{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,18]],"date-time":"2026-08-18T05:15:14Z","timestamp":1787030114135,"version":"build-2736575974"},"reference-count":36,"publisher":"MDPI AG","issue":"5","license":[{"start":{"date-parts":[[2025,5,17]],"date-time":"2025-05-17T00:00:00Z","timestamp":1747440000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100012166","name":"National Key R&amp;D Program of China","doi-asserted-by":"publisher","award":["2021YFD1800300"],"award-info":[{"award-number":["2021YFD1800300"]}],"id":[{"id":"10.13039\/501100012166","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Viruses"],"abstract":"<jats:p>Foot and mouth disease virus (FMDV) is a highly pathogenic virus that mainly infects cloven hooved animals, such as pigs. The establishment of a rapid, sensitive and accurate point-of-care detection method is critical for the timely identification and elimination of infected pigs for controlling this disease. In this study, a RT-RAA-CRISPR\/Cas13a method was developed for the detection of FMDV serotype O in pigs. Six pairs of RT-RAA primers were designed based on the conserved gene sequence of FMDV serotype O, and the optimal amplification primers and reaction temperatures were screened. The CRISPR-derived RNA (crRNA) was further designed based on the optimal target band sequence and the most efficient crRNA was screened. The results revealed that FMDV-O-F4\/R4 was the optimal primer set, and the optimal temperature for the RT-RAA reaction was 37 \u00b0C. Moreover, crRNA4 exhibited the strongest detection signal among the six crRNAs. The established RT-RAA-CRISPR\/Cas13a method demonstrated high specificity and no cross-reactivity with other common swine pathogens such as Senecavirus A (SVA), porcine reproductive and respiratory virus (PRRSV), porcine epidemic diarrhea virus (PEDV), porcine circovirus type 2 (PCV2), classical swine fever virus (CSFV), and pseudorabies virus (PRV), additionally, it was observed to be highly sensitive, with a detection limit of 19.1 copies\/\u00b5L. The repeatability of this method was also observed to be good. This method could produce stable fluorescence and exhibited good repeatability when three independent experiments yielded the same results. A validation test using three types of simulated clinical samples (including swab, tissue, and serum samples) revealed a 100% concordance rate. The detection results could be visualized via a fluorescence reader or lateral flow strips (LFSs). Thus, a highly specific and sensitive RT-RAA-CRISPR\/Cas13a detection method was developed and is expected to be applied for the rapid detection of FMDV serotype O in situ.<\/jats:p>","DOI":"10.3390\/v17050721","type":"journal-article","created":{"date-parts":[[2025,5,20]],"date-time":"2025-05-20T06:54:28Z","timestamp":1747724068000},"page":"721","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":3,"title":["Establishment and Implementation of the Point-of-Care RT-RAA-CRISPR\/Cas13a Diagnostic Test for Foot-And-Mouth Disease Virus Serotype O in Pigs"],"prefix":"10.3390","volume":"17","author":[{"given":"Ping","family":"Meng","sequence":"first","affiliation":[{"name":"China Animal Health and Epidemiology Center, Qingdao 266011, China"},{"name":"College of Veterinary Medicine, Qingdao Agricultural University, Qingdao 266109, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Bo","family":"Ni","sequence":"additional","affiliation":[{"name":"China Animal Health and Epidemiology Center, Qingdao 266011, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Chenyu","family":"Li","sequence":"additional","affiliation":[{"name":"College of Veterinary Medicine, Shanxi Agricultural University, Jinzhong 030031, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zhou","family":"Sha","sequence":"additional","affiliation":[{"name":"China Animal Health and Epidemiology Center, Qingdao 266011, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Chunju","family":"Liu","sequence":"additional","affiliation":[{"name":"China Animal Health and Epidemiology Center, Qingdao 266011, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Weijie","family":"Ren","sequence":"additional","affiliation":[{"name":"China Animal Health and Epidemiology Center, Qingdao 266011, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Rong","family":"Wei","sequence":"additional","affiliation":[{"name":"China Animal Health and Epidemiology Center, Qingdao 266011, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3078-0613","authenticated-orcid":false,"given":"Fuxiao","family":"Liu","sequence":"additional","affiliation":[{"name":"College of Veterinary Medicine, Qingdao Agricultural University, Qingdao 266109, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jinming","family":"Li","sequence":"additional","affiliation":[{"name":"China Animal Health and Epidemiology Center, Qingdao 266011, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zhiliang","family":"Wang","sequence":"additional","affiliation":[{"name":"China Animal Health and Epidemiology Center, Qingdao 266011, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2025,5,17]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"116","DOI":"10.1186\/1297-9716-44-116","article-title":"Foot-and-mouth disease: Past, present and future","volume":"44","author":"Jamal","year":"2013","journal-title":"Vet. Res."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"465","DOI":"10.1128\/CMR.17.2.465-493.2004","article-title":"Foot-and-mouth disease","volume":"17","author":"Grubman","year":"2004","journal-title":"Clin. Microbiol. Rev."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1038\/s41426-018-0206-y","article-title":"Epidemiological and serological surveillance of hand-foot-and-mouth disease in Shanghai, China, 2012\u20132016","volume":"7","author":"Wang","year":"2018","journal-title":"Emerg. Microbes Infect."},{"key":"ref_4","doi-asserted-by":"crossref","unstructured":"Gaboiphiwe, K., Kabelo, T.I., Mosholombe, P.T., Hyera, J., Fana, E.M., Masisi, K., and Lebani, K. (2024). A Review of the Utility of Established Cell Lines for Isolation and Propagation of the Southern African Territories Serotypes of Foot-and-Mouth Disease Virus. Viruses, 17.","DOI":"10.3390\/v17010039"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"6487","DOI":"10.1128\/JVI.79.10.6487-6504.2005","article-title":"Comparative genomics of foot-and-mouth disease virus","volume":"79","author":"Carrillo","year":"2005","journal-title":"J. Virol."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"175","DOI":"10.20506\/rst.35.1.2426","article-title":"Genomics and outbreaks: Foot and mouth disease","volume":"35","author":"Freimanis","year":"2016","journal-title":"Rev. Sci. Tech. Int. Off. Epizoot."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"56","DOI":"10.1186\/s13567-022-01072-7","article-title":"Evaluation of immunogenicity and cross-reactive responses of vaccines prepared from two chimeric serotype O foot-and-mouth disease viruses in pigs and cattle","volume":"53","author":"Li","year":"2022","journal-title":"Vet. Res."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"109940","DOI":"10.1016\/j.virol.2023.109940","article-title":"Epidemiological and genetic characteristics of foot-and-mouth disease virus in China from 2010 to 2022","volume":"589","author":"Ren","year":"2024","journal-title":"Virology"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"301","DOI":"10.1017\/S095026880100646X","article-title":"Natural aerosol transmission of foot-and-mouth disease virus to pigs: Minimal infectious dose for strain O1 Lausanne","volume":"128","author":"Alexandersen","year":"2002","journal-title":"Epidemiol. Infect."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"1681","DOI":"10.1016\/j.vaccine.2008.01.038","article-title":"High potency vaccines induce protection against heterologous challenge with foot-and-mouth disease virus","volume":"26","author":"Brehm","year":"2008","journal-title":"Vaccine"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"278","DOI":"10.1007\/s11250-023-03674-5","article-title":"A review of foot-and-mouth disease status and control measures in Botswana","volume":"55","author":"Kabelo","year":"2023","journal-title":"Trop. Anim. Health Prod."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"729","DOI":"10.1136\/vr.149.24.729","article-title":"Descriptive epidemiology of the 2001 foot-and-mouth disease epidemic in Great Britain: The first five months","volume":"149","author":"Gibbens","year":"2001","journal-title":"Vet. Rec."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"301","DOI":"10.1016\/j.jviromet.2007.09.010","article-title":"Rapid and differential diagnosis of foot-and-mouth disease, swine vesicular disease, and vesicular stomatitis by a new multiplex RT-PCR assay","volume":"147","author":"Romero","year":"2008","journal-title":"J. Virol. Methods"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"253","DOI":"10.1016\/j.jviromet.2011.05.026","article-title":"Validation of a recombinant integrin \u03b1v\u03b26\/monoclonal antibody based antigen ELISA for the diagnosis of foot-and-mouth disease","volume":"175","author":"Ferris","year":"2011","journal-title":"J. Virol. Methods"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"497","DOI":"10.1111\/tbed.13051","article-title":"The development of two field-ready reverse transcription loop-mediated isothermal amplification assays for the rapid detection of Seneca Valley virus 1","volume":"66","author":"Armson","year":"2019","journal-title":"Transbound. Emerg. Dis."},{"key":"ref_16","doi-asserted-by":"crossref","unstructured":"Shimmon, G., Kotecha, A., Ren, J., Asfor, A.S., Newman, J., Berryman, S., Cottam, E.M., Gold, S., Tuthill, T.J., and King, D.P. (2018). Generation and characterisation of recombinant FMDV antibodies: Applications for advancing diagnostic and laboratory assays. PLoS ONE, 13.","DOI":"10.1371\/journal.pone.0201853"},{"key":"ref_17","doi-asserted-by":"crossref","unstructured":"Wang, H., Ding, X., Sun, W., Chen, Z., Bai, L., Liang, H., Liu, Y., Zhang, W., Wang, G., and Yang, G. (2022). Recombinase polymerase amplification assay for rapid detection of Seneca Valley Virus. Anal. Biochem., 642.","DOI":"10.1016\/j.ab.2022.114564"},{"key":"ref_18","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_19","doi-asserted-by":"crossref","first-page":"439","DOI":"10.1126\/science.aaq0179","article-title":"Multiplexed and portable nucleic acid detection platform with Cas13, Cas12a, and Csm6","volume":"360","author":"Gootenberg","year":"2018","journal-title":"Science"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"825","DOI":"10.1038\/nsmb.3466","article-title":"Guide-bound structures of an RNA-targeting A-cleaving CRISPR-Cas13a enzyme","volume":"24","author":"Knott","year":"2017","journal-title":"Nat. Struct. Mol. Biol."},{"key":"ref_21","doi-asserted-by":"crossref","unstructured":"Zhao, L., Qiu, M., Li, X., Yang, J., and Li, J. (2022). CRISPR-Cas13a system: A novel tool for molecular diagnostics. Front. Microbiol., 13.","DOI":"10.3389\/fmicb.2022.1060947"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"438","DOI":"10.1126\/science.aam9321","article-title":"Nucleic acid detection with CRISPR-Cas13a\/C2c2","volume":"356","author":"Gootenberg","year":"2017","journal-title":"Science"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"20754","DOI":"10.1002\/anie.202005398","article-title":"CRISPR Mediated Biosensing Toward Understanding Cellular Biology and Point-of-Care Diagnosis","volume":"59","author":"Dai","year":"2020","journal-title":"Angew. Chem. (Int. Ed. Engl.)"},{"key":"ref_24","doi-asserted-by":"crossref","unstructured":"Wang, M., Zhang, R., and Li, J. (2020). CRISPR\/cas systems redefine nucleic acid detection: Principles and methods. Biosens. Bioelectron., 165.","DOI":"10.1016\/j.bios.2020.112430"},{"key":"ref_25","doi-asserted-by":"crossref","unstructured":"Yin, L., Man, S., Ye, S., Liu, G., and Ma, L. (2021). CRISPR-Cas based virus detection: Recent advances and perspectives. Biosens. Bioelectron., 193.","DOI":"10.1016\/j.bios.2021.113541"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"199130","DOI":"10.1016\/j.virusres.2023.199130","article-title":"Rapid detection of Nipah virus using the one-pot RPA-CRISPR\/Cas13a assay","volume":"332","author":"Miao","year":"2023","journal-title":"Virus Res."},{"key":"ref_27","doi-asserted-by":"crossref","unstructured":"Stenfeldt, C., Diaz-San Segundo, F., de Los Santos, T., Rodriguez, L.L., and Arzt, J. (2016). The Pathogenesis of Foot-and-Mouth Disease in Pigs. Front. Vet. Sci., 3.","DOI":"10.3389\/fvets.2016.00041"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"123","DOI":"10.1016\/j.virol.2016.12.021","article-title":"Adenovirus-vectored foot-and-mouth disease vaccine confers early and full protection against FMDV O1 Manisa in swine","volume":"502","author":"Medina","year":"2017","journal-title":"Virology"},{"key":"ref_29","doi-asserted-by":"crossref","unstructured":"Ren, H.R., Li, M.T., Wang, Y.M., Jin, Z., and Zhang, J. (2021). The risk factor assessment of the spread of foot-and-mouth disease in mainland China. J. Theor. Biol., 512.","DOI":"10.1016\/j.jtbi.2020.110558"},{"key":"ref_30","doi-asserted-by":"crossref","unstructured":"Chen, W., Wang, W., Wang, X., Li, Z., Wu, K., Li, X., Li, Y., Yi, L., Zhao, M., and Ding, H. (2022). Advances in the differential molecular diagnosis of vesicular disease pathogens in swine. Front. Microbiol., 13.","DOI":"10.3389\/fmicb.2022.1019876"},{"key":"ref_31","doi-asserted-by":"crossref","unstructured":"Yeo, S., Yang, M., Nyachoti, M., Rauh, R., Callahan, J.D., and Nfon, C. (2020). Detection of Foot-and-Mouth Disease Virus in Swine Meat Juice. Pathogens, 9.","DOI":"10.3390\/pathogens9060424"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"132077","DOI":"10.1016\/j.jhazmat.2023.132077","article-title":"Field-deployable assay based on CRISPR-Cas13a coupled with RT-RPA in one tube for the detection of SARS-CoV-2 in wastewater","volume":"459","author":"Yang","year":"2023","journal-title":"J. Hazard. Mater."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"270","DOI":"10.1038\/nature19802","article-title":"Two distinct RNase activities of CRISPR-C2c2 enable guide-RNA processing and RNA detection","volume":"538","author":"Knight","year":"2016","journal-title":"Nature"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"790","DOI":"10.1166\/jbn.2019.2742","article-title":"CRISPR-Cas13a Nanomachine Based Simple Technology for Avian Influenza A (H7N9) Virus On-Site Detection","volume":"15","author":"Liu","year":"2019","journal-title":"J. Biomed. Nanotechnol."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"2986","DOI":"10.1038\/s41596-019-0210-2","article-title":"SHERLOCK: Nucleic acid detection with CRISPR nucleases","volume":"14","author":"Kellner","year":"2019","journal-title":"Nat. Protoc."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"5858","DOI":"10.1002\/jmv.28096","article-title":"Highly sensitive and rapid detection of SARS-CoV-2 via a portable CRISPR-Cas13a-based lateral flow assay","volume":"94","author":"Liu","year":"2022","journal-title":"J. Med. Virol."}],"container-title":["Viruses"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1999-4915\/17\/5\/721\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,9]],"date-time":"2025-10-09T17:34:36Z","timestamp":1760031276000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1999-4915\/17\/5\/721"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,5,17]]},"references-count":36,"journal-issue":{"issue":"5","published-online":{"date-parts":[[2025,5]]}},"alternative-id":["v17050721"],"URL":"https:\/\/doi.org\/10.3390\/v17050721","relation":{},"ISSN":["1999-4915"],"issn-type":[{"value":"1999-4915","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,5,17]]}}}