{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T12:44:45Z","timestamp":1785415485818,"version":"3.56.0"},"reference-count":32,"publisher":"MDPI AG","issue":"7","license":[{"start":{"date-parts":[[2026,4,2]],"date-time":"2026-04-02T00:00:00Z","timestamp":1775088000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001809","name":"National Natural Science Foundation of China","doi-asserted-by":"publisher","award":["32360889"],"award-info":[{"award-number":["32360889"]}],"id":[{"id":"10.13039\/501100001809","id-type":"DOI","asserted-by":"publisher"}]},{"award":["32360889"],"award-info":[{"award-number":["32360889"]}],"id":[{"id":"https:\/\/ror.org\/01h0zpd94","id-type":"ROR","asserted-by":"publisher"}]},{"name":"Qinghai Provincial Science and Technology Department","award":["2023-NK-135"],"award-info":[{"award-number":["2023-NK-135"]}]},{"award":["2023-NK-135"],"award-info":[{"award-number":["2023-NK-135"]}],"id":[{"id":"https:\/\/ror.org\/03vz7b375","id-type":"ROR","asserted-by":"publisher"}]},{"name":"Qinghai Province \u201cKunlun Talents \u22c5 Highend Innovation and Entrepreneurial Talents\u201d Top Talent Project"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Animals"],"abstract":"<jats:p>Fascioliasis, a globally prevalent zoonosis, severely threatens public health and livestock security. Current diagnostic approaches, hindered by the need for sophisticated instrumentation and specialized expertise, are inadequate for on-site surveillance in resource-constrained settings. This study developed a rapid, visual detection assay for Fasciola hepatica via recombinase-aided amplification (RAA) integrated with CRISPR\/Cas12b, addressing critical equipment and operational constraints. Targeting a specific mitochondrial DNA fragment of F. hepatica, recombinant plasmid standards were constructed, RAA primers and sgRNA optimized, and three detection modalities (real-time fluorescence, UV lamp, test strip) integrated. Clinical validation against PCR demonstrated 45 min turnaround time, F. hepatica-specific positivity, and real-time fluorescence sensitivity of 2.6 copies\/\u03bcL. Results showed high concordance with PCR and qPCR, with substantially reduced assay duration and streamlined workflow. This highly sensitive, specific, multi-visualized method overcomes limitations of conventional techniques, offering an efficient, field-deployable tool for fascioliasis surveillance and control in grassroots and pastoral regions.<\/jats:p>","DOI":"10.3390\/ani16071093","type":"journal-article","created":{"date-parts":[[2026,4,2]],"date-time":"2026-04-02T11:51:03Z","timestamp":1775130663000},"page":"1093","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["A Rapid Visual Detection Method for Fasciola hepatica Based on RAA-CRISPR\/Cas12b"],"prefix":"10.3390","volume":"16","author":[{"ORCID":"https:\/\/orcid.org\/0009-0002-2422-2946","authenticated-orcid":false,"given":"Jiangying","family":"Li","sequence":"first","affiliation":[{"name":"The Academy of Animal and Veterinary Sciences, Qinghai University, Xining 810016, China"},{"name":"Qinghai Provincial Key Laboratory of Pathogen Diagnosis for Animal Disease and Green Technical Research for Prevention and Control, Xining 810016, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tao","family":"Zhang","sequence":"additional","affiliation":[{"name":"The Academy of Animal and Veterinary Sciences, Qinghai University, Xining 810016, China"},{"name":"Qinghai Provincial Key Laboratory of Pathogen Diagnosis for Animal Disease and Green Technical Research for Prevention and Control, Xining 810016, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jingkai","family":"Ai","sequence":"additional","affiliation":[{"name":"The Academy of Animal and Veterinary Sciences, Qinghai University, Xining 810016, China"},{"name":"Qinghai Provincial Key Laboratory of Pathogen Diagnosis for Animal Disease and Green Technical Research for Prevention and Control, Xining 810016, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zijuan","family":"Zhao","sequence":"additional","affiliation":[{"name":"The Academy of Animal and Veterinary Sciences, Qinghai University, Xining 810016, China"},{"name":"Qinghai Provincial Key Laboratory of Pathogen Diagnosis for Animal Disease and Green Technical Research for Prevention and Control, Xining 810016, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zhi","family":"Li","sequence":"additional","affiliation":[{"name":"The Academy of Animal and Veterinary Sciences, Qinghai University, Xining 810016, China"},{"name":"Qinghai Provincial Key Laboratory of Pathogen Diagnosis for Animal Disease and Green Technical Research for Prevention and Control, Xining 810016, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yong","family":"Fu","sequence":"additional","affiliation":[{"name":"The Academy of Animal and Veterinary Sciences, Qinghai University, Xining 810016, China"},{"name":"Qinghai Provincial Key Laboratory of Pathogen Diagnosis for Animal Disease and Green Technical Research for Prevention and Control, Xining 810016, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Dan","family":"Jia","sequence":"additional","affiliation":[{"name":"The Academy of Animal and Veterinary Sciences, Qinghai University, Xining 810016, China"},{"name":"Qinghai Provincial Key Laboratory of Pathogen Diagnosis for Animal Disease and Green Technical Research for Prevention and Control, Xining 810016, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hong","family":"Duo","sequence":"additional","affiliation":[{"name":"The Academy of Animal and Veterinary Sciences, Qinghai University, Xining 810016, China"},{"name":"Qinghai Provincial Key Laboratory of Pathogen Diagnosis for Animal Disease and Green Technical Research for Prevention and Control, Xining 810016, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xiuying","family":"Shen","sequence":"additional","affiliation":[{"name":"The Academy of Animal and Veterinary Sciences, Qinghai University, Xining 810016, China"},{"name":"Qinghai Provincial Key Laboratory of Pathogen Diagnosis for Animal Disease and Green Technical Research for Prevention and Control, Xining 810016, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ru","family":"Meng","sequence":"additional","affiliation":[{"name":"Xining Animal Disease Prevention and Control Center, Xining 810016, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5696-2546","authenticated-orcid":false,"given":"Yingna","family":"Jian","sequence":"additional","affiliation":[{"name":"The Academy of Animal and Veterinary Sciences, Qinghai University, Xining 810016, China"},{"name":"Qinghai Provincial Key Laboratory of Pathogen Diagnosis for Animal Disease and Green Technical Research for Prevention and Control, Xining 810016, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5076-9211","authenticated-orcid":false,"given":"Xueyong","family":"Zhang","sequence":"additional","affiliation":[{"name":"The Academy of Animal and Veterinary Sciences, Qinghai University, Xining 810016, China"},{"name":"Qinghai Provincial Key Laboratory of Pathogen Diagnosis for Animal Disease and Green Technical Research for Prevention and Control, Xining 810016, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,4,2]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"195","DOI":"10.1016\/j.pt.2021.12.001","article-title":"On the arrival of fasciolosis in the Americas","volume":"38","author":"Alba","year":"2022","journal-title":"Trends Parasitol."},{"key":"ref_2","doi-asserted-by":"crossref","unstructured":"Cancela, M., Ru\u00e9talo, N., Dell\u2019Oca, N., da Silva, E., Smircich, P., Rinaldi, G., Roche, L., Carmona, C., Alvarez-Val\u00edn, F., and Zaha, A. (2010). Survey of transcripts expressed by the invasive juvenile stage of the liver fluke Fasciola hepatica. BMC Genom., 11.","DOI":"10.1186\/1471-2164-11-227"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"260","DOI":"10.1186\/s13071-023-05890-2","article-title":"Comparison of Mini-FLOTAC, Flukefinder\u00ae and sedimentation techniques for detection and quantification of Fasciola hepatica and Calicophoron daubneyi eggs using spiked and naturally infected bovine faecal samples","volume":"16","author":"Bosco","year":"2023","journal-title":"Parasites Vectors"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"e0008819","DOI":"10.1128\/cmr.00088-19","article-title":"Human and Animal Fascioliasis: Origins and Worldwide Evolving Scenario","volume":"35","author":"Valero","year":"2022","journal-title":"Clin. Microbiol. Rev."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"333","DOI":"10.1136\/vr.102720","article-title":"Efficacy of treatment of cattle for liver fluke at housing: Influence of differences in flukicidal activity against juvenile Fasciola hepatica","volume":"176","author":"Forbes","year":"2015","journal-title":"Vet. Rec."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"35","DOI":"10.1016\/j.pt.2020.09.007","article-title":"Insights into Fasciola hepatica Juveniles: Crossing the Fasciolosis Rubicon","volume":"37","year":"2021","journal-title":"Trends Parasitol."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"4940","DOI":"10.1021\/pr1000785","article-title":"Comparative proteomic analysis of triclabendazole response in the liver fluke Fasciola hepatica","volume":"9","author":"Chemale","year":"2010","journal-title":"J. Proteome Res."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"321","DOI":"10.1016\/j.ijpara.2017.12.001","article-title":"Advances in Fasciola hepatica research using \u2018omics\u2019 technologies","volume":"48","author":"Cwiklinski","year":"2018","journal-title":"Int. J. Parasitol."},{"key":"ref_9","doi-asserted-by":"crossref","unstructured":"Dorey, A., Cwiklinski, K., Rooney, J., De Marco Verissimo, C., L\u00f3pez Corrales, J., Jewhurst, H., Fazekas, B., Calvani, N.E.D., Hamon, S., and Gaughan, S. (2021). Autonomous Non Antioxidant Roles for Fasciola hepatica Secreted Thioredoxin-1 and Peroxiredoxin-1. Front. Cell. Infect. Microbiol., 11.","DOI":"10.3389\/fcimb.2021.667272"},{"key":"ref_10","doi-asserted-by":"crossref","unstructured":"Ricafrente, A., Nguyen, H., Tran, N., and Donnelly, S. (2021). An Evaluation of the Fasciola hepatica miRnome Predicts a Targeted Regulation of Mammalian Innate Immune Responses. Front. Immunol., 11.","DOI":"10.3389\/fimmu.2020.608686"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"383","DOI":"10.1139\/cjm-2021-0329","article-title":"Recombinase polymerase amplification in the molecular diagnosis of microbiological targets and its applications","volume":"68","author":"Mota","year":"2022","journal-title":"Can. J. Microbiol."},{"key":"ref_12","doi-asserted-by":"crossref","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":"15","author":"James","year":"2015","journal-title":"Expert Rev. Mol. Diagn."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"451","DOI":"10.1038\/s41594-023-00932-w","article-title":"Phase separation properties of RPA combine high-affinity ssDNA binding with dynamic condensate functions at telomeres","volume":"30","author":"Spegg","year":"2023","journal-title":"Nat. Struct. Mol. Biol."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"234","DOI":"10.1016\/j.molcel.2014.03.011","article-title":"CRISPR-Cas systems: Prokaryotes upgrade to adaptive immunity","volume":"54","author":"Barrangou","year":"2014","journal-title":"Mol. Cell"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"2721","DOI":"10.1002\/jcb.27690","article-title":"Current progress in CRISPR-based diagnostic platforms","volume":"120","author":"Khambhati","year":"2019","journal-title":"J. Cell. Biochem."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"885","DOI":"10.1038\/s41589-023-01534-9","article-title":"Fast and sensitive CRISPR detection by minimized interference of target amplification","volume":"20","author":"Tong","year":"2024","journal-title":"Nat. Chem. Biol."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"71","DOI":"10.1038\/s41576-022-00570-w","article-title":"New cuts for CRISPR effectors","volume":"24","author":"Burgess","year":"2023","journal-title":"Nat. Rev. Genet."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"102350","DOI":"10.1016\/j.nano.2020.102350","article-title":"CRISPR: A new paradigm of theranostics","volume":"33","author":"Yadav","year":"2021","journal-title":"Nanomedicine"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"523","DOI":"10.1016\/j.jgg.2019.11.003","article-title":"Engineering guide RNA to reduce the off-target effects of CRISPR","volume":"46","author":"Wu","year":"2019","journal-title":"J. Genet. Genom."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"266","DOI":"10.1016\/j.vetpar.2011.01.059","article-title":"A TaqMan real-time PCR-based assay for the identification of Fasciola spp.","volume":"179","author":"Alasaad","year":"2011","journal-title":"Vet. Parasitol."},{"key":"ref_21","unstructured":"(2025). Diagnostic Techniques for the Yak Fascioliasis; Group Standard (Standard No. T\/CVMA 320-2025)."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"1633","DOI":"10.1021\/acssynbio.4c00161","article-title":"Multiplexed Detection Strategies for Biosensors Based on the CRISPR-Cas System","volume":"13","author":"Wei","year":"2024","journal-title":"ACS Synth. Biol."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"2274","DOI":"10.1111\/tbed.13882","article-title":"Genetic diversity and relationships of the liver fluke Fasciola hepatica (Trematoda) with native and introduced definitive and intermediate hosts","volume":"68","author":"Sabourin","year":"2021","journal-title":"Transbound. Emerg. Dis."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"88","DOI":"10.1126\/science.aav7271","article-title":"Functionally diverse type V CRISPR-Cas systems","volume":"363","author":"Yan","year":"2019","journal-title":"Science"},{"key":"ref_25","doi-asserted-by":"crossref","unstructured":"Huang, S., Du, L., Liu, S., Yang, Q., Lei, C., Wang, H., Yang, L., and Yang, X. (2024). Development and Validation of RAA-CRISPR\/Cas12a-Based Assay for Detecting Porcine Rotavirus. Animals, 14.","DOI":"10.3390\/ani14233387"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"136912","DOI":"10.1016\/j.ijbiomac.2024.136912","article-title":"Rapid and multiple visual detection of Fasciola hepatica in feces via recombinase polymerase amplification integrated with CRISPR\/Cas12a technology","volume":"282","author":"Yang","year":"2024","journal-title":"Int. J. Biol. Macromol."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"110412","DOI":"10.1016\/j.vetpar.2025.110412","article-title":"Rapid and sensitive detection of Trichomonas gallinae using RAA-CRISPR-Cas12a","volume":"334","author":"Zhou","year":"2025","journal-title":"Vet. Parasitol."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"123334","DOI":"10.1016\/j.talanta.2022.123334","article-title":"Design of high performance fluorescent probe-based test strips for hydrogensulfite determination by chemical grafting","volume":"243","author":"Lu","year":"2022","journal-title":"Talanta"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"10643","DOI":"10.1038\/s41598-023-37347-y","article-title":"Gold conjugated nanobodies in a signal-enhanced lateral flow test strip for rapid detection of SARS-CoV-2 S1 antigen in saliva samples","volume":"13","author":"Maher","year":"2023","journal-title":"Sci. Rep."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"e0347522","DOI":"10.1128\/spectrum.03475-22","article-title":"Development of CRISPR\/Cas12b-Based Multiple Cross Displacement Amplification Technique for the Detection of Mycobacterium tuberculosis Complex in Clinical Settings","volume":"11","author":"Yang","year":"2023","journal-title":"Microbiol. Spectr."},{"key":"ref_31","first-page":"718","article-title":"CRISPR-Cas in its prime","volume":"20","author":"Zlotorynski","year":"2019","journal-title":"Nat. Rev. Mol. Cell Biol."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1089\/crispr.2024.29172.editorial","article-title":"CRISPR Momentum in the Clinic and the Field","volume":"7","author":"Barrangou","year":"2024","journal-title":"CRISPR J."}],"container-title":["Animals"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2076-2615\/16\/7\/1093\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,4,2]],"date-time":"2026-04-02T12:06:26Z","timestamp":1775131586000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2076-2615\/16\/7\/1093"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,4,2]]},"references-count":32,"journal-issue":{"issue":"7","published-online":{"date-parts":[[2026,4]]}},"alternative-id":["ani16071093"],"URL":"https:\/\/doi.org\/10.3390\/ani16071093","relation":{},"ISSN":["2076-2615"],"issn-type":[{"value":"2076-2615","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,4,2]]}}}