{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,19]],"date-time":"2026-08-19T17:41:37Z","timestamp":1787161297674,"version":"build-2736575974"},"reference-count":62,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2025,5,14]],"date-time":"2025-05-14T00:00:00Z","timestamp":1747180800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Cell. Infect. Microbiol."],"abstract":"<jats:sec>\n                    <jats:title>Introduction<\/jats:title>\n                    <jats:p>Dengue virus (DENV) is the most rapidly spreading arbovirus globally, with over half of the world\u2019s population at risk of infection. Early and rapid detection is crucial to ensure timely patient care, reduce healthcare burden, and prevent severe disease progression. However, conventional nucleic acid amplification techniques are often unsuitable for low-resource settings due to their equipment and procedural demands.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Methods<\/jats:title>\n                    <jats:p>We evaluated a real-time reverse transcription recombinase polymerase amplification (RT-RPA) assay for the sensitive and specific detection of DENV serotype 2 (DENV2). The assay was tested using both Twista fluorometer and lateral flow detection (LFD) formats. Analytical sensitivity was determined by probit regression, while specificity was assessed against unrelated viruses and other flaviviruses. Clinical validation was performed using serum, cell culture, and FTA\u00ae card samples. Assay robustness was evaluated under varying temperatures and after freeze-thaw cycles.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Results<\/jats:title>\n                    <jats:p>The RT-RPA assay reliably amplified DENV2 at concentrations as low as 50 copies per reaction, with LOD\u2089\u2085 estimated at 38.48 copies (Twista) and 50.37 copies (LFD). No cross-reactivity was observed with respiratory syncytial virus, influenza, rabbit herpes virus, West Nile virus, or other DENV serotypes (DENV1, DENV3, DENV4). The assay successfully detected multiple DENV2 strains and maintained performance across 33\u00b0C\u201340\u00b0C and after repeated freeze-thaw cycles. RNA extracted from FTA\u00ae cards was successfully amplified. Clinical validation confirmed accurate detection in serum and cell culture samples, while DENV3-positive blood samples tested negative, reinforcing specificity.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Discussion<\/jats:title>\n                    <jats:p>The RT-RPA\/LFD assay offers a rapid, sensitive, and specific tool for DENV2 detection, compatible with low-resource and field-based settings. Its simplicity, robustness, and portability make it a promising approach for point-of-care diagnostics and outbreak surveillance in endemic regions.<\/jats:p>\n                  <\/jats:sec>","DOI":"10.3389\/fcimb.2025.1578549","type":"journal-article","created":{"date-parts":[[2025,5,14]],"date-time":"2025-05-14T01:37:05Z","timestamp":1747186625000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":9,"title":["Development of a rapid point-of-care dengue virus type 2 infection diagnostic assay using recombinase polymerase amplification and lateral flow device"],"prefix":"10.3389","volume":"15","author":[{"given":"Meagan A.","family":"Prescott","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Myra T.","family":"Koesdjojo","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"David T.","family":"Mandrell","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Manoj K.","family":"Pastey","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1965","published-online":{"date-parts":[[2025,5,14]]},"reference":[{"key":"B1","doi-asserted-by":"publisher","first-page":"503","DOI":"10.1017\/S0950268804003541","article-title":"Serological and virological features of dengue fever and dengue haemorrhagic fever in Thailand from 1999 to 2002","volume":"133","author":"Ananta Preecha","year":"2005","journal-title":"Epidemiol. Infect."},{"key":"B2","doi-asserted-by":"publisher","first-page":"473","DOI":"10.4269\/ajtmh.2011.10-0521","article-title":"Health economics of dengue: a systematic literature review and expertpanel\u2019s assessment","volume":"84","author":"Beatty","year":"2011","journal-title":"Am. J. Trop. Med. Hyg"},{"key":"B3","doi-asserted-by":"publisher","DOI":"10.1002\/jmv.70219","article-title":"RT-RPA assisted CRISPR\/cas12a based one-pot rapid and visual detection of the pan-dengue virus","volume":"97","author":"Bhardwaj","year":"2025","journal-title":"J. Med. Virol."},{"key":"B4","doi-asserted-by":"publisher","first-page":"504","DOI":"10.1038\/nature12060","article-title":"The global distribution and burden of dengue","volume":"496","author":"Bhatt","year":"2013","journal-title":"Nature"},{"key":"B5","doi-asserted-by":"publisher","first-page":"804","DOI":"10.1128\/CVI.05717-11","article-title":"Comparison of seven commercial antigen and antibody enzyme-linked immunosorbent assays for detection of acute dengue infection","volume":"19","author":"Blacksell","year":"2012","journal-title":"Clin. Vaccine Immunol."},{"key":"B6","doi-asserted-by":"publisher","first-page":"52","DOI":"10.1093\/clinchem\/hvab194","article-title":"Lateral flow assays in infectious disease diagnosis","volume":"68","author":"Boehringer","year":"2021","journal-title":"Clin. Chem."},{"key":"B7","doi-asserted-by":"publisher","DOI":"10.1016\/j.jviromet.2021.114351","article-title":"Rapid reverse transcriptase recombinase polymerase amplification assay for flaviviruses using non-infectious in vitro transcribed RNA as positive controls","volume":"299","author":"Bonnet","year":"2022","journal-title":"J. Virological Methods"},{"key":"B8","doi-asserted-by":"publisher","first-page":"30","DOI":"10.1016\/j.diagmicrobio.2015.05.004","article-title":"Development of a pan-serotype reverse transcription loop-mediated isothermal amplification assay for the detection of dengue virus","volume":"83","author":"Dauner","year":"2015","journal-title":"Diagn. Microbiol. Infect. Dis."},{"key":"B9","doi-asserted-by":"publisher","first-page":"34","DOI":"10.1016\/j.jviromet.2008.09.023","article-title":"A retrospective survey of dengue virus infection in fatal cases from an epidemic in Brazil","author":"de Ara\u00fajo","year":"2009","journal-title":"J. Virol Methods"},{"key":"B10","doi-asserted-by":"publisher","first-page":"360","DOI":"10.4269\/ajtmh.14-0139","article-title":"Economic cost and burden of dengue in the Philippines","volume":"92","author":"Edillo","year":"2015","journal-title":"Am. J. Trop. Med. Hyg"},{"key":"B11","doi-asserted-by":"publisher","first-page":"990","DOI":"10.1086\/382280","article-title":"Relationship of preexisting dengue virus (DV) neutralizing antibody levels to viremia and severity of disease in a prospective cohort study of DV infection in Thailand","volume":"189","author":"Endy","year":"2004","journal-title":"J. Infect. Dis."},{"key":"B12","doi-asserted-by":"publisher","first-page":"1972","DOI":"10.1128\/CVI.00535-12","article-title":"Low sensitivity of NS1 protein tests evidenced during a dengue type 2 virus outbreak in Santos, Brazil","volume":"19","author":"Felix","year":"2012","journal-title":"Clin. Vaccine Immunol."},{"key":"B13","doi-asserted-by":"publisher","first-page":"347","DOI":"10.1590\/S0074-02761990000300012","article-title":"Serological diagnosis of dengue by an ELISA inhibition method (EIM)","volume":"85","author":"Fern\u00e1ndez","year":"1990","journal-title":"Mem Inst Oswaldo Cruz"},{"key":"B14","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pntd.0000617","article-title":"Serotype-specific differences in the risk of dengue hemorrhagic fever: an analysis of data collected in Bangkok, Thailand from 1994 to 2006","volume":"4","author":"Fried","year":"2010","journal-title":"PloS Negl. Trop. Dis."},{"key":"B15","doi-asserted-by":"publisher","first-page":"910","DOI":"10.4269\/ajtmh.2007.77.910","article-title":"Analysis of repeat hospital admissions for dengue to estimate the frequency of third or fourth dengue infections resulting in admissions and dengue hemorrhagic fever, and serotype sequences","volume":"77","author":"Gibbons","year":"2007","journal-title":"Am. J. Trop. Med. Hyg"},{"key":"B16","doi-asserted-by":"publisher","first-page":"f6280","DOI":"10.1136\/bmj.f6280","article-title":"Burden of dengue fever is higher than previously thought","volume":"347","author":"Gulland","year":"2013","journal-title":"BMJ"},{"key":"B17","doi-asserted-by":"publisher","first-page":"1445","DOI":"10.1007\/s00705-013-1645-3","article-title":"Secondary infection as a risk factor for dengue hemorrhagic fever\/dengue shock syndrome: an historical perspective and role of antibody-dependent enhancement of infection","volume":"158","author":"Guzman","year":"2013","journal-title":"Arch. Virol"},{"key":"B18","doi-asserted-by":"publisher","first-page":"407","DOI":"10.1590\/S1020-49892006001100007","article-title":"Dengue in the Americas and Southeast Asia: do they differ","volume":"20","author":"Halstead","year":"2006","journal-title":"Rev. Panam Salud Publica"},{"key":"B19","doi-asserted-by":"publisher","first-page":"436","DOI":"10.3201\/eid1503.080923","article-title":"Evaluation of commercially available anti-dengue virus immunoglobulin M tests","volume":"15","author":"Hunsperger","year":"2009","journal-title":"Emerg Infect. Dis."},{"key":"B20","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pntd.0003171","article-title":"Evaluation of commercially available diagnostic tests for the detection of dengue virus NS1 antigen and anti-dengue virus IgM antibody","volume":"8","author":"Hunsperger","year":"2014","journal-title":"PloS Negl. Trop. Dis."},{"key":"B21","doi-asserted-by":"publisher","first-page":"4977","DOI":"10.1128\/JCM.43.10.4977-4983.2005","article-title":"Serotype-specific detection of dengue viruses in a fourplex real-time reverse transcriptase PCR assay","volume":"43","author":"Johnson","year":"2005","journal-title":"J. Clin. Microbiol"},{"key":"B22","doi-asserted-by":"publisher","DOI":"10.3390\/bios11070206","article-title":"Dengue detection: advances in diagnostic tools from conventional technology to point of care","volume":"11","author":"Kabir","year":"2021","journal-title":"Biosensors"},{"key":"B23","doi-asserted-by":"publisher","DOI":"10.1016\/j.mtbio.2024.101188","article-title":"Lateral flow assays: Progress and evolution of recent trends in point-of-care applications","volume":"28","author":"Kakkar","year":"2024","journal-title":"Mater Today Bio"},{"key":"B24","doi-asserted-by":"publisher","first-page":"545","DOI":"10.1128\/jcm.30.3.545-551.1992","article-title":"Rapid detection and typing of dengue viruses from clinical samples by using reverse transcriptase-polymerase chain reaction","volume":"30","author":"Lanciotti","year":"1992","journal-title":"J. Clin. Microbiol"},{"key":"B25","doi-asserted-by":"publisher","first-page":"2019","DOI":"10.1039\/D0LC01222J","article-title":"A microfluidic-integrated lateral flow recombinase polymerase amplification (MI-IF-RPA) assay for rapid COVID-19 detection","volume":"21","author":"Liu","year":"2021","journal-title":"Lab. Chip"},{"key":"B26","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pntd.0006381","article-title":"Development and validation of four one-step real-time RT-LAMP assays for specific detection of each dengue virus serotype","volume":"12","author":"Lopez-Jimena","year":"2018","journal-title":"PloS Negl. Trop. Dis."},{"key":"B27","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pgph.0002598","article-title":"Facing the escalating burden of dengue: Challenges and perspectives","volume":"3","author":"Malavige","year":"2023","journal-title":"PloS Glob Public Health"},{"key":"B28","doi-asserted-by":"publisher","first-page":"15D.2","DOI":"10.1002\/9780471729259.mc15d02s27","article-title":"Dengue virus: isolation, propagation, quantification, and storage","volume":"15","author":"Medina","year":"2012","journal-title":"Curr. Protoc. Microbiol"},{"key":"B29","doi-asserted-by":"publisher","first-page":"185","DOI":"10.1111\/jtm.12018","article-title":"Detection of dengue virus nonstructural protein 1 (NS1) by using ELISA as a useful laboratorydiagnostic method for dengue virus infection of international travelers","volume":"20","author":"Moi","year":"2013","journal-title":"J. Travel Med."},{"key":"B30","doi-asserted-by":"publisher","DOI":"10.1186\/1743-422X-11-164","article-title":"Evaluation of four commercial real-time RT-PCR kits for the detection of dengue viruses in clinical samples","volume":"11","author":"Najioullah","year":"2014","journal-title":"Virol J."},{"key":"B31","doi-asserted-by":"publisher","DOI":"10.1186\/s12879-014-0570-8","article-title":"Dengue NS1 antigen as a marker of severe clinical disease","volume":"14","author":"Paranavitane","year":"2014","journal-title":"BMC Infect. Dis."},{"key":"B32","doi-asserted-by":"publisher","first-page":"S30","DOI":"10.1038\/nrmicro2459","article-title":"Evaluation of diagnostic tests: dengue","volume":"8","author":"Peeling","year":"2010","journal-title":"Nat. Rev. Microbiol"},{"key":"B33","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pbio.0040204","article-title":"DNA detection using recombination proteins","volume":"4","author":"Piepenburg","year":"2006","journal-title":"PloS Biol."},{"key":"B34","doi-asserted-by":"publisher","first-page":"1328","DOI":"10.1111\/j.1365-3156.2008.02151.x","article-title":"Clinical and laboratory features that distinguish dengue from other febrile illnesses in endemic populations","volume":"13","author":"Potts","year":"2008","journal-title":"Trop. Med. Int. Health"},{"key":"B35","doi-asserted-by":"publisher","first-page":"246","DOI":"10.1016\/j.mjafi.2012.07.017","article-title":"Reverse transcription loop-mediated isothermal amplification (RT-LAMP) for diagnosis of dengue","volume":"69","author":"Sahni","year":"2013","journal-title":"Med. J. Armed Forces India"},{"key":"B36","doi-asserted-by":"publisher","DOI":"10.7883\/yoken.JJID.2014.441","article-title":"Detection of dengue virus nonstructural protein 1 (NS1) in urine samples using ELISA as a laboratory diagnostic method for dengue virus infection","volume":"68","author":"Saito","year":"2015","journal-title":"Jpn J. Infect. Dis"},{"key":"B37","doi-asserted-by":"publisher","DOI":"10.4269\/ajtmh.14-0755","article-title":"Economic burden of dengue virus infection at the household level among residents of puerto maldonado, Peru","volume":"93","author":"Salmon-Mulanovich","year":"2015","journal-title":"Am. J. Trop. Med. Hyg"},{"key":"B38","doi-asserted-by":"publisher","first-page":"653","DOI":"10.1093\/oxfordjournals.aje.a113932","article-title":"Risk factors in dengue shock syndrome: a prospective epidemiologic study in Rayong, Thailand. I. The 1980 outbreak","volume":"120","author":"Sangkawibha","year":"1984","journal-title":"Am. J. Epidemiol."},{"key":"B39","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pntd.0002311","article-title":"Analytical and clinical performance of the CDC real time RT-PCR assay for detection and typing of dengue virus","volume":"7","author":"Santiago","year":"2013","journal-title":"PloS Negl. Trop. Dis."},{"key":"B40","doi-asserted-by":"publisher","first-page":"1235","DOI":"10.4269\/ajtmh.14-0002","article-title":"Economic and disease burden of dengue illness in India","volume":"91","author":"Shepard","year":"2014","journal-title":"Am. J. Trop. Med. Hyg"},{"key":"B41","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pntd.0002055","article-title":"Economic and disease burden of dengue in Southeast Asia","volume":"7","author":"Shepard","year":"2013","journal-title":"PloS Negl. Trop. Dis."},{"key":"B42","doi-asserted-by":"publisher","first-page":"669","DOI":"10.4103\/ijmr.IJMR_1219_18","article-title":"Concurrent dengue infections: Epidemiology & clinical implications","volume":"154","author":"Sirisena","year":"2021","journal-title":"Indian J. Med. Res."},{"key":"B43","doi-asserted-by":"publisher","first-page":"3591","DOI":"10.1007\/s00216-019-01840-zTeoh","article-title":"Visual signal generation for the detection of influenza viruses by duplex recombinase polymerase amplification with lateral flow dipsticks","volume":"411","author":"Sun","year":"2019","journal-title":"Anal. Bioanal Chem."},{"key":"B44","doi-asserted-by":"publisher","DOI":"10.1128\/jcm.02648-14","article-title":"Early detection of dengue virus by use of reverse transcription-recombinase polymerase amplification","volume":"53","author":"Teoh","year":"2015","journal-title":"J. Clin. Microbiol."},{"key":"B45","doi-asserted-by":"publisher","first-page":"990","DOI":"10.4269\/ajtmh.2008.78.990","article-title":"Influence of the dengue serotype, previous dengue infection, and plasma viral load on clinical presentation and outcome during a dengue-2 and dengue-4 co-epidemic","volume":"78","author":"Thomas","year":"2008","journal-title":"Am. J. Trop. Med. Hyg"},{"key":"B46","doi-asserted-by":"publisher","first-page":"515","DOI":"10.1007\/s11910-022-01213-7","article-title":"Neurological complications of dengue fever","volume":"22","author":"Trivedi","year":"2022","journal-title":"Curr. Neurol. Neurosci. Rep."},{"key":"B47","volume-title":"Appendix to the TwistAmpTM reaction kit manuals","author":"Twist","year":"2018"},{"key":"B48","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pntd.0003547","article-title":"Economic and disease burden of dengue in Mexico","volume":"9","author":"Undurraga","year":"2015","journal-title":"PloS Negl. Trop. Dis."},{"key":"B49","doi-asserted-by":"publisher","first-page":"2","DOI":"10.1086\/315215","article-title":"Dengue viremia titer, antibody response pattern, and virus serotype correlate with disease severity","volume":"181","author":"Vaughn","year":"2000","journal-title":"J. Infect. Dis."},{"key":"B50","doi-asserted-by":"publisher","DOI":"10.1128\/jcm.00203-18.https:\/\/doi.org\/10.1128\/jcm.00203-18","article-title":"Potential point-of-Care testing for dengue virus in the field","volume":"56","author":"Wang","year":"2018","journal-title":"J. Clin. Microbiol"},{"key":"B51","doi-asserted-by":"publisher","first-page":"7221","DOI":"10.1016\/j.vaccine.2011.07.022","article-title":"The pathogenesis of dengue","volume":"29","author":"Whitehorn","year":"2011","journal-title":"Vaccine"},{"key":"B52","volume-title":"Global strategy for dengue prevention and control. edited by WHO","year":"2012"},{"key":"B53","unstructured":"2025"},{"key":"B54","volume-title":"Dengue: guidelines for diagnosis, treatment, prevention and control. edited by WHO","year":"2009"},{"key":"B55","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pntd.0000996","article-title":"Dengue in Thailand and Cambodia: an assessment of the degree of underrecognized disease burden based on reported cases","volume":"5","author":"Wichmann","year":"2011","journal-title":"PloS Negl. Trop. Dis."},{"key":"B56","doi-asserted-by":"publisher","first-page":"2794","DOI":"10.1128\/JCM.39.8.2794-2798.2001","article-title":"Detection of dengue viral RNA using a nucleic acid sequence-based amplification assay","volume":"39","author":"Wu","year":"2001","journal-title":"J. Clin. Microbiol"},{"key":"B57","doi-asserted-by":"publisher","first-page":"101413","DOI":"10.1016\/j.mcp.2019.06.003","article-title":"Rapid and visual detection of dengue virus using recombinase polymerase amplification method combined with lateral flow dipstick","volume":"46","author":"Xi","year":"2019","journal-title":"Mol. Cell. Probes"},{"key":"B58","doi-asserted-by":"publisher","DOI":"10.3389\/fmicb.2021.629533","article-title":"One-step reverse-transcription recombinase polymerase amplification using lateral flow strips for the detection of coxsackievirus A6","volume":"12","author":"Xie","year":"2021","journal-title":"Front. Microbiol."},{"key":"B59","doi-asserted-by":"publisher","first-page":"406","DOI":"10.1016\/j.ijid.2020.03.075","article-title":"Rapid visual detection of dengue virus by combining reverse transcription recombinase-aided amplification with lateral-flow dipstick assay","volume":"95","author":"Xiong","year":"2020","journal-title":"Int. J. Infect. diseases: IJID: Off. Publ. Int. Soc. Infect. Dis."},{"key":"B60","doi-asserted-by":"publisher","first-page":"203","DOI":"10.1016\/S1386-6532(01)00250-5","article-title":"Virus isolation as one of the diagnostic methods for dengue virus infection","volume":"24","author":"Yamada","year":"2002","journal-title":"J. Clin. Virol"},{"key":"B61","doi-asserted-by":"publisher","DOI":"10.1016\/j.jviromet.2021.114094","article-title":"Rapid and sensitive detection of hepatitis B virus by lateral flow recombinase polymerase amplification assay","volume":"291","author":"Zhang","year":"2021","journal-title":"J. Virological Methods"},{"key":"B62","doi-asserted-by":"publisher","DOI":"10.3389\/fcimb.2024.1475922","article-title":"Application of recombinase polymerase amplification with lateral flow assay to pathogen point-of-care diagnosis","volume":"14","author":"Zhao","year":"2024","journal-title":"Front. Cell Infect. Microbiol"}],"container-title":["Frontiers in Cellular and Infection Microbiology"],"original-title":[],"link":[{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fcimb.2025.1578549\/full","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,5,14]],"date-time":"2025-05-14T01:37:08Z","timestamp":1747186628000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fcimb.2025.1578549\/full"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,5,14]]},"references-count":62,"alternative-id":["10.3389\/fcimb.2025.1578549"],"URL":"https:\/\/doi.org\/10.3389\/fcimb.2025.1578549","relation":{},"ISSN":["2235-2988"],"issn-type":[{"value":"2235-2988","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,5,14]]},"article-number":"1578549"}}