{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,14]],"date-time":"2026-10-14T04:48:39Z","timestamp":1791953319796,"version":"4.3.4"},"reference-count":29,"publisher":"MDPI AG","issue":"19","license":[{"start":{"date-parts":[[2026,10,8]],"date-time":"2026-10-08T00:00:00Z","timestamp":1791417600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"},{"start":{"date-parts":[[2026,10,8]],"date-time":"2026-10-08T00:00:00Z","timestamp":1791417600000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Animals"],"abstract":"<jats:p>Avian metapneumovirus (aMPV) is the main etiological agent of turkey rhinotracheitis, causing respiratory distress and economic losses in turkeys. While its impact in broilers and laying hens is relatively limited, aMPV subtype C (aMPV\/C) is an important pathogen in ducks and waterfowl, leading to respiratory distress and severe egg-drop syndrome. To overcome the limitations of current diagnostics, such as heavy reliance on laboratory infrastructure and prolonged turnaround times, we established a rapid detection assay integrating recombinase polymerase amplification (RPA) with CRISPR\/Cas12a and a lateral flow dipstick (LFD), targeting the conserved N gene of aMPV\/C. Reaction parameters were systematically optimized, with 30 nmol\/L Cas12a and 40 nmol\/L crRNA identified as the optimal concentrations. Rigorous specificity evaluation against seven common avian pathogens revealed no cross-reactivity. The limit of detection (LOD) was determined to be as low as 1.3 \u00d7 101 copies\/\u03bcL, outperforming conventional PCR. Validation using clinical samples demonstrated a 98.85% concordance rate with quantitative PCR (qPCR). This platform enables visual detection of aMPV\/C within 1 h with minimal equipment requirements. Combining high sensitivity, absolute specificity, and operational simplicity, this assay shows promise as a practical tool for point-of-care diagnostics and epidemiological surveillance of aMPV\/C.<\/jats:p>","DOI":"10.3390\/ani16193154","type":"journal-article","created":{"date-parts":[[2026,10,8]],"date-time":"2026-10-08T15:16:52Z","timestamp":1791472612000},"page":"3154","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Visual and Equipment-Minimized Detection of Avian Metapneumovirus Subtype C by RT-RPA-CRISPR\/Cas12a Coupled with Lateral Flow Dipstick"],"prefix":"10.3390","volume":"16","author":[{"given":"Qizhang","family":"Liang","sequence":"first","affiliation":[{"name":"Fujian Academy of Agricultural Sciences\/Fujian Provincial Key Laboratory for Avian Diseases Control and Prevention, Institute of Animal Husbandry and Veterinary Medicine, Fuzhou 350013, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shoukang","family":"Ma","sequence":"additional","affiliation":[{"name":"College of Animal Sciences, Fujian Agriculture and Forestry University, Fuzhou 350002, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hongxing","family":"Li","sequence":"additional","affiliation":[{"name":"College of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou 350002, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2336-6681","authenticated-orcid":false,"given":"Weiwei","family":"Wang","sequence":"additional","affiliation":[{"name":"Fujian Academy of Agricultural Sciences\/Fujian Provincial Key Laboratory for Avian Diseases Control and Prevention, Institute of Animal Husbandry and Veterinary Medicine, Fuzhou 350013, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4005-8050","authenticated-orcid":false,"given":"Qiuling","family":"Fu","sequence":"additional","affiliation":[{"name":"Fujian Academy of Agricultural Sciences\/Fujian Provincial Key Laboratory for Avian Diseases Control and Prevention, Institute of Animal Husbandry and Veterinary Medicine, Fuzhou 350013, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nansong","family":"Jiang","sequence":"additional","affiliation":[{"name":"Fujian Academy of Agricultural Sciences\/Fujian Provincial Key Laboratory for Avian Diseases Control and Prevention, Institute of Animal Husbandry and Veterinary Medicine, Fuzhou 350013, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0000-7997-3275","authenticated-orcid":false,"given":"Rongchang","family":"Liu","sequence":"additional","affiliation":[{"name":"Fujian Academy of Agricultural Sciences\/Fujian Provincial Key Laboratory for Avian Diseases Control and Prevention, Institute of Animal Husbandry and Veterinary Medicine, Fuzhou 350013, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0004-9579-9691","authenticated-orcid":false,"given":"Guanghua","family":"Fu","sequence":"additional","affiliation":[{"name":"Fujian Academy of Agricultural Sciences\/Fujian Provincial Key Laboratory for Avian Diseases Control and Prevention, Institute of Animal Husbandry and Veterinary Medicine, Fuzhou 350013, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hongmei","family":"Chen","sequence":"additional","affiliation":[{"name":"Fujian Academy of Agricultural Sciences\/Fujian Provincial Key Laboratory for Avian Diseases Control and Prevention, Institute of Animal Husbandry and Veterinary Medicine, Fuzhou 350013, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Longfei","family":"Cheng","sequence":"additional","affiliation":[{"name":"Fujian Academy of Agricultural Sciences\/Fujian Provincial Key Laboratory for Avian Diseases Control and Prevention, Institute of Animal Husbandry and Veterinary Medicine, Fuzhou 350013, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2011-5609","authenticated-orcid":false,"given":"Chunhe","family":"Wan","sequence":"additional","affiliation":[{"name":"Fujian Academy of Agricultural Sciences\/Fujian Provincial Key Laboratory for Avian Diseases Control and Prevention, Institute of Animal Husbandry and Veterinary Medicine, Fuzhou 350013, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yu","family":"Huang","sequence":"additional","affiliation":[{"name":"Fujian Academy of Agricultural Sciences\/Fujian Provincial Key Laboratory for Avian Diseases Control and Prevention, Institute of Animal Husbandry and Veterinary Medicine, Fuzhou 350013, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,10,8]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"2912","DOI":"10.1099\/jgv.0.000959","article-title":"ICTV Virus Taxonomy Profile: Pneumoviridae","volume":"98","author":"Rima","year":"2017","journal-title":"J. Gen. Virol."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"1093","DOI":"10.1007\/s11250-014-0591-8","article-title":"First evidence of avian metapneumovirus subtype A infection in turkeys in Egypt","volume":"46","author":"Franzo","year":"2014","journal-title":"Trop. Anim. Health Prod."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"1408","DOI":"10.1099\/vir.0.009688-0","article-title":"Fusion protein is the main determinant of metapneumovirus host tropism","volume":"90","author":"Schrauwen","year":"2009","journal-title":"J. Gen. Virol."},{"key":"ref_4","doi-asserted-by":"crossref","unstructured":"Brown, P.A., Lemaitre, E., Briand, F.X., Courtillon, C., Guionie, O., All\u00e9e, C., Toquin, D., Bayon-Auboyer, M.H., Jestin, V., and Eterradossi, N. (2014). Molecular comparisons of full length metapneumovirus (MPV) genomes, including newly determined French AMPV-C and -D isolates, further supports possible subclassification within the MPV Genus. PLoS ONE, 9.","DOI":"10.1371\/journal.pone.0102740"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"97","DOI":"10.1007\/s11262-005-5850-3","article-title":"European and American subgroup C isolates of avian metapneumovirus belong to different genetic lineages","volume":"32","author":"Toquin","year":"2006","journal-title":"Virus Genes"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"1092","DOI":"10.3201\/eid1907.121126","article-title":"Avian metapneumovirus subgroup C infection in chickens, China","volume":"19","author":"Wei","year":"2013","journal-title":"Emerg. Infect. Dis."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"74","DOI":"10.1186\/s13567-014-0074-y","article-title":"Isolation and characterization of a subtype C avian metapneumovirus circulating in Muscovy ducks in China","volume":"45","author":"Sun","year":"2014","journal-title":"Vet. Res."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"e00284-19","DOI":"10.1128\/MRA.00284-19","article-title":"Genome Sequence of a Divergent Avian Metapneumovirus from a Monk Parakeet (Myiopsitta monachus)","volume":"8","author":"Retallack","year":"2019","journal-title":"Microbiol. Resour. Announc."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"191","DOI":"10.1007\/s00705-024-06122-3","article-title":"Molecular characterization of avian metapneumovirus subtype C associated with hydrosalpinx fluid and egg drop syndrome in Jinding ducks","volume":"169","author":"Yu","year":"2024","journal-title":"Arch. Virol."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"2590254","DOI":"10.1080\/21505594.2025.2590254","article-title":"Novel avian metapneumovirus subtype C variant is a newly emerged pathogen causing hydrosalpinx fluid syndrome in Sheldrake ducks in China","volume":"16","author":"Fu","year":"2025","journal-title":"Virulence"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"61","DOI":"10.1016\/j.jviromet.2017.10.010","article-title":"Single reaction, real time RT-PCR detection of all known avian and human metapneumoviruses","volume":"251","author":"Lemaitre","year":"2018","journal-title":"J. Virol. Methods"},{"key":"ref_12","doi-asserted-by":"crossref","unstructured":"Bai, Y., Wu, X., Liu, J., Wang, Z., Dong, M., Li, T., Dai, Z., Li, H., Xie, Q., and Zhang, X. (2024). Establishment and Application Prospect of Reverse Transcriptase Recombinase-Aided Amplification Assay for Subgroup C Avian Metapneumovirus. Vet. Sci., 11.","DOI":"10.3390\/vetsci11030122"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"283","DOI":"10.1080\/03079457.2013.788130","article-title":"Development of a real-time RT-PCR assay for the simultaneous identification, quantitation and differentiation of avian metapneumovirus subtypes A and B","volume":"42","author":"Cecchinato","year":"2013","journal-title":"Avian Pathol."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"104608","DOI":"10.1016\/j.psj.2024.104608","article-title":"Establishment and application of a one-step multiplex real-time PCR assay for detection of A, B, and C subtypes of avian metapneumovirus","volume":"104","author":"Su","year":"2025","journal-title":"Poult. Sci."},{"key":"ref_15","first-page":"17","article-title":"Development and application of a SYBR green I based real-time quantitative PCR method for avian metapneumovirus subtype C","volume":"40","author":"Chen","year":"2018","journal-title":"China Poult."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"150","DOI":"10.1016\/j.jviromet.2006.09.022","article-title":"Laboratory evaluation of a quantitative real-time reverse transcription PCR assay for the detection and identification of the four subgroups of avian metapneumovirus","volume":"139","author":"Guionie","year":"2007","journal-title":"J. Virol. Methods"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"110151","DOI":"10.1016\/j.vetimm.2020.110151","article-title":"Development of A recombinant nucleocapsid based indirect ELISA for the detection of antibodies to avian metapneumovirus subtypes, A, B, and C","volume":"231","author":"Xu","year":"2021","journal-title":"Vet. Immunol. Immunopathol."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"2217","DOI":"10.1094\/PDIS-09-19-1988-RE","article-title":"Genome-Informed Recombinase Polymerase Amplification Assay Coupled with a Lateral Flow Device for In-Field Detection of Dickeya Species","volume":"104","author":"Boluk","year":"2020","journal-title":"Plant Dis."},{"key":"ref_19","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_20","doi-asserted-by":"crossref","first-page":"870","DOI":"10.1038\/s41587-020-0513-4","article-title":"CRISPR-Cas12-based detection of SARS-CoV-2","volume":"38","author":"Broughton","year":"2020","journal-title":"Nat. Biotechnol."},{"key":"ref_21","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_22","doi-asserted-by":"crossref","unstructured":"Miao, F., Zhang, J., Li, N., Chen, T., Wang, L., Zhang, F., Mi, L., Zhang, J., Wang, S., and Wang, Y. (2019). Rapid and Sensitive Recombinase Polymerase Amplification Combined with Lateral Flow Strip for Detecting African Swine Fever Virus. Front Microbiol., 10.","DOI":"10.3389\/fmicb.2019.01004"},{"key":"ref_23","doi-asserted-by":"crossref","unstructured":"Mo, X., Wang, X., Zhu, Z., Yu, Y., Chang, D., Zhang, X., Li, D., Sun, F., Zhou, L., and Xu, J. (2021). Quality Management for Point-Of-Care Testing of Pathogen Nucleic Acids: Chinese Expert Consensus. Front Cell Infect. Microbiol., 11.","DOI":"10.3389\/fcimb.2021.755508"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"589","DOI":"10.1016\/j.molcel.2018.11.021","article-title":"Mechanistic Insights into the cis- and trans-Acting DNase Activities of Cas12a","volume":"73","author":"Swarts","year":"2019","journal-title":"Mol. Cell"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"144966","DOI":"10.1016\/j.ijbiomac.2025.144966","article-title":"Rapid field visual detection of avian metapneumovirus by integrating MIRA pre-amplification with CRISPR-Cas13a to enhance sensitivity and specificity: Innovative technologies well-suited for real-time large-scale epidemiological surveillance","volume":"318","author":"Dong","year":"2025","journal-title":"Int. J. Biol. Macromol."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"106612","DOI":"10.1016\/j.psj.2026.106612","article-title":"Development of an RT-ERA-CRISPR\/Cas12a assay for duck Tembusu virus","volume":"105","author":"Huang","year":"2026","journal-title":"Poult. Sci."},{"key":"ref_27","doi-asserted-by":"crossref","unstructured":"Tian, C., Feng, L., Zhou, X., Huang, K., Wang, F., Luo, R., Meng, F., Yang, H., Qiao, C., and Wang, X. (2025). A Portable One-Tube Assay Integrating RT-RPA and CRISPR\/Cas12a for Rapid Visual Detection of Eurasian Avian-like H1N1 Swine Influenza Virus in the Field. Viruses, 18.","DOI":"10.3390\/v18010047"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"106681","DOI":"10.1016\/j.psj.2026.106681","article-title":"Field-deployable multiplex RAA-CRISPR\/Cas12a platform rapidly and simultaneously detects seven Eimeria species in chickens","volume":"105","author":"Guo","year":"2026","journal-title":"Poult. Sci."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"e70921","DOI":"10.1002\/jmv.70921","article-title":"Dual-Mode CRISPR\/Cas13a Assay for the Detection of Human Metapneumovirus in Clinical Respiratory Samples","volume":"98","author":"Zheng","year":"2026","journal-title":"J. Med. Virol."}],"container-title":["Animals"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2076-2615\/16\/19\/3154\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,10,14]],"date-time":"2026-10-14T04:17:52Z","timestamp":1791951472000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2076-2615\/16\/19\/3154"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,10,8]]},"references-count":29,"journal-issue":{"issue":"19","published-online":{"date-parts":[[2026,10]]}},"alternative-id":["ani16193154"],"URL":"https:\/\/doi.org\/10.3390\/ani16193154","relation":{},"ISSN":["2076-2615"],"issn-type":[{"value":"2076-2615","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,10,8]]}}}