{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,31]],"date-time":"2026-08-31T23:07:28Z","timestamp":1788217648199,"version":"build-2803163510"},"reference-count":47,"publisher":"MDPI AG","issue":"12","license":[{"start":{"date-parts":[[2025,12,10]],"date-time":"2025-12-10T00:00:00Z","timestamp":1765324800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100002631","name":"Gachon University","doi-asserted-by":"publisher","award":["GCU-202502820001"],"award-info":[{"award-number":["GCU-202502820001"]}],"id":[{"id":"10.13039\/501100002631","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Biosensors"],"abstract":"<jats:p>The West Nile Virus (WNV), transmitted by Culex mosquitoes as a major vector, has been reported worldwide. Also, West Nile neuroinvasive disease (WNND) caused by WNV lineage 1a and 2 neuroinvasive infections has been constantly reported with high fatality rates. Nevertheless, there are no treatments and vaccinations, so diagnosis in the early stages is important. Recently, a molecular diagnostic technique using DNA endonuclease-targeted CRISPR trans reporter (DETECTR) with the CRISPR-Cas12a system integrated with isothermal nucleic acid amplification has newly emerged. In this study, we designed a 2-Step WNV DETECTR with reverse transcription\u2013recombinase polymerase amplification (RT-RPA) for rapid and sensitive WNV diagnosis. It successfully detected down to 1.0 \u00d7 102 RNA copies for both WNV lineage 1a and 2 with demonstrating similar sensitivity to qRT-PCR without cross-reactivity to other viruses. Additionally, we designed a 1-Step WNV DETECTR, incorporating all processing steps into a single tube, capable of detecting down to 1.0 \u00d7 103 RNA copies for both lineages. Furthermore, we developed a more streamlined method, the 1-Step with Filter WNV DETECTR, which achieved detection limits comparable to the 2-Step method, while reducing the processing time by 5 min. This study also explored the potential of the Punch-it\u2122 NA-Sample Kit as an efficient alternative lysis method by comparing the detection differences across various lysis methods. Through this method, we achieved rapid and simple amplification and detection processes suitable for field diagnostics with high specificity and sufficient sensitivity. Therefore, DETECTR methods presented themselves as promising alternatives to conventional diagnostic tools, potentially overcoming financial and technical constraints in diverse medical settings.<\/jats:p>","DOI":"10.3390\/bios15120807","type":"journal-article","created":{"date-parts":[[2025,12,10]],"date-time":"2025-12-10T16:11:18Z","timestamp":1765383078000},"page":"807","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":5,"title":["A Novel Diagnostic Tool for West Nile Virus Lineage 1a and 2 Using a CRISPR-Cas12a System"],"prefix":"10.3390","volume":"15","author":[{"given":"Soo Bin","family":"Hwang","sequence":"first","affiliation":[{"name":"Department of Life Science, Gachon University, 1342 Seongnam-Daero, Seongnam-Si 13120, Gyeonggi-Do, Republic of Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7900-5012","authenticated-orcid":false,"given":"Yoon-Jae","family":"Song","sequence":"additional","affiliation":[{"name":"Department of Life Science, Gachon University, 1342 Seongnam-Daero, Seongnam-Si 13120, Gyeonggi-Do, Republic of Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3024-3439","authenticated-orcid":false,"given":"Pil-Gu","family":"Park","sequence":"additional","affiliation":[{"name":"Department of Life Science, Gachon University, 1342 Seongnam-Daero, Seongnam-Si 13120, Gyeonggi-Do, Republic of Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2025,12,10]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"471","DOI":"10.4269\/ajtmh.1940.s1-20.471","article-title":"A neurotropic virus isolated from the blood of a native of Uganda","volume":"20","author":"Smithburn","year":"1940","journal-title":"Am. J. Trop. Med."},{"key":"ref_2","first-page":"29","article-title":"Epidemiology of West Nile in Europe and in the Mediterranean basin","volume":"4","author":"Calistri","year":"2010","journal-title":"Open Virol. J."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"376230","DOI":"10.1155\/2015\/376230","article-title":"The global ecology and epidemiology of West Nile virus","volume":"2015","author":"Chancey","year":"2015","journal-title":"BioMed Res. Int."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"637","DOI":"10.1007\/s00705-004-0463-z","article-title":"West Nile virus in the vertebrate world","volume":"150","author":"Pensaert","year":"2005","journal-title":"Arch. Virol."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"1236","DOI":"10.1056\/NEJMoa030969","article-title":"Transmission of West Nile virus through blood transfusion in the United States in 2002","volume":"349","author":"Pealer","year":"2003","journal-title":"N. Engl. J. Med."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"2196","DOI":"10.1056\/NEJMoa022987","article-title":"Transmission of West Nile virus from an organ donor to four transplant recipients","volume":"348","author":"Iwamoto","year":"2003","journal-title":"N. Engl. J. Med."},{"key":"ref_7","first-page":"1135","article-title":"Intrauterine West Nile virus infection\u2014New York, 2002","volume":"51","author":"Nguyen","year":"2002","journal-title":"MMWR Morb. Mortal. Wkly. Rep."},{"key":"ref_8","first-page":"877","article-title":"Possible West Nile virus transmission to an infant through breast-feeding\u2014Michigan, 2002","volume":"51","author":"Stobierski","year":"2002","journal-title":"MMWR Morb. Mortal. Wkly. Rep."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"e666","DOI":"10.1542\/peds.2006-2107","article-title":"Transmission of West Nile virus through human breast milk seems to be rare","volume":"119","author":"Hinckley","year":"2007","journal-title":"Pediatrics"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"606","DOI":"10.3390\/v6020606","article-title":"Clinical manifestations and outcomes of West Nile virus infection","volume":"6","author":"Sejvar","year":"2014","journal-title":"Viruses"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"519","DOI":"10.1016\/S1473-3099(02)00368-7","article-title":"West nile virus","volume":"2","author":"Campbell","year":"2002","journal-title":"Lancet Infect. Dis."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"547","DOI":"10.1016\/S1473-3099(04)01128-4","article-title":"West Nile virus: Where are we now?","volume":"4","author":"Granwehr","year":"2004","journal-title":"Lancet Infect. Dis."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"61","DOI":"10.1089\/153036604773083004","article-title":"The epidemic of West Nile virus in the United States, 2002","volume":"4","author":"Marfin","year":"2004","journal-title":"Vector-Borne Zoonotic Dis."},{"key":"ref_14","doi-asserted-by":"crossref","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. Rev."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"6719","DOI":"10.1128\/JVI.75.14.6719-6728.2001","article-title":"Essential role of cyclization sequences in flavivirus RNA replication","volume":"75","author":"Khromykh","year":"2001","journal-title":"J. Virol."},{"key":"ref_16","doi-asserted-by":"crossref","unstructured":"Westaway, E., Mackenzie, J., and Khromykh, A. (2002). Replication and gene function in Kunjin virus. Japanese Encephalitis and West Nile Viruses, Springer.","DOI":"10.1007\/978-3-642-59403-8_16"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"23","DOI":"10.1016\/S0065-3527(03)59002-9","article-title":"Molecular biology of flaviviruses","volume":"59","author":"Lindenbach","year":"2003","journal-title":"Adv. Virus Res."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"2396","DOI":"10.1128\/JVI.80.5.2396-2404.2006","article-title":"A single amino acid substitution in the West Nile virus nonstructural protein NS2A disables its ability to inhibit alpha\/beta interferon induction and attenuates virus virulence in mice","volume":"80","author":"Liu","year":"2006","journal-title":"J. Virol."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"8004","DOI":"10.1128\/JVI.79.13.8004-8013.2005","article-title":"Inhibition of alpha\/beta interferon signaling by the NS4B protein of flaviviruses","volume":"79","author":"Ashour","year":"2005","journal-title":"J. Virol."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"877","DOI":"10.3201\/eid1506.081515","article-title":"Lineage 2 West Nile virus as cause of fatal neurologic disease in horses, South Africa","volume":"15","author":"Venter","year":"2009","journal-title":"Emerg. Infect. Dis."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"67","DOI":"10.1051\/vetres\/2010039","article-title":"West Nile virus and its emergence in the United States of America","volume":"41","author":"Murray","year":"2010","journal-title":"Vet. Res."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"153","DOI":"10.1111\/j.1749-6632.2001.tb02693.x","article-title":"Kunjin virus: An Australian variant of West Nile?","volume":"951","author":"Hall","year":"2001","journal-title":"Ann. N. Y. Acad. Sci."},{"key":"ref_23","doi-asserted-by":"crossref","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":"ref_24","doi-asserted-by":"crossref","first-page":"618","DOI":"10.3201\/eid1204.051379","article-title":"Lineage 1 and 2 strains of encephalitic West Nile virus, central Europe","volume":"12","author":"Bakonyi","year":"2006","journal-title":"Emerg. Infect. Dis."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"747","DOI":"10.4269\/ajtmh.13-0405","article-title":"Vector competence of Culex neavei and Culex quinquefasciatus (Diptera: Culicidae) from Senegal for lineages 1, 2, Koutango and a putative new lineage of West Nile virus","volume":"90","author":"Fall","year":"2014","journal-title":"Am. J. Trop. Med. Hyg."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"748","DOI":"10.1177\/104063871002200515","article-title":"Two new real-time quantitative reverse transcription polymerase chain reaction assays with unique target sites for the specific and sensitive detection of lineages 1 and 2 West Nile virus strains","volume":"22","author":"Eiden","year":"2010","journal-title":"J. Vet. Diagn. Investig."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"436","DOI":"10.1126\/science.aar6245","article-title":"CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity","volume":"360","author":"Chen","year":"2018","journal-title":"Science"},{"key":"ref_28","doi-asserted-by":"crossref","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":"ref_29","doi-asserted-by":"crossref","first-page":"20","DOI":"10.1038\/s41421-018-0028-z","article-title":"CRISPR-Cas12a-assisted nucleic acid detection","volume":"4","author":"Li","year":"2018","journal-title":"Cell Discov."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"e0010666","DOI":"10.1371\/journal.pntd.0010666","article-title":"A CRISPR-Cas12a-based diagnostic method for multiple genotypes of severe fever with thrombocytopenia syndrome virus","volume":"16","author":"Park","year":"2022","journal-title":"PLoS Neglected Trop. Dis."},{"key":"ref_31","doi-asserted-by":"crossref","unstructured":"Kwak, N., Park, B.J., and Song, Y.-J. (2023). A CRISPR-Cas12a-Based Diagnostic Method for Japanese Encephalitis Virus Genotypes I, III, and V. Biosensors, 13.","DOI":"10.3390\/bios13080769"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"870","DOI":"10.1038\/s41587-020-0513-4","article-title":"CRISPR\u2013Cas12-based detection of SARS-CoV-2","volume":"38","author":"Broughton","year":"2020","journal-title":"Nat. Biotechnol."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"74","DOI":"10.1186\/s12967-021-02741-5","article-title":"One-tube SARS-CoV-2 detection platform based on RT-RPA and CRISPR\/Cas12a","volume":"19","author":"Sun","year":"2021","journal-title":"J. Transl. Med."},{"key":"ref_34","doi-asserted-by":"crossref","unstructured":"Park, B.J., Park, M.S., Lee, J.M., and Song, Y.J. (2021). Specific detection of influenza A and B viruses by CRISPR-Cas12a-based assay. Biosensors, 11.","DOI":"10.3390\/bios11030088"},{"key":"ref_35","doi-asserted-by":"crossref","unstructured":"Fall, G., Di Paola, N., Faye, M., Dia, M., Freire, C.C.d.M., Loucoubar, C., Zanotto, P.M.d.A., Faye, O., and Sall, A.A. (2017). Biological and phylogenetic characteristics of West African lineages of West Nile virus. PLoS Neglected Trop. Dis., 11.","DOI":"10.1371\/journal.pntd.0006078"},{"key":"ref_36","doi-asserted-by":"crossref","unstructured":"Campeau, E., Ruhl, V.E., Rodier, F., Smith, C.L., Rahmberg, B.L., Fuss, J.O., Campisi, J., Yaswen, P., Cooper, P.K., and Kaufman, P.D. (2009). A versatile viral system for expression and depletion of proteins in mammalian cells. PLoS ONE, 4.","DOI":"10.1371\/journal.pone.0006529"},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"8463","DOI":"10.1128\/JVI.72.11.8463-8471.1998","article-title":"A third-generation lentivirus vector with a conditional packaging system","volume":"72","author":"Dull","year":"1998","journal-title":"J. Virol."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"10","DOI":"10.1128\/JVI.00878-19","article-title":"Novel small molecule targeting the hemagglutinin stalk of influenza viruses","volume":"93","author":"Kim","year":"2019","journal-title":"J. Virol."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"444","DOI":"10.1126\/science.aas8836","article-title":"Field-deployable viral diagnostics using CRISPR-Cas13","volume":"360","author":"Myhrvold","year":"2018","journal-title":"Science"},{"key":"ref_40","doi-asserted-by":"crossref","unstructured":"Li, Y., Shi, Z., Hu, A., Cui, J., Yang, K., Liu, Y., Deng, G., Zhu, C., and Zhu, L. (2022). Rapid one-tube RPA-CRISPR\/Cas12 detection platform for methicillin-resistant Staphylococcus aureus. Diagnostics, 12.","DOI":"10.3390\/diagnostics12040829"},{"key":"ref_41","doi-asserted-by":"crossref","unstructured":"Aman, R., Mahas, A., Marsic, T., Hassan, N., and Mahfouz, M.M. (2020). Efficient, rapid, and sensitive detection of plant RNA viruses with one-pot RT-RPA\u2013CRISPR\/Cas12a assay. Front. Microbiol., 11.","DOI":"10.3389\/fmicb.2020.610872"},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"325","DOI":"10.1038\/s42003-022-03275-2","article-title":"Characterization of Cme and Yme thermostable Cas12a orthologs","volume":"5","author":"Fuchs","year":"2022","journal-title":"Commun. Biol."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"10","DOI":"10.1128\/mBio.00135-13","article-title":"Rapid detection of HIV-1 proviral DNA for early infant diagnosis using recombinase polymerase amplification","volume":"4","author":"Boyle","year":"2013","journal-title":"mBio"},{"key":"ref_44","doi-asserted-by":"crossref","unstructured":"Abd El Wahed, A., El-Deeb, A., El-Tholoth, M., Abd El Kader, H., Ahmed, A., Hassan, S., Hoffmann, B., Haas, B., Shalaby, M.A., and Hufert, F.T. (2013). A portable reverse transcription recombinase polymerase amplification assay for rapid detection of foot-and-mouth disease virus. PLoS ONE, 8.","DOI":"10.1371\/journal.pone.0071642"},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"111","DOI":"10.1042\/EBC20150012","article-title":"Lateral flow assays","volume":"60","author":"Koczula","year":"2016","journal-title":"Essays Biochem."},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"31","DOI":"10.1039\/C8AN01621F","article-title":"Review: A comprehensive summary of a decade development of the recombinase polymerase amplification","volume":"144","author":"Li","year":"2018","journal-title":"Analyst"},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"1454","DOI":"10.1038\/s42003-024-07173-7","article-title":"Iterative crRNA design and a PAM-free strategy enabled an ultra-specific RPA-CRISPR\/Cas12a detection platform","volume":"7","author":"Mao","year":"2024","journal-title":"Commun. Biol."}],"container-title":["Biosensors"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2079-6374\/15\/12\/807\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,12,10]],"date-time":"2025-12-10T16:18:10Z","timestamp":1765383490000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2079-6374\/15\/12\/807"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,12,10]]},"references-count":47,"journal-issue":{"issue":"12","published-online":{"date-parts":[[2025,12]]}},"alternative-id":["bios15120807"],"URL":"https:\/\/doi.org\/10.3390\/bios15120807","relation":{},"ISSN":["2079-6374"],"issn-type":[{"value":"2079-6374","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,12,10]]}}}