{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,27]],"date-time":"2026-10-27T16:55:37Z","timestamp":1793120137421,"version":"4.5.0"},"reference-count":21,"publisher":"MDPI AG","issue":"11","license":[{"start":{"date-parts":[[2025,11,20]],"date-time":"2025-11-20T00:00:00Z","timestamp":1763596800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"National Key Research and Development Program of China","award":["2021YFF0703600"],"award-info":[{"award-number":["2021YFF0703600"]}]},{"name":"Science and Technology Innovation Project of the Chinese Academy of Agricultural Sciences","award":["CAAS-ASTIP-2021-ISAPS"],"award-info":[{"award-number":["CAAS-ASTIP-2021-ISAPS"]}]},{"DOI":"10.13039\/100007847","name":"Natural Science Foundation of Jilin Province of China","doi-asserted-by":"crossref","award":["20230101172JC"],"award-info":[{"award-number":["20230101172JC"]}],"id":[{"id":"10.13039\/100007847","id-type":"DOI","asserted-by":"crossref"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Veterinary Sciences"],"abstract":"<jats:p>Feline herpesvirus type-1 (FHV-1), a double-stranded DNA virus, which is a highly infectious upper respiratory tract infection of felids, particularly in kittens. Droplet digital PCR (ddPCR) provides an absolute quantification method with high sensitivity and accuracy. This study aimed to develop a highly sensitive and accurate ddPCR assay for the detection of FHV-1. We designed primers and a probe targeting the FHV-1 glycoprotein D (gD) gene and evaluated the assay\u2019s limit of detection (LOD), sensitivity, repeatability, and specificity in comparison to quantitative real-time PCR (qPCR). The developed ddPCR assay demonstrated a strong linear dynamic range (R2 \u2265 0.99) and an exceptionally low LOD of 0.18 copies\/\u03bcL, which was significantly more sensitive than the method qPCR (LOD ~10 copies\/\u03bcL). Additionally, the assay exhibited high specificity with no cross-reactivity against other common feline pathogens (feline calicivirus, FCV; feline panleukopenia virus, FPV; feline infectious peritonitis virus, FIPV; Bordetella bronchiseptica and Chlamydia felis) and displayed outstanding repeatability (inter-run CV &lt; 1.35). When applied to 118 clinical samples, the ddPCR assay achieved a significantly higher positive detection rate (27.4%) compared to qPCR (14.8%). In conclusion, we have successfully established a reliable ddPCR assay for the absolute quantification of FHV-1, providing a superior tool for laboratory diagnosis and research.<\/jats:p>","DOI":"10.3390\/vetsci12111107","type":"journal-article","created":{"date-parts":[[2025,11,20]],"date-time":"2025-11-20T13:25:27Z","timestamp":1763645127000},"page":"1107","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Development and Validation of a Droplet Digital PCR Assay for Detection of Feline Herpesvirus Type-1"],"prefix":"10.3390","volume":"12","author":[{"given":"Yaxi","family":"Zhou","sequence":"first","affiliation":[{"name":"Key Laboratory of Special Animal Epidemic Disease, Ministry of Agriculture, Institute of Special Animal and Plant Sciences, Chinese Academy of Agriculture Sciences, Changchun 130112, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Danni","family":"Wu","sequence":"additional","affiliation":[{"name":"Key Laboratory of Special Animal Epidemic Disease, Ministry of Agriculture, Institute of Special Animal and Plant Sciences, Chinese Academy of Agriculture Sciences, Changchun 130112, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mengle","family":"Tang","sequence":"additional","affiliation":[{"name":"Key Laboratory of Special Animal Epidemic Disease, Ministry of Agriculture, Institute of Special Animal and Plant Sciences, Chinese Academy of Agriculture Sciences, Changchun 130112, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zihan","family":"Ye","sequence":"additional","affiliation":[{"name":"Key Laboratory of Special Animal Epidemic Disease, Ministry of Agriculture, Institute of Special Animal and Plant Sciences, Chinese Academy of Agriculture Sciences, Changchun 130112, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Erkai","family":"Feng","sequence":"additional","affiliation":[{"name":"Key Laboratory of Special Animal Epidemic Disease, Ministry of Agriculture, Institute of Special Animal and Plant Sciences, Chinese Academy of Agriculture Sciences, Changchun 130112, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Haili","family":"Zhang","sequence":"additional","affiliation":[{"name":"State Key Laboratory for Diagnosis and Treatment of Severe Zoonotic Infectious Diseases, Changchun 130062, China"},{"name":"Key Laboratory for Zoonosis Research of the Ministry of Education, Institute of Zoonosis, Changchun 130062, China"},{"name":"College of Veterinary Medicine, Jilin University, Changchun 130062, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Guoliang","family":"Luo","sequence":"additional","affiliation":[{"name":"Key Laboratory of Special Animal Epidemic Disease, Ministry of Agriculture, Institute of Special Animal and Plant Sciences, Chinese Academy of Agriculture Sciences, Changchun 130112, China"},{"name":"State Key Laboratory for Diagnosis and Treatment of Severe Zoonotic Infectious Diseases, Changchun 130062, China"},{"name":"Key Laboratory for Zoonosis Research of the Ministry of Education, Institute of Zoonosis, Changchun 130062, China"},{"name":"College of Veterinary Medicine, Jilin University, Changchun 130062, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zhenjun","family":"Wang","sequence":"additional","affiliation":[{"name":"Key Laboratory of Special Animal Epidemic Disease, Ministry of Agriculture, Institute of Special Animal and Plant Sciences, Chinese Academy of Agriculture Sciences, Changchun 130112, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Chunxia","family":"Wang","sequence":"additional","affiliation":[{"name":"Key Laboratory of Special Animal Epidemic Disease, Ministry of Agriculture, Institute of Special Animal and Plant Sciences, Chinese Academy of Agriculture Sciences, Changchun 130112, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lina","family":"Liu","sequence":"additional","affiliation":[{"name":"Key Laboratory of Special Animal Epidemic Disease, Ministry of Agriculture, Institute of Special Animal and Plant Sciences, Chinese Academy of Agriculture Sciences, Changchun 130112, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yuening","family":"Cheng","sequence":"additional","affiliation":[{"name":"Key Laboratory of Special Animal Epidemic Disease, Ministry of Agriculture, Institute of Special Animal and Plant Sciences, Chinese Academy of Agriculture Sciences, Changchun 130112, China"},{"name":"State Key Laboratory for Diagnosis and Treatment of Severe Zoonotic Infectious Diseases, Changchun 130062, China"},{"name":"Key Laboratory for Zoonosis Research of the Ministry of Education, Institute of Zoonosis, Changchun 130062, China"},{"name":"College of Veterinary Medicine, Jilin University, Changchun 130062, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2025,11,20]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"177","DOI":"10.1007\/s00705-024-06093-5","article-title":"Spectrum Detection and Analysis of the Epidemiological Characteristics of Infectious Pathogens in the Feline Respiratory Tract","volume":"169","author":"Ju","year":"2024","journal-title":"Arch. Virol."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"e0085224","DOI":"10.1128\/msystems.00852-24","article-title":"A \u201cplus One\u201d Strategy Impacts Replication of Felid Alphaherpesvirus 1, Mycoplasma and Chlamydia, and the Metabolism of Coinfected Feline Cells","volume":"9","author":"Klose","year":"2024","journal-title":"mSystems"},{"key":"ref_3","doi-asserted-by":"crossref","unstructured":"Wu, Q., Wu, H., He, S., Liu, Y., Chen, Y., Qi, X., Gu, X., Wen, Y., Jin, X., and Jin, Y. (2022). Feline Herpesvirus Infection and Pathology in Captive Snow Leopard. Sci. Rep., 12.","DOI":"10.1038\/s41598-022-08994-4"},{"key":"ref_4","doi-asserted-by":"crossref","unstructured":"Deng, M., Liang, H., Xu, Y., Shi, Q., Bao, F., Mei, C., Dai, Z., and Huang, X. (2024). Identification, Genetic Characterization, and Pathogenicity of Three Feline Herpesvirus Type 1 Isolates from Domestic Cats in China. Vet. Sci., 11.","DOI":"10.3390\/vetsci11070285"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"29","DOI":"10.1111\/lam.13702","article-title":"Quantitative Differential Analysis of Norovirus Outbreak Samples Using RT-ddPCR","volume":"75","author":"Song","year":"2022","journal-title":"Lett. Appl. Microbiol."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"98","DOI":"10.1016\/j.ymeth.2017.11.014","article-title":"Exploring Viral Reservoir: The Combining Approach of Cell Sorting and Droplet Digital PCR","volume":"134\u2013135","author":"Gibellini","year":"2018","journal-title":"Methods"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"8604","DOI":"10.1021\/ac202028g","article-title":"High-Throughput Droplet Digital PCR System for Absolute Quantitation of DNA Copy Number","volume":"83","author":"Hindson","year":"2011","journal-title":"Anal. Chem."},{"key":"ref_8","doi-asserted-by":"crossref","unstructured":"Quan, P.-L., Sauzade, M., and Brouzes, E. (2018). dPCR: A Technology Review. Sensors, 18.","DOI":"10.3390\/s18041271"},{"key":"ref_9","doi-asserted-by":"crossref","unstructured":"Li, H., Bai, R., Zhao, Z., Tao, L., Ma, M., Ji, Z., Jian, M., Ding, Z., Dai, X., and Bao, F. (2018). Application of Droplet Digital PCR to Detect the Pathogens of Infectious Diseases. Biosci. Rep., 38.","DOI":"10.1042\/BSR20181170"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"1621","DOI":"10.1128\/JCM.00211-17","article-title":"Applications of Digital PCR for Clinical Microbiology","volume":"55","author":"Kuypers","year":"2017","journal-title":"J. Clin. Microbiol."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"174","DOI":"10.1097\/QCO.0b013e3283262fe9","article-title":"New Diagnostic Methods for Tuberculosis","volume":"22","author":"Nyendak","year":"2009","journal-title":"Curr. Opin. Infect. Dis."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"9236","DOI":"10.1073\/pnas.96.16.9236","article-title":"Digital PCR","volume":"96","author":"Vogelstein","year":"1999","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"2638","DOI":"10.1158\/1055-9965.EPI-14-0503","article-title":"Quantification of Circulating miRNAs by Droplet Digital PCR: Comparison of EvaGreen- and TaqMan-Based Chemistries","volume":"23","author":"Miotto","year":"2014","journal-title":"Cancer Epidemiol. Biomarkers Prev."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"1003","DOI":"10.1021\/ac202578x","article-title":"Evaluation of a Droplet Digital Polymerase Chain Reaction Format for DNA Copy Number Quantification","volume":"84","author":"Pinheiro","year":"2012","journal-title":"Anal. Chem."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"45","DOI":"10.1016\/j.jviromet.2011.09.017","article-title":"Digital PCR Provides Absolute Quantitation of Viral Load for an Occult RNA Virus","volume":"179","author":"White","year":"2012","journal-title":"J. Virol. Methods"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"19675","DOI":"10.7448\/IAS.17.4.19675","article-title":"Validation of an Ultrasensitive Digital Droplet PCR Assay for HIV-2 Plasma RNA Quantification","volume":"17","author":"Ruelle","year":"2014","journal-title":"J. Int. AIDS Soc."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"6474","DOI":"10.1021\/ac103230c","article-title":"Evaluation of Digital PCR for Absolute DNA Quantification","volume":"83","author":"Sanders","year":"2011","journal-title":"Anal. Chem."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"28","DOI":"10.1016\/j.meegid.2014.03.028","article-title":"Comparison of a Quantitative Real-Time PCR Assay and Droplet Digital PCR for Copy Number Analysis of the CCL4L Genes","volume":"25","author":"Bharuthram","year":"2014","journal-title":"Infect. Genet. Evol."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"192","DOI":"10.1016\/j.watres.2019.04.056","article-title":"Variably Improved Microbial Source Tracking with Digital Droplet PCR","volume":"159","author":"Nshimyimana","year":"2019","journal-title":"Water Res."},{"key":"ref_20","doi-asserted-by":"crossref","unstructured":"Belmonte, F.R., Martin, J.L., Frescura, K., Damas, J., Pereira, F., Tarnopolsky, M.A., and Kaufman, B.A. (2016). Digital PCR Methods Improve Detection Sensitivity and Measurement Precision of Low Abundance mtDNA Deletions. Sci. Rep., 6.","DOI":"10.1038\/srep25186"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"4163","DOI":"10.1039\/C6LC01075J","article-title":"Robust Scalable High Throughput Production of Monodisperse Drops","volume":"16","author":"Amstad","year":"2016","journal-title":"Lab. Chip"}],"container-title":["Veterinary Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2306-7381\/12\/11\/1107\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,11,22]],"date-time":"2025-11-22T05:32:26Z","timestamp":1763789546000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2306-7381\/12\/11\/1107"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,11,20]]},"references-count":21,"journal-issue":{"issue":"11","published-online":{"date-parts":[[2025,11]]}},"alternative-id":["vetsci12111107"],"URL":"https:\/\/doi.org\/10.3390\/vetsci12111107","relation":{},"ISSN":["2306-7381"],"issn-type":[{"value":"2306-7381","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,11,20]]}}}