{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,18]],"date-time":"2026-08-18T04:35:01Z","timestamp":1787027701848,"version":"build-2736575974"},"reference-count":40,"publisher":"MDPI AG","issue":"2","license":[{"start":{"date-parts":[[2026,1,26]],"date-time":"2026-01-26T00:00:00Z","timestamp":1769385600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100012166","name":"National Key R&D Program of China","doi-asserted-by":"crossref","award":["2024YFD200030303"],"award-info":[{"award-number":["2024YFD200030303"]}],"id":[{"id":"10.13039\/501100012166","id-type":"DOI","asserted-by":"crossref"}]},{"name":"State Key Laboratory for Managing Biotic and Chemical Threats to Quality and Safety of the Agro-products","award":["2021DG700024-KF202507"],"award-info":[{"award-number":["2021DG700024-KF202507"]}]},{"name":"Science and Technology Cooperation Project of Zhejiang Province","award":["2024C04018"],"award-info":[{"award-number":["2024C04018"]}]},{"name":"Team of Science and Technology for Livestock and Poultry Farming Project in Huangyan"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Biosensors"],"abstract":"<jats:p>Duck plague virus (DPV), a highly contagious \u03b1-herpesvirus in the livestock and poultry environment, poses a significant threat to the healthy growth of ducks, potentially causing substantial economic losses. Effective control of DPV requires the development of specific diagnostic tools. A new fluorescent biosensor (R-C-CHA) was developed to detect virulent strains of DPV. It combined recombinase polymerase amplification (RPA), a CRISPR\/Cas12a system, and catalytic hairpin assembly (CHA) for signal enhancement. The RPA primers were specifically designed to target the conserved DPV-CHv UL2 gene region, allowing for the rapid, efficient amplification of the target nucleic acids in isothermal conditions. The CRISPR\/Cas12a system was used for sequence-specific recognition, activating its lateral cleavage activity. Furthermore, the CHA cascade reaction was utilized for enzyme-free fluorescent signal amplification. The results showed that the R-C-CHA biosensor completed the detection process in 40 min with a detection limit of 0.02 fg\/\u03bcL, which was an approximate five-fold improvement compared to traditional RPA-CRISPR\/Cas12a biosensors. The R-C-CHA biosensor also demonstrated perfect consistency with clinical detection and polymerase chain reaction (PCR) diagnosis, highlighting its strong potential for rapid detection in livestock and poultry farming settings.<\/jats:p>","DOI":"10.3390\/bios16020073","type":"journal-article","created":{"date-parts":[[2026,1,26]],"date-time":"2026-01-26T16:13:48Z","timestamp":1769444028000},"page":"73","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":3,"title":["A RPA-CRISPR\/Cas12a-Powered Catalytic Hairpin Assembly Fluorescence Biosensor for Duck Plague Virus Virulent Strain Detection"],"prefix":"10.3390","volume":"16","author":[{"given":"Yue","family":"Wu","sequence":"first","affiliation":[{"name":"State Key Laboratory for Quality and Safety of Agro-Products, Institute of Animal Husbandry and Veterinary Science, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jiaxin","family":"Wan","sequence":"additional","affiliation":[{"name":"College of Biological and Environmental Science, Zhejiang Wanli University, Ningbo 315100, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xingbo","family":"Wang","sequence":"additional","affiliation":[{"name":"State Key Laboratory for Quality and Safety of Agro-Products, Institute of Animal Husbandry and Veterinary Science, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yunjie","family":"Shen","sequence":"additional","affiliation":[{"name":"College of Biological and Environmental Science, Zhejiang Wanli University, Ningbo 315100, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xiangjun","family":"Li","sequence":"additional","affiliation":[{"name":"College of Information Engineering, China Jiliang University, Hangzhou 314423, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Weidong","family":"Zhou","sequence":"additional","affiliation":[{"name":"State Key Laboratory for Quality and Safety of Agro-Products, Institute of Animal Husbandry and Veterinary Science, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0223-1331","authenticated-orcid":false,"given":"Yinchu","family":"Zhu","sequence":"additional","affiliation":[{"name":"State Key Laboratory for Quality and Safety of Agro-Products, Institute of Animal Husbandry and Veterinary Science, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xing","family":"Xu","sequence":"additional","affiliation":[{"name":"State Key Laboratory for Quality and Safety of Agro-Products, Institute of Animal Husbandry and Veterinary Science, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,1,26]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"105092","DOI":"10.1016\/j.psj.2025.105092","article-title":"Duck plague virus UL47 gene affects the release and cell-to-cell spread of the virus and its deletion strains can provide strong protection for ducks","volume":"104","author":"Cui","year":"2025","journal-title":"Poult. Sci."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"105892","DOI":"10.1016\/j.psj.2025.105892","article-title":"Comparative genomic analysis of duck plague virus reveals evolutionary divergence between vaccine and contemporary field isolates in Thailand","volume":"104","author":"Kruasuwan","year":"2025","journal-title":"Poult. Sci."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"105908","DOI":"10.1016\/j.psj.2025.105908","article-title":"Pioneering discovery: US3-phosphorylated sites on VP22 orchestrate duck plague virus release and pathogenicity","volume":"104","author":"Wu","year":"2025","journal-title":"Poult. Sci."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"105848","DOI":"10.1016\/j.psj.2025.105848","article-title":"The UL24 gene affects duck plague virus replication, and its deletion attenuates virulence, enabling use as a vaccine","volume":"104","author":"Cao","year":"2025","journal-title":"Poult. Sci."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"105399","DOI":"10.1016\/j.psj.2025.105399","article-title":"Duck plague virus gG is secreted, nonstructural glycoprotein, not essential for viral replication and responsible for the virulence","volume":"104","author":"Qiu","year":"2025","journal-title":"Poult. Sci."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"105693","DOI":"10.1016\/j.psj.2025.105693","article-title":"Deciphering pUL10\u2032s mastery in duck plague virus virulence: Dual coordination of host immunity and viral replication","volume":"104","author":"Li","year":"2025","journal-title":"Poult. Sci."},{"key":"ref_7","doi-asserted-by":"crossref","unstructured":"Chen, W., Han, S., Li, R., Li, S., Cao, Z., Pan, Q., and Li, G. (2025). Distribution and potential involvement of PTEN in the innate immune response during viral infections in Cherry Valley ducks. Front. Immunol., 16.","DOI":"10.3389\/fimmu.2025.1570872"},{"key":"ref_8","doi-asserted-by":"crossref","unstructured":"Ruan, P., Feng, X., Cheng, A., Wang, M., Zhang, W., Wu, Y., Yang, Q., Tian, B., Ou, X., and Sun, D. (2022). Evaluation of safety and immunogenicity of duck-plague virus gC\/gE double gene deletion. Front. Immunol., 13.","DOI":"10.3389\/fimmu.2022.963009"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"102555","DOI":"10.1016\/j.psj.2023.102555","article-title":"Establishment and application of a PCR assay for the identification of virulent and attenuated duck plague virus DNA in cotton swabs","volume":"102","author":"Wu","year":"2023","journal-title":"Poult. Sci."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"114788","DOI":"10.1016\/j.jviromet.2023.114788","article-title":"Development of the isothermal recombinase polymerase amplification assays for rapid detection of the genus Capripoxvirus","volume":"320","author":"Liu","year":"2023","journal-title":"J. Virol. Methods"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"730","DOI":"10.1016\/j.tibtech.2018.12.005","article-title":"CRISPR\/Cas Systems towards Next-Generation Biosensing","volume":"37","author":"Li","year":"2019","journal-title":"Trends Biotechnol."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"117525","DOI":"10.1016\/j.trac.2024.117525","article-title":"Magnetic particles-integrated CRISPR\/Cas systems for biosensing","volume":"171","author":"Wang","year":"2024","journal-title":"TrAC Trends Anal. Chem."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"118497","DOI":"10.1016\/j.trac.2025.118497","article-title":"CRISPR\/Cas system-based strategies for pathogenic bacteria detection","volume":"194","author":"Zheng","year":"2026","journal-title":"TrAC Trends Anal. Chem."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"5178","DOI":"10.1021\/acs.analchem.3c05364","article-title":"Enhancing Point-of-Care Diagnosis of African Swine Fever Virus (ASFV) DNA with the CRISPR-Cas12a-Assisted Triplex Amplified Assay","volume":"96","author":"Zhu","year":"2024","journal-title":"Anal. Chem."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"117154","DOI":"10.1016\/j.bios.2025.117154","article-title":"CIMNE-CRISPR: A novel amplification-free diagnostic for rapid early detection of African Swine Fever Virus","volume":"273","author":"Pal","year":"2025","journal-title":"Biosens. Bioelectron."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"839","DOI":"10.1126\/science.aav4294","article-title":"Programmed DNA destruction by miniature CRISPR-Cas14 enzymes","volume":"362","author":"Harrington","year":"2018","journal-title":"Science"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"465","DOI":"10.1038\/s41586-024-07815-0","article-title":"Structural basis for the activity of the type VII CRISPR\u2013Cas system","volume":"633","author":"Yang","year":"2024","journal-title":"Nature"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"1166","DOI":"10.1038\/s41467-025-56516-3","article-title":"Tunable control of Cas12 activity promotes universal and fast one-pot nucleic acid detection","volume":"16","author":"Cheng","year":"2025","journal-title":"Nat. Commun."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"126441","DOI":"10.1016\/j.talanta.2024.126441","article-title":"Rapid, portable, and sensitive detection of CaMV35S by RPA-CRISPR\/Cas12a-G4 colorimetric assays with high accuracy deep learning object recognition and classification","volume":"278","author":"Li","year":"2024","journal-title":"Talanta"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"17567","DOI":"10.1021\/acs.analchem.4c03288","article-title":"Optimization and Clinical Application Potential of Single Nucleotide Polymorphism Detection Method Based on CRISPR\/Cas12a and Recombinase Polymerase Amplification","volume":"96","author":"Wang","year":"2024","journal-title":"Anal. Chem."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"132793","DOI":"10.1016\/j.jhazmat.2023.132793","article-title":"Portable biosensor combining CRISPR\/Cas12a and loop-mediated isothermal amplification for antibiotic resistance gene ermB in wastewater","volume":"462","author":"Mao","year":"2024","journal-title":"J. Hazard. Mater."},{"key":"ref_22","doi-asserted-by":"crossref","unstructured":"Wu, Y., Xu, X., Zhu, Y., Wan, J., Wang, X., Zhou, X., Li, X., and Zhou, W. (2025). Research Progress on Multiplexed Pathogen Detection Using Optical Biosensors. Biosensors, 15.","DOI":"10.3390\/bios15060378"},{"key":"ref_23","doi-asserted-by":"crossref","unstructured":"Zhou, X., Wang, S., Ma, Y., Jiang, Y., Li, Y., Shi, J., Deng, G., Tian, G., Kong, H., and Wang, X. (2024). On-Site and Visual Detection of the H5 Subtype Avian Influenza Virus Based on RT-RPA and CRISPR\/Cas12a. Viruses, 16.","DOI":"10.3390\/v16050753"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"318","DOI":"10.1038\/nature06451","article-title":"Programming biomolecular self-assembly pathways","volume":"451","author":"Yin","year":"2008","journal-title":"Nature"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"128043","DOI":"10.1016\/j.talanta.2025.128043","article-title":"Catalytic hairpin assembly assists CRISPR\/Cas12a-mediated high-sensitivity detection of aflatoxin B1","volume":"293","author":"Liu","year":"2025","journal-title":"Talanta"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"118082","DOI":"10.1016\/j.bios.2025.118082","article-title":"DNA tetrahedral nanostructures-loaded DNA microcapsule-enhanced catalytic hairpin assembly for electrochemiluminescence detection of serotonin","volume":"292","author":"Gao","year":"2026","journal-title":"Biosens. Bioelectron."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"113809","DOI":"10.1016\/j.bios.2021.113809","article-title":"Sustainable and cascaded catalytic hairpin assembly for amplified sensing of microRNA biomarkers in living cells","volume":"197","author":"Li","year":"2022","journal-title":"Biosens. Bioelectron."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"137122","DOI":"10.1016\/j.jhazmat.2025.137122","article-title":"DNAzyme-mediated isothermal catalytic hairpin assembly for rapid and enzyme-free amplified detection of lead(II) ion","volume":"486","author":"Zheng","year":"2025","journal-title":"J. Hazard. Mater."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"328","DOI":"10.1186\/s12951-023-02092-x","article-title":"A sensitive and rapid electrochemical biosensor for sEV-miRNA detection based on domino-type localized catalytic hairpin assembly","volume":"21","author":"Li","year":"2023","journal-title":"J. Nanobiotechnol."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"124855","DOI":"10.1016\/j.talanta.2023.124855","article-title":"A colloidal gold test strip based on catalytic hairpin assembly for the clinical detection of influenza a virus nucleic acid","volume":"265","author":"Yao","year":"2023","journal-title":"Talanta"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"135296","DOI":"10.1016\/j.snb.2024.135296","article-title":"A CRISPR\/Cas13a-powered catalytic hairpin assembly evanescent wave fluorescence biosensor for target amplification-free SARS-CoV-2 detection","volume":"405","author":"Song","year":"2024","journal-title":"Sens. Actuators B Chem."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"8377","DOI":"10.1093\/nar\/gkac578","article-title":"Enhancement of CRISPR\/Cas12a trans-cleavage activity using hairpin DNA reporters","volume":"50","author":"Rossetti","year":"2022","journal-title":"Nucleic Acids Res."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"147576","DOI":"10.1016\/j.ijbiomac.2025.147576","article-title":"Duck plague virus UL13 hijacks an RNA-binding protein, interferon induced protein with tetratricopeptide repeats 5, to promote replication","volume":"328","author":"Lin","year":"2025","journal-title":"Int. J. Biol. Macromol."},{"key":"ref_34","first-page":"19","article-title":"Development of a polymerase chain reaction assay for differentiation of duck plague virus virulent strains from attenuated strains","volume":"38","author":"Xie","year":"2017","journal-title":"Prog. Vet. Med."},{"key":"ref_35","first-page":"1439","article-title":"First survey of the occurrence of duck enteritis virus (DEV) in free-ranging Polish water birds","volume":"12","author":"Grzegorz","year":"2013","journal-title":"Arch. Virol."},{"key":"ref_36","unstructured":"Wan, J.X., Zhu, Y.C., Xu, X., Wang, X.B., Zhou, W.D., and Wu, Y. (2025). Development of a rapid detection method for a virulent strain of duck enteritis virus based on real-time fluorescence recombinase polymerase amplification. Chin. J. Biotechnol."},{"key":"ref_37","doi-asserted-by":"crossref","unstructured":"Qiu, X., Xu, S., Liu, X., Ren, H., Han, L., and Li, Z. (2022). CRISPR\/Cas12a-Based Diagnostic Platform Accurately Detects Nocardia farcinica Targeting a Novel Species-Specific Gene. Front. Cell. Infect. Microbiol., 12.","DOI":"10.3389\/fcimb.2022.884411"},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"e70079","DOI":"10.1111\/jfd.70079","article-title":"CRISPR-Enhanced RAA-SHERLOCK Assay for Point-of-Care Detection of Cyprinid Her-pesvirus-3: Development, Validation and Clinical Application","volume":"31","author":"Soliman","year":"2025","journal-title":"J. Fish Dis."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"142304","DOI":"10.1016\/j.ijbiomac.2025.142304","article-title":"Spatial double-layer hydrogels enabled visual detection of Cladobotryum mycophilum based on recombinase-aided amplification\u2014CRISPR\/Cas12a","volume":"308","author":"Kong","year":"2025","journal-title":"Int. J. Biol. Macromol."},{"key":"ref_40","doi-asserted-by":"crossref","unstructured":"Yao, K., Peng, D., Jiang, C., Zhao, W., Li, G., Huang, W., Kong, L., Gao, H., Zheng, J., and Peng, H. (2022). Rapid and Visual Detection of Heterodera schachtii Using Recombinase Polymerase Amplification Combined with Cas12a-Mediated Technology. Int. J. Mol. Sci., 22.","DOI":"10.3390\/ijms222212577"}],"container-title":["Biosensors"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2079-6374\/16\/2\/73\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,1,28]],"date-time":"2026-01-28T05:23:27Z","timestamp":1769577807000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2079-6374\/16\/2\/73"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,1,26]]},"references-count":40,"journal-issue":{"issue":"2","published-online":{"date-parts":[[2026,2]]}},"alternative-id":["bios16020073"],"URL":"https:\/\/doi.org\/10.3390\/bios16020073","relation":{},"ISSN":["2079-6374"],"issn-type":[{"value":"2079-6374","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,1,26]]}}}