{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,31]],"date-time":"2026-07-31T11:41:30Z","timestamp":1785498090987,"version":"3.56.0"},"reference-count":35,"publisher":"MDPI AG","issue":"5","license":[{"start":{"date-parts":[[2026,2,26]],"date-time":"2026-02-26T00:00:00Z","timestamp":1772064000000},"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":["2025YFD1800402"],"award-info":[{"award-number":["2025YFD1800402"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Feline infectious peritonitis (FIP) is a highly lethal disease caused by feline infectious peritonitis virus (FIPV), which poses significant diagnostic challenges in clinical practice. FIPV is divided into two serotypes (serotype I and serotype II) based on distinct serological responses driven by substantial sequence divergence in the spike (S) protein. Serotype I predominates in Europe and North America, whereas serotype II is more common in Asia. In this study, we developed a triplex reverse transcription quantitative PCR (RT-qPCR) assay for simultaneous detection and serotyping of FIPV. Primers and TaqMan probes were designed to target the conserved nucleocapsid (N) gene and serotype-specific regions within the S gene. After systematic optimization of reaction conditions, the final assay employed an annealing temperature of 64 \u00b0C and optimized primer\u2013probe concentrations. The assay exhibited excellent linearity (R2 &gt; 0.99 for all targets), with amplification efficiencies ranging from 97.39% to 109.97%. No cross-reactivity was observed with other common feline pathogens, confirming high specificity. The limit of detection was as low as 0.5 copies\/\u00b5L, and intra-assay repeatability showed coefficients of variation below 2.1%. Clinical validation using 63 feline samples revealed an overall FIPV positivity rate of 21.63%, with serotype II (17.46%) markedly more prevalent than serotype I (3.17%). Collectively, this triplex RT-qPCR assay demonstrates high sensitivity, exceptional specificity, and robust reproducibility, making it a valuable tool for rapid clinical diagnosis through the simultaneous detection of feline coronavirus (FCoV) and serotyping of FIPV.<\/jats:p>","DOI":"10.3390\/ijms27052204","type":"journal-article","created":{"date-parts":[[2026,2,26]],"date-time":"2026-02-26T13:58:03Z","timestamp":1772114283000},"page":"2204","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Development and Validation of a Triplex RT-qPCR Assay for Rapid Clinical Diagnosis and Serotyping of Feline Infectious Peritonitis Virus"],"prefix":"10.3390","volume":"27","author":[{"given":"Ruilong","family":"Xiao","sequence":"first","affiliation":[{"name":"College of Veterinary Medicine, Beijing University of Agriculture, Beijing 102206, China"},{"name":"Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yanhe","family":"Chen","sequence":"additional","affiliation":[{"name":"Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0002-2828-9021","authenticated-orcid":false,"given":"Ying","family":"Huang","sequence":"additional","affiliation":[{"name":"Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Chunhao","family":"Tao","sequence":"additional","affiliation":[{"name":"Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xinxin","family":"Jin","sequence":"additional","affiliation":[{"name":"Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yingjia","family":"Gu","sequence":"additional","affiliation":[{"name":"Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2782-6510","authenticated-orcid":false,"given":"Weifeng","family":"Yuan","sequence":"additional","affiliation":[{"name":"Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Wenjin","family":"Song","sequence":"additional","affiliation":[{"name":"Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zhen","family":"Wang","sequence":"additional","affiliation":[{"name":"Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Huanrong","family":"Li","sequence":"additional","affiliation":[{"name":"College of Veterinary Medicine, Beijing University of Agriculture, Beijing 102206, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hong","family":"Jia","sequence":"additional","affiliation":[{"name":"Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,2,26]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"199059","DOI":"10.1016\/j.virusres.2023.199059","article-title":"An Updated Review of Feline Coronavirus: Mind the Two Biotypes","volume":"326","author":"Gao","year":"2023","journal-title":"Virus Res."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"505","DOI":"10.1177\/0300985814522077","article-title":"Feline Infectious Peritonitis: Still an Enigma?","volume":"51","author":"Kipar","year":"2014","journal-title":"Vet. Pathol."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"17","DOI":"10.1016\/j.jcpa.2025.11.003","article-title":"Diagnosis of Feline Infectious Peritonitis Using the Cell Tube Block Technique\u2014A Pilot Study","volume":"223","author":"Lopes","year":"2025","journal-title":"J. Comp. Pathol."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"415","DOI":"10.1099\/vir.0.016485-0","article-title":"Feline Infectious Peritonitis: Insights into Feline Coronavirus Pathobiogenesis and Epidemiology Based on Genetic Analysis of the Viral 3c Gene","volume":"91","author":"Chang","year":"2010","journal-title":"J. Gen. Virol."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"102209","DOI":"10.1016\/j.cimid.2024.102209","article-title":"Viral Pathogens in Domestic Cats in Southern Italy: A Retrospective Analysis in Sicily, 2020\u20132022","volume":"111","author":"Mira","year":"2024","journal-title":"Comp. Immunol. Microbiol. Infect. Dis."},{"key":"ref_6","doi-asserted-by":"crossref","unstructured":"Wang, Y.-T., Chueh, L.-L., and Wan, C.-H. (2014). An Eight-Year Epidemiologic Study Based on Baculovirus-Expressed Type-Specific Spike Proteins for the Differentiation of Type I and II Feline Coronavirus Infections. BMC Vet. Res., 10.","DOI":"10.1186\/s12917-014-0186-7"},{"key":"ref_7","doi-asserted-by":"crossref","unstructured":"Vasinioti, V.I., Lucente, M.S., Catella, C., Buonavoglia, C., Decaro, N., Pratelli, A., and Capozza, P. (2026). Feline Infectious Peritonitis: A Challenging Diagnostic and Therapeutic Labyrinth. Animals, 16.","DOI":"10.3390\/ani16010128"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"17","DOI":"10.1016\/j.vetimm.2016.01.002","article-title":"Natural Resistance to Experimental Feline Infectious Peritonitis Virus Infection Is Decreased Rather than Increased by Positive Genetic Selection","volume":"171","author":"Pedersen","year":"2016","journal-title":"Vet. Immunol. Immunopathol."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"233","DOI":"10.1016\/j.vetmic.2008.11.010","article-title":"Genetic Diversity and Correlation with Feline Infectious Peritonitis of Feline Coronavirus Type I and II: A 5-Year Study in Taiwan","volume":"136","author":"Lin","year":"2009","journal-title":"Vet. Microbiol."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"115214","DOI":"10.1016\/j.jviromet.2025.115214","article-title":"Development of Rapid and Simple FCoV RNA Detection Systems Using RT-PCR and RT-RPA Combined with STH-PAS to Diagnose FIP in Cats","volume":"338","author":"Doki","year":"2025","journal-title":"J. Virol. Methods"},{"key":"ref_11","doi-asserted-by":"crossref","unstructured":"Hao, F., Tao, C., Xiao, R., Huang, Y., Yuan, W., Wang, Z., and Jia, H. (2025). Development of a Multiplex Real-Time PCR Assay for the Detection of Eight Pathogens Associated with Bovine Respiratory Disease Complex from Clinical Samples. Microorganisms, 13.","DOI":"10.3390\/microorganisms13071629"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"142683","DOI":"10.1016\/j.ijbiomac.2025.142683","article-title":"Development of a Monoclonal Antibody-Based Colloidal Gold Immunochromatographic Strip for Rapid Detection of Feline Coronavirus","volume":"309","author":"Zhang","year":"2025","journal-title":"Int. J. Biol. Macromol."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"235","DOI":"10.1016\/j.vetmic.2008.05.019","article-title":"Differential Role for Low pH and Cathepsin-Mediated Cleavage of the Viral Spike Protein during Entry of Serotype II Feline Coronaviruses","volume":"132","author":"Regan","year":"2008","journal-title":"Vet. Microbiol."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"60","DOI":"10.1186\/s13567-017-0467-9","article-title":"Limitations of Using Feline Coronavirus Spike Protein Gene Mutations to Diagnose Feline Infectious Peritonitis","volume":"48","author":"Barker","year":"2017","journal-title":"Vet. Res."},{"key":"ref_15","doi-asserted-by":"crossref","unstructured":"Liu, Y., Zhu, Z., Du, J., Zhu, X., Pan, C., Yin, C., and Sun, W. (2024). Development of Multiplex Real-Time PCR for Simultaneous Detection of SARS-CoV-2, CCoV, and FIPV. Front. Vet. Sci., 11.","DOI":"10.3389\/fvets.2024.1337690"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"105709","DOI":"10.1016\/j.rvsc.2025.105709","article-title":"Strategies for Combating FIPV Infection: Antiviral Agents and Vaccines","volume":"192","author":"Gao","year":"2025","journal-title":"Res. Vet. Sci."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"1037","DOI":"10.1038\/s41598-025-30674-2","article-title":"Analysis of Risk Factors, Clinical Data, Treatment Outcomes for Cats with Feline Infectious Peritonitis Using GS-441524 (2020\u20132024)","volume":"16","year":"2025","journal-title":"Sci. Rep."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"124344","DOI":"10.1016\/j.ijpharm.2024.124344","article-title":"Design of Novel Chiral Self-Assembling Peptides to Explore the Efficiency and Mechanism of mRNA-FIPV Vaccine Delivery Vehicles","volume":"660","author":"Lu","year":"2024","journal-title":"Int. J. Pharm."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"126576","DOI":"10.1016\/j.ijpharm.2026.126576","article-title":"A Label-Free Multi-Parameter Screening Method for Predicting the Stability of mRNA-LNP Formulations","volume":"691","author":"Sun","year":"2026","journal-title":"Int. J. Pharm."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"110864","DOI":"10.1016\/j.vetmic.2025.110864","article-title":"Beyond Macrophages: FIPV Tropism Includes T and B Lymphocytes","volume":"313","author":"Balakumar","year":"2025","journal-title":"Vet. Microbiol."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"7589098","DOI":"10.1155\/tbed\/7589098","article-title":"Circulation and Genetic Characterizations of Coronaviruses from Companion Animals in Chengdu, Southwest China: One-Year Postpandemic","volume":"2025","author":"Liao","year":"2025","journal-title":"Transbound. Emerg. Dis."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"e0083925","DOI":"10.1128\/jvi.00839-25","article-title":"Mutations in Feline Infectious Peritonitis Virus Nonstructural Protein 14\/16 Methyltransferase Attenuate the Pathogenicity of the Virus in Cats","volume":"99","author":"Jiao","year":"2025","journal-title":"J. Virol."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"002130","DOI":"10.1099\/jgv.0.002130","article-title":"Coronaviruses in Wild Rodent and Eulipotyphlan Small Mammals: A Review of Diversity, Ecological Implications and Surveillance Considerations","volume":"106","author":"Jeeves","year":"2025","journal-title":"J. Gen. Virol."},{"key":"ref_24","doi-asserted-by":"crossref","unstructured":"Maeda, C., Ono, Y., Takahashi, K., Mori, M., Suzuki, M., Tamamura, N., Sun, Y., Itoh, T., Tanaka, H., and Kawabata, H. (2026). Six-Color Multiplex Digital PCR Assays for Comprehensive Screening and Identification of Multiple Driver Mutations Associated with Pancreatic Carcinogenesis. Clin. Chem., Advance article.","DOI":"10.1093\/clinchem\/hvaf181"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"149634","DOI":"10.1016\/j.ijbiomac.2025.149634","article-title":"Development and Validation of a Recombinant N Protein-Based Indirect ELISA for Serological Detection of Feline Infectious Peritonitis Virus","volume":"338","author":"Xing","year":"2025","journal-title":"Int. J. Biol. Macromol."},{"key":"ref_26","doi-asserted-by":"crossref","unstructured":"Ouyang, H., Liu, J., Yin, Y., Cao, S., Yan, R., Ren, Y., Zhou, D., Li, Q., Li, J., and Liao, X. (2022). Epidemiology and Comparative Analyses of the S Gene on Feline Coronavirus in Central China. Pathogens, 11.","DOI":"10.3390\/pathogens11040460"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"228","DOI":"10.1038\/s41586-025-09340-0","article-title":"Feline Infectious Peritonitis Epizootic Caused by a Recombinant Coronavirus","volume":"645","author":"Attipa","year":"2025","journal-title":"Nature"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"348","DOI":"10.1016\/j.vetmic.2007.04.031","article-title":"Differentiation of Feline Coronavirus Type I and II Infections by Virus Neutralization Test","volume":"124","author":"Shiba","year":"2007","journal-title":"Vet. Microbiol."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"115224","DOI":"10.1016\/j.jviromet.2025.115224","article-title":"Establishment of an Indirect ELISA for Feline Coronavirus Antibody Detection and Serotype Discrimination","volume":"338","author":"Lu","year":"2025","journal-title":"J. Virol. Methods"},{"key":"ref_30","doi-asserted-by":"crossref","unstructured":"Burgener, C.J., Barker, E.N., Soare, T., Soare, D.-G., Spiri, A.M., Malbon, A.J., Meli, M.L., and Kipar, A. (2025). Deciphering Viral Replication Dynamics in Feline Infectious Peritonitis: A Quantitative Approach. Viruses, 17.","DOI":"10.3390\/v17020279"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"331","DOI":"10.1186\/s12985-025-02954-w","article-title":"The Establishment of a Universal Standard for Both Viral Antigen and Nucleid Acid Detection Based on Digital PCR","volume":"22","author":"Li","year":"2025","journal-title":"Virol. J."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"e0192125","DOI":"10.1128\/mbio.01921-25","article-title":"Rethinking the Drivers of Coronavirus Virulence and Pathogenesis; toward an Understanding of the Dynamic World of Mutations, Indels, and Recombination within the Alphacoronaviruses","volume":"16","author":"Frazier","year":"2025","journal-title":"mBio"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"3863","DOI":"10.1128\/JVI.78.8.3863-3871.2004","article-title":"Live, Attenuated Coronavirus Vaccines through the Directed Deletion of Group-Specific Genes Provide Protection against Feline Infectious Peritonitis","volume":"78","author":"Haijema","year":"2004","journal-title":"J. Virol."},{"key":"ref_34","doi-asserted-by":"crossref","unstructured":"Serra, F., Canzanella, S., Brandi, S., Picazio, G., Pugliese, A.M., Del Sorbo, L., Miletti, G., Ragosta, E., Sannino, E., and Fiorito, F. (2025). Linking Pollution and Viral Risk: Detection of Dioxins and Coronaviruses in Cats and Dogs. Viruses, 17.","DOI":"10.3390\/v17091271"},{"key":"ref_35","unstructured":"Green, M.R., and Joseph, S. (2012). Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press. [4th ed.]."}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/27\/5\/2204\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,2,28]],"date-time":"2026-02-28T05:19:57Z","timestamp":1772255997000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/27\/5\/2204"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,2,26]]},"references-count":35,"journal-issue":{"issue":"5","published-online":{"date-parts":[[2026,3]]}},"alternative-id":["ijms27052204"],"URL":"https:\/\/doi.org\/10.3390\/ijms27052204","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,2,26]]}}}