{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,3]],"date-time":"2026-09-03T15:28:49Z","timestamp":1788449329474,"version":"build-2803163510"},"reference-count":49,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2021,2,23]],"date-time":"2021-02-23T00:00:00Z","timestamp":1614038400000},"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:p>West Nile virus (WNV) causes West Nile fever and encephalitis worldwide. Currently, there are no effective drugs or vaccines available in the market to treat WNV infection in humans. Hence, it is of paramount importance to detect WNV early for the success of the disease control programs and timely clinical management in endemic areas. In the present paper, we report the development of real-time reverse transcription recombinase polymerase amplification (RT-RPA) assay for rapid and real-time detection of WNV targeting the envelope (env) gene of the virus. The RPA reaction was performed successfully at 39\u00b0C for 15\u00a0min in a real-time thermal cycler. The sensitivity of this assay was found similar to that of the quantitative real-time RT PCR (RT-qPCR) assay, which could detect 10 copies of the gene. The efficacy of the assay was evaluated with a panel of 110 WN suspected human samples showing the signs of retinitis, febrile illness and acute posterior uveitis. In comparison with RT-qPCR, RT-RPA showed a specificity of 100% (CI, 95.07\u2013100%) and sensitivity of 96.15% (CI, 80.36\u201399.90%) with a negative (NPV) and positive predictive value (PPV) of 98.65 and 100%, respectively. The level of agreement between RT-RPA and reference RT-qPCR assay was shown to be very high. The turnaround time of real-time RPA assay is about 10-20 times faster than the RT-qPCR, which confirms its utility in the rapid and sensitive diagnosis of WNV infection. To the best of our knowledge, this is the first report which deals with the development of real-time RT-RPA assay for simple, rapid, sensitive, and specific detection of WNV in human clinical samples. The present RT-RPA assay proves to be a powerful tool that can be used for the rapid diagnosis of a large number of patient samples in endemic settings.<\/jats:p>","DOI":"10.3389\/fcimb.2020.619071","type":"journal-article","created":{"date-parts":[[2021,2,23]],"date-time":"2021-02-23T04:44:25Z","timestamp":1614055465000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":7,"title":["Development and Evaluation of Real-Time Reverse Transcription Recombinase Polymerase Amplification Assay for Rapid and Sensitive Detection of West Nile Virus in Human Clinical Samples"],"prefix":"10.3389","volume":"10","author":[{"given":"Priyanka Singh","family":"Tomar","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sanjay","family":"Kumar","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sapan","family":"Patel","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jyoti S.","family":"Kumar","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1965","published-online":{"date-parts":[[2021,2,23]]},"reference":[{"key":"B1","doi-asserted-by":"publisher","first-page":"1","DOI":"10.2807\/1560-7917-ES2018.23.43.1800545","article-title":"Increase in human West Nile and Usutu virus infections, Austria 2018","volume":"23","author":"Aberle","year":"2018","journal-title":"Euro Surveill."},{"key":"B2","doi-asserted-by":"publisher","first-page":"152","DOI":"10.1016\/j.jcv.2014.06.003","article-title":"West Nile Encephalitis outbreak in Kerala, India","volume":"61","author":"Anukumar","year":"2011","journal-title":"J. Clin. Virol."},{"key":"B3","first-page":"733","article-title":"Preliminary observation on antibody patterns against certain viruses among inhabitants of Bombay City. Indian","volume":"6","author":"Banker","year":"1952","journal-title":"J. @ Med. Res."},{"key":"B4","doi-asserted-by":"publisher","first-page":"875","DOI":"10.1099\/vir.0.82403-0","article-title":"West Nile virus isolates from India: evidence for a distinct genetic lineage","volume":"88","author":"Bondre","year":"2007","journal-title":"J. Gen. Virol."},{"key":"B5","doi-asserted-by":"publisher","DOI":"10.3389\/fmicb.2016.00554","article-title":"Visual detection of West Nile Virus using reverse transcription loop-mediated isothermal amplification combined with a vertical flow visualization strip","volume":"7","author":"Cao","year":"2016","journal-title":"Front. Microbiol."},{"key":"B6","article-title":"West Nile virus","year":"2019"},{"key":"B7","first-page":"185","article-title":"NASBA and other transcription-based amplification methods for research and diagnostic microbiology","volume":"10","author":"Chan","year":"1999","journal-title":"Clin. Microbiol."},{"key":"B8","doi-asserted-by":"publisher","first-page":"2562","DOI":"10.1155\/2015\/376230","article-title":"The Global ecology and epidemiology of West Nile Virus","volume":"47","author":"Chancey","year":"2015","journal-title":"BioMed. Res. Int."},{"key":"B9","doi-asserted-by":"crossref","first-page":"37","DOI":"10.1177\/001316446002000104","article-title":"A coefficient of agreement for nominal scales.Educ","volume":"20","author":"Cohen","year":"1960","journal-title":"Psychol. Meas."},{"key":"B10","doi-asserted-by":"publisher","first-page":"635","DOI":"10.1128\/CMR.00045-12","article-title":"West Nile Virus: biology, transmission, and human infection","volume":"25","author":"Colpitts","year":"2012","journal-title":"Clin. Microbiol. Review"},{"key":"B11","doi-asserted-by":"publisher","first-page":"91","DOI":"10.1038\/350091a0","article-title":"Nucleic acid sequence-based amplification","volume":"350","author":"Compton","year":"1991","journal-title":"Nature"},{"key":"B12","doi-asserted-by":"publisher","first-page":"947","DOI":"10.1373\/clinchem.2015.245829","article-title":"Recombinase polymerase Amplification for diagnostic applications","volume":"62","author":"Daher","year":"2016","journal-title":"Clin.Chem."},{"key":"B13","doi-asserted-by":"publisher","first-page":"10","DOI":"10.2807\/1560-7917.ES.2015.20.44.30053","article-title":"Development and deployment of a rapid recombinase polymerase amplification Ebola virus detection assay in Guinea in 2015","volume":"20","author":"Faye","year":"2015","journal-title":"Euro Surveill."},{"key":"B14","doi-asserted-by":"publisher","first-page":"6103","DOI":"10.1128\/JVI.02042-09","article-title":"Interplay of RNA elements in the dengue virus 5\u2019 and 3\u2019 ends required for viral RNA replication","volume":"84","author":"Friebe","year":"2010","journal-title":"J. Virol."},{"key":"B15","doi-asserted-by":"publisher","first-page":"189","DOI":"10.1007\/s00705-017-3321-5","article-title":"Polymerase spiral reaction (PSR): a novel, visual isothermal amplification method for detection of canine parvovirus 2 genomic DNA","volume":"20","author":"Gupta","year":"2017","journal-title":"Arch. Virol."},{"key":"B16","doi-asserted-by":"publisher","first-page":"1204","DOI":"10.1371\/journal.pone.0101419.t001","article-title":"Recombinations in staphylococcal cassette chromosome mec elements compromise the molecular detection of methicillin resistance in Staphylococcus aureus","volume":"24","author":"Hill","year":"2014","journal-title":"PLoS One"},{"key":"B17","doi-asserted-by":"publisher","first-page":"827","DOI":"10.1111\/j.1348-0421.1994.tb01866.x","article-title":"Detection of West Nile and Japanese encephalitis viral genome sequences in cerebrospinal fluid from acute encephalitis cases in Karachi, Pakistan","volume":"38","author":"Igarashi","year":"1994","journal-title":"Microbiol. Immunol."},{"key":"B18","doi-asserted-by":"publisher","first-page":"1475","DOI":"10.1586\/14737159.2015.1090877","article-title":"Recombinase polymerase amplification: Emergence as a critical molecular technology for rapid, low-resource diagnostics","volume":"11","author":"James","year":"2015","journal-title":"Expert Rev. Mol. Diagn."},{"key":"B19","doi-asserted-by":"publisher","first-page":"947","DOI":"10.3201\/eid1705.100479","article-title":"West Nile virus infection Assam, India","volume":"5","author":"Khan","year":"2011","journal-title":"Emerg. Infect. Dis."},{"key":"B20","doi-asserted-by":"publisher","first-page":"323","DOI":"10.1126\/science.1201010","article-title":"Globalization, land use, and the invasion of West Nile virus","volume":"34","author":"Kilpatrick","year":"2011","journal-title":"Science"},{"key":"B21","doi-asserted-by":"publisher","first-page":"1247","DOI":"10.1016\/j.foodcont.2016.10.041","article-title":"Development of a multiplex real-time recombinase polymerase amplification (RPA) assay for rapid quantitative detection of Campylobacter coli and jejuni from eggs and chicken products","volume":"73","author":"Kim","year":"2017","journal-title":"Food Control"},{"key":"B22","first-page":"991","article-title":"Flaviviridae: the viruses and their replication","volume-title":"Fields virology","author":"Knipe","year":"2001"},{"key":"B23","doi-asserted-by":"publisher","first-page":"775","DOI":"10.1016\/j.mcp.2014.03.005","article-title":"Development and comparative evaluation of SYBR Green I-based one-step real-time RT-PCR assay for detection and quantification of West Nile virus in human patients","volume":"11","author":"Kumar","year":"2014","journal-title":"Mol. Cell. Probes."},{"key":"B24","doi-asserted-by":"publisher","first-page":"e0004472","DOI":"10.1371\/journal.pntd.0004472","article-title":"Development and evaluation of reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay coupled with a portable device for rapid diagnosis of Ebola virus disease in Guinea","volume":"10","author":"Kurosaki","year":"2016","journal-title":"PLoS Negl. Trop. Dis."},{"key":"B25","doi-asserted-by":"publisher","first-page":"4506","DOI":"10.1128\/JCM.39.12.4506-4513.2001","article-title":"Nucleic acid sequence based amplification assays for rapid detection of West Nile and St. Louis encephalitis viruses","volume":"39","author":"Lanciotti","year":"2001","journal-title":"J.\u00a0Clin. Microbiol."},{"key":"B26","doi-asserted-by":"publisher","first-page":"2333","DOI":"10.1126\/science.286.5448.2333","article-title":"Origin of the West Nile virus responsible for an outbreak of encephalitis in the northeastern United States","volume":"286","author":"Lanciotti","year":"1999","journal-title":"Science"},{"key":"B27","doi-asserted-by":"publisher","first-page":"4066","DOI":"10.1128\/JCM.38.11.4066-4071.2000","article-title":"Rapid Detection of West Nile virus from human clinical specimens, field-collected mosquitoes, and avian samples by a Taqman reverse transcriptase-PCR assay","volume":"11","author":"Lanciotti","year":"2000","journal-title":"J. Clin. Microbiol."},{"key":"B28","doi-asserted-by":"publisher","first-page":"880","DOI":"10.3390\/foods9010027","article-title":"Recombinase Polymerase Amplification (RPA) Combined with Lateral Flow Immunoassay for Rapid Detection of\u00a0Salmonella\u00a0in Food","volume":"27","author":"Li","year":"2019","journal-title":"Foods"},{"key":"B29","doi-asserted-by":"publisher","first-page":"811","DOI":"10.3390\/v3060811","article-title":"West Nile virus: immunity and pathogenesis","volume":"3","author":"Lim","year":"2011","journal-title":"Viruses"},{"key":"B30","doi-asserted-by":"publisher","first-page":"1713","DOI":"10.1086\/322700","article-title":"West Nile encephalitis: an emerging disease in the United States","volume":"33","author":"Marfin","year":"2001","journal-title":"Clin. Infect. Dis."},{"key":"B31","doi-asserted-by":"publisher","first-page":"1823","DOI":"10.1128\/JCM.38.5.1823-1826.2000","article-title":"Standardization of immunoglobulin M capture enzyme-linked immunosorbent assays for routine diagnosis of arboviral infections","volume":"38","author":"Martin","year":"2000","journal-title":"J. Clin. Microbiol."},{"key":"B32","doi-asserted-by":"publisher","first-page":"1372","DOI":"10.3201\/eid2308.170384","article-title":"West Nile virus outbreak in Houston and Harris Texas, USA 2014","volume":"23","author":"Martinez","year":"2017","journal-title":"Emer. Infect. Dis."},{"key":"B33","doi-asserted-by":"publisher","first-page":"24","DOI":"10.1016\/j.jviromet.2014.04.015","article-title":"Rapid and sensitive detection of Little cherry virus 2 using isothermal reverse-transcription recombinase polymerase amplification","volume":"205","author":"Mekuria","year":"2014","journal-title":"J. Virol. Methods"},{"key":"B34","doi-asserted-by":"publisher","first-page":"248","DOI":"10.1126\/science.1089316","article-title":"Structure of West Nile virus","volume":"302","author":"Mukhopadhyay","year":"2003","journal-title":"Science"},{"key":"B35","doi-asserted-by":"publisher","first-page":"1","DOI":"10.1093\/nar\/28.12.e63","article-title":"Loop-mediated isothermal amplification of DNA","volume":"28","author":"Notomi","year":"2000","journal-title":"Nucleic Acids Res."},{"key":"B36","doi-asserted-by":"publisher","first-page":"2119","DOI":"10.3201\/eid2012.140921","article-title":"Putative New West Nile virus lineage in Uranotaeniaunguiculata mosquitoes, Austria","volume":"20","author":"Pachler","year":"2014","journal-title":"Emerg. Infect. Dis."},{"key":"B37","doi-asserted-by":"publisher","first-page":"105","DOI":"10.3201\/eid1710.110525","article-title":"Ongoing outbreak of West Nile virus infections in humans in Greece, July-August 2010","volume":"34","author":"Papa","year":"2010","journal-title":"EuroSurveill."},{"key":"B38","first-page":"101","article-title":"West Nile virus: the Indian scenario","volume":"118","author":"Paramasivan","year":"2003","journal-title":"Indian J. Med. Res."},{"key":"B39","doi-asserted-by":"publisher","first-page":"257","DOI":"10.1128\/JCM.42.1.257-263.2004","article-title":"Real-time reverse transcription loop-mediated isothermal amplification for rapid detection of West Nile virus","volume":"42","author":"Parida","year":"2004","journal-title":"J. Clin. Microbiol."},{"key":"B40","doi-asserted-by":"publisher","first-page":"1","DOI":"10.1371\/journal.pntd.0004953","article-title":"A field-deployable reverse transcription recombinase polymerase amplification assay for rapid detection of the chikungunya virus","volume":"10","author":"Patel","year":"2016","journal-title":"PLos Neglect Trop. Dis"},{"key":"B41","unstructured":"Methods for multiplexing recombinase polymerase amplification\n            PiepenburgO.\n            WilliamsC. H.\n            ArmesN. A.\n          2011"},{"key":"B42","doi-asserted-by":"publisher","first-page":"53","DOI":"10.1016\/j.ijid.2011.09.020","article-title":"Molecular detection and characterization of WNV associated with multifocal retinitis in patients from southern India. International","volume":"16","author":"Shukla","year":"2012","journal-title":"J. Infect. Dis."},{"key":"B43","doi-asserted-by":"publisher","first-page":"865","DOI":"10.1136\/bmj.326.7394.865","article-title":"West Nile encephalitis","volume":"326","author":"Solomon","year":"2003","journal-title":"B.M.J."},{"key":"B44","doi-asserted-by":"crossref","first-page":"120","DOI":"10.4269\/ajtmh.2004.71.120","article-title":"Real-time reverse transcriptase-polymerase chain reaction quantification of West Nile virus transmitted by Culex pipiensquinquefasciatus","volume":"71","author":"Vanlandingham","year":"2004","journal-title":"Am. J. Trop. Med. Hyg."},{"key":"B45","doi-asserted-by":"publisher","first-page":"16","DOI":"10.1016\/j.jcv.2015.05.004","article-title":"Diagnostics in- a-suitcase: development of a portable and rapid assay for the detection of the emerging avian influenza A (H7N9) virus","volume":"69","author":"Wahed","year":"2015","journal-title":"J. Clin. Virol."},{"key":"B46","doi-asserted-by":"publisher","first-page":"65","DOI":"10.1128\/JCM.42.1.65\u201372.2004","article-title":"Detection of Human Anti-Flavivirus Antibodies with a West Nile Virus Recombinant Antigen Microsphere Immunoassay","volume":"42","author":"Wong","year":"2004","journal-title":"J. Clin. Microbiol."},{"key":"B47","doi-asserted-by":"publisher","first-page":"300","DOI":"10.1016\/j.mcp.2016.08.011","article-title":"Rapid and specific detection of porcine parvovirus by isothermal recombinase polymerase amplification assays","volume":"30","author":"Yang","year":"2016","journal-title":"Mol. Cell. Probes."},{"key":"B48","doi-asserted-by":"publisher","first-page":"1","DOI":"10.1186\/s12985-017-0792-7","article-title":"Development of real-time and lateral flow dipstick recombinase polymerase amplification assays for rapid detection of goat pox virus and sheep pox virus","volume":"131","author":"Yang","year":"2017","journal-title":"J. Virol."},{"key":"B49","doi-asserted-by":"publisher","first-page":"290","DOI":"10.1155\/2017\/8403642","article-title":"Development of isothermal recombinase polymerase amplification assay for rapid detection of porcine circovirus type 2","volume":"47","author":"Yang","year":"2017","journal-title":"BioMed. Res. Int."}],"container-title":["Frontiers in Cellular and Infection Microbiology"],"original-title":[],"link":[{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fcimb.2020.619071\/full","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2021,2,23]],"date-time":"2021-02-23T04:44:45Z","timestamp":1614055485000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fcimb.2020.619071\/full"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2021,2,23]]},"references-count":49,"alternative-id":["10.3389\/fcimb.2020.619071"],"URL":"https:\/\/doi.org\/10.3389\/fcimb.2020.619071","relation":{},"ISSN":["2235-2988"],"issn-type":[{"value":"2235-2988","type":"electronic"}],"subject":[],"published":{"date-parts":[[2021,2,23]]},"article-number":"619071"}}