of Lao PDR, 2006-2010","volume":"12","author":"J Castonguay-Vanier","year":"2018","journal-title":"PLoS Negl Trop Dis"},{"issue":"7","key":"pone.0330459.ref014","doi-asserted-by":"crossref","first-page":"101088","DOI":"10.1016\/j.lanmic.2025.101088","article-title":"Evaluating the performance of common reference laboratory tests for acute dengue diagnosis: a systematic review and meta-analysis of RT-PCR, NS1 ELISA, and IgM ELISA","volume":"6","author":"K Pillay","year":"2025","journal-title":"Lancet Microbe"},{"issue":"9","key":"pone.0330459.ref015","article-title":"Evaluation of rapid diagnostic tests to detect dengue virus infections in Taiwan","volume":"15","author":"L-T Liu","year":"2020","journal-title":"PLoS One"},{"key":"pone.0330459.ref016","doi-asserted-by":"crossref","first-page":"104587","DOI":"10.1016\/j.jcv.2020.104587","article-title":"Use of a simplified sample processing step without RNA extraction for direct SARS-CoV-2 RT-PCR detection","volume":"132","author":"R Barza","year":"2020","journal-title":"J Clin Virol"},{"issue":"5","key":"pone.0330459.ref017","doi-asserted-by":"crossref","first-page":"1091","DOI":"10.3390\/diagnostics12051091","article-title":"Clinical Evaluation of Nasopharyngeal, Oropharyngeal, Nasal Swabs, and Saliva for the Detection of SARS-CoV-2 by Direct RT-PCR","volume":"12","author":"SA Kiryanov","year":"2022","journal-title":"Diagnostics (Basel)"},{"key":"pone.0330459.ref018","doi-asserted-by":"crossref","first-page":"104579","DOI":"10.1016\/j.jcv.2020.104579","article-title":"Extraction-free SARS-CoV-2 detection by rapid RT-qPCR universal for all primary respiratory materials","volume":"130","author":"N L\u00fcbke","year":"2020","journal-title":"J Clin Virol"},{"key":"pone.0330459.ref019","doi-asserted-by":"crossref","first-page":"114199","DOI":"10.1016\/j.jviromet.2021.114199","article-title":"SARS-CoV-2 detection by extraction-free qRT-PCR for massive and rapid COVID-19 diagnosis during a pandemic in Armenia","volume":"295","author":"D Avetyan","year":"2021","journal-title":"J Virol Methods"},{"issue":"10","key":"pone.0330459.ref020","doi-asserted-by":"crossref","DOI":"10.1371\/journal.pbio.3000896","article-title":"Direct RT-qPCR detection of SARS-CoV-2 RNA from patient nasopharyngeal swabs without an RNA extraction step","volume":"18","author":"EA Bruce","year":"2020","journal-title":"PLoS Biol"},{"issue":"10","key":"pone.0330459.ref021","doi-asserted-by":"crossref","first-page":"2825","DOI":"10.1007\/s00705-021-05165-0","article-title":"Evaluation of extraction-free RT-qPCR methods for SARS-CoV-2 diagnostics","volume":"166","author":"A Domnich","year":"2021","journal-title":"Arch Virol"},{"key":"pone.0330459.ref022","doi-asserted-by":"crossref","first-page":"264","DOI":"10.1016\/j.ijid.2021.09.046","article-title":"Evaluation of extraction-free RT-PCR methods for faster and cheaper detection of SARS-CoV-2 using two commercial systems","volume":"112","author":"F Morecchiato","year":"2021","journal-title":"Int J Infect Dis"},{"issue":"4","key":"pone.0330459.ref023","doi-asserted-by":"crossref","first-page":"291","DOI":"10.1101\/gr.1.4.291","article-title":"Direct PCR amplification of HCV RNA from human serum","volume":"1","author":"A Ravaggi","year":"1992","journal-title":"PCR Methods Appl"},{"issue":"1","key":"pone.0330459.ref024","doi-asserted-by":"crossref","first-page":"54","DOI":"10.1002\/jmv.1890440111","article-title":"Rapid detection of virus genome from imported dengue fever and dengue hemorrhagic fever patients by direct polymerase chain reaction","volume":"44","author":"K Morita","year":"1994","journal-title":"J Med Virol"},{"issue":"2","key":"pone.0330459.ref025","doi-asserted-by":"crossref","first-page":"282","DOI":"10.1016\/j.jviromet.2009.10.011","article-title":"Detection of noroviruses in fecal specimens by direct RT-PCR without RNA purification","volume":"163","author":"N Nishimura","year":"2010","journal-title":"J Virol Methods"},{"issue":"6","key":"pone.0330459.ref026","doi-asserted-by":"crossref","first-page":"1534","DOI":"10.4269\/ajtmh.19-0138","article-title":"A direct from blood\/plasma reverse transcription\u2013polymerase chain reaction for dengue virus detection in point-of-care settings","volume":"100","author":"N Mehta","year":"2019","journal-title":"Am J Trop Med Hyg"},{"key":"pone.0330459.ref027","author":"MB Nathan","year":"2009"},{"issue":"1","key":"pone.0330459.ref028","doi-asserted-by":"crossref","first-page":"61","DOI":"10.1016\/j.jcv.2009.02.010","article-title":"Development and validation of real-time one-step reverse transcription-PCR for the detection and typing of dengue viruses","volume":"45","author":"I Leparc-Goffart","year":"2009","journal-title":"J Clin Virol"},{"issue":"2","key":"pone.0330459.ref029","doi-asserted-by":"crossref","first-page":"159","DOI":"10.3390\/v12020159","article-title":"Lyophilized Matrix Containing Ready-to-Use Primers and Probe Solution for Standardization of Real-Time PCR and RT-qPCR Diagnostics in Virology","volume":"12","author":"L Thirion","year":"2020","journal-title":"Viruses"},{"issue":"1","key":"pone.0330459.ref030","doi-asserted-by":"crossref","first-page":"4812","DOI":"10.1038\/s41467-020-18611-5","article-title":"Massive and rapid COVID-19 testing is feasible by extraction-free SARS-CoV-2 RT-PCR","volume":"11","author":"I Smyrlaki","year":"2020","journal-title":"Nature Communications"},{"key":"pone.0330459.ref031","doi-asserted-by":"crossref","first-page":"114086","DOI":"10.1016\/j.jviromet.2021.114086","article-title":"Evaluation of three extraction-free SARS-CoV-2 RT-PCR assays: A feasible alternative approach with low technical requirements","volume":"291","author":"B Visseaux","year":"2021","journal-title":"J Virol Methods"}],"container-title":["PLOS One"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/dx.plos.org\/10.1371\/journal.pone.0330459","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,8,18]],"date-time":"2025-08-18T17:39:40Z","timestamp":1755538780000},"score":1,"resource":{"primary":{"URL":"https:\/\/dx.plos.org\/10.1371\/journal.pone.0330459"}},"subtitle":[],"editor":[{"given":"Nilanka","family":"Perera","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"editor"}]}],"short-title":[],"issued":{"date-parts":[[2025,8,18]]},"references-count":31,"journal-issue":{"issue":"8","published-online":{"date-parts":[[2025,8,18]]}},"URL":"https:\/\/doi.org\/10.1371\/journal.pone.0330459","relation":{},"ISSN":["1932-6203"],"issn-type":[{"value":"1932-6203","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,8,18]]}}}