{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,3,9]],"date-time":"2026-03-09T19:43:10Z","timestamp":1773085390264,"version":"3.50.1"},"reference-count":44,"publisher":"MDPI AG","issue":"12","license":[{"start":{"date-parts":[[2024,11,29]],"date-time":"2024-11-29T00:00:00Z","timestamp":1732838400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Guangxi Science and Technology Bureau","award":["AB21238003"],"award-info":[{"award-number":["AB21238003"]}]},{"name":"Guangxi Science and Technology Bureau","award":["Z2022198"],"award-info":[{"award-number":["Z2022198"]}]},{"name":"Guangxi Agricultural and Rural Bureau","award":["AB21238003"],"award-info":[{"award-number":["AB21238003"]}]},{"name":"Guangxi Agricultural and Rural Bureau","award":["Z2022198"],"award-info":[{"award-number":["Z2022198"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Pathogens"],"abstract":"<jats:p>Canine coronavirus (CCoV), canine respiratory coronavirus (CRCoV), canine adenovirus type 2 (CAV-2), and canine norovirus (CNV) are important pathogens for canine viral gastrointestinal and respiratory diseases. Especially, co-infections with these viruses exacerbate the damages of diseases. In this study, four pairs of primers and probes were designed to specifically amplify the conserved regions of the CCoV M gene, CRCoV N gene, CAV-2 hexon gene, and CNV RdRp gene. After optimizing different reaction conditions, a quadruplex RT-qPCR was established for the detection of CCoV, CRCoV, CAV-2, and CNV. The specificity, sensitivity, and repeatability of the established assay were evaluated. Then, the assay was used to test 1688 clinical samples from pet hospitals in Guangxi province of China during 2022\u20132024 to validate its clinical applicability. In addition, these samples were also assessed using the reported reference RT-qPCR assays, and the agreements between the developed and reference assays were determined. The results indicated that the quadruplex RT-qPCR could specifically test only CCoV, CRCoV, CAV-2, and CNV, without cross-reaction with other canine viruses. The assay had high sensitivity with limits of detection (LODs) of 1.0 \u00d7 102 copies\/reaction for CCoV, CRCoV, CAV-2, and CNV. The repeatability was excellent, with intra-assay variability of 0.19\u20131.31% and inter-assay variability of 0.10\u20130.88%. The positivity rates of CCoV, CRCoV, CAV-2, and CNV using the developed assay were 8.59% (145\/1688), 8.65% (146\/1688), 2.84% (48\/1688), and 1.30% (22\/1688), respectively, while the positivity rates using the reference assays were 8.47% (143\/1688), 8.53% (144\/1688), 2.78% (47\/1688), and 1.24% (21\/1688), respectively, with agreements of more than 99.53% between two methods. In conclusion, a quadruplex RT-qPCR with high sensitivity, specificity, and repeatability was developed for rapid, and accurate detection of CCoV, CRCoV, CAV-2, and CNV.<\/jats:p>","DOI":"10.3390\/pathogens13121054","type":"journal-article","created":{"date-parts":[[2024,12,2]],"date-time":"2024-12-02T03:50:57Z","timestamp":1733111457000},"page":"1054","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":4,"title":["Establishment of a Quadruplex RT-qPCR for the Detection of Canine Coronavirus, Canine Respiratory Coronavirus, Canine Adenovirus Type 2, and Canine Norovirus"],"prefix":"10.3390","volume":"13","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-1023-9635","authenticated-orcid":false,"given":"Kaichuang","family":"Shi","sequence":"first","affiliation":[{"name":"School of Basic Medical Sciences, Youjiang Medical University for Nationalities, Baise 533000, China"},{"name":"College of Animal Science and Technology, Guangxi University, Nanning 530005, China"},{"name":"Guangxi Center for Animal Disease Control and Prevention, Nanning 530001, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yandi","family":"Shi","sequence":"additional","affiliation":[{"name":"College of Animal Science and Technology, Guangxi University, Nanning 530005, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yuwen","family":"Shi","sequence":"additional","affiliation":[{"name":"College of Animal Science and Technology, Guangxi University, Nanning 530005, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9625-0527","authenticated-orcid":false,"given":"Feng","family":"Long","sequence":"additional","affiliation":[{"name":"Guangxi Center for Animal Disease Control and Prevention, Nanning 530001, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yanwen","family":"Yin","sequence":"additional","affiliation":[{"name":"Guangxi Center for Animal Disease Control and Prevention, Nanning 530001, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yi","family":"Pan","sequence":"additional","affiliation":[{"name":"School of Basic Medical Sciences, Youjiang Medical University for Nationalities, Baise 533000, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7489-0005","authenticated-orcid":false,"given":"Zongqiang","family":"Li","sequence":"additional","affiliation":[{"name":"College of Animal Science and Technology, Guangxi University, Nanning 530005, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Shuping","family":"Feng","sequence":"additional","affiliation":[{"name":"Guangxi Center for Animal Disease Control and Prevention, Nanning 530001, China"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2024,11,29]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"Haake, C., Cook, S., Pusterla, N., and Murphy, B. (2020). Coronavirus infections in companion animals: Virology, epidemiology, clinical and pathologic features. Viruses, 12.","DOI":"10.3390\/v12091023"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"815","DOI":"10.1016\/j.cvsm.2008.02.008","article-title":"Canine respiratory coronavirus: An emerging pathogen in the canine infectious respiratory disease complex","volume":"38","author":"Erles","year":"2008","journal-title":"Vet. Clin. N. Am. Small Anim. Pract."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"86","DOI":"10.1016\/j.jcpa.2020.02.005","article-title":"Aetiology of canine infectious respiratory disease complex and prevalence of its pathogens in Europe","volume":"176","author":"Day","year":"2020","journal-title":"J. Comp. Pathol."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"980","DOI":"10.3201\/eid1606.091861","article-title":"Novel norovirus in dogs with diarrhea","volume":"16","author":"Mesquita","year":"2010","journal-title":"Emerg. Infect. Dis."},{"key":"ref_5","doi-asserted-by":"crossref","unstructured":"Yondo, A., Kalantari, A.A., Fernandez-Marrero, I., McKinney, A., Naikare, H.K., and Velayudhan, B.T. (2023). Predominance of canine parainfluenza virus and Mycoplasma in canine infectious respiratory disease complex in dogs. Pathogens, 12.","DOI":"10.20944\/preprints202310.0863.v1"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"31","DOI":"10.1016\/j.vetmic.2017.10.019","article-title":"European surveillance of emerging pathogens associated with canine infectious respiratory disease","volume":"212","author":"Mitchell","year":"2017","journal-title":"Vet. Microbiol."},{"key":"ref_7","doi-asserted-by":"crossref","unstructured":"Hao, X., Liu, R., He, Y., Xiao, X., Xiao, W., Zheng, Q., Lin, X., Tao, P., Zhou, P., and Li, S. (2019). Multiplex PCR methods for detection of several viruses associated with canine respiratory and enteric diseases. PLoS ONE, 14.","DOI":"10.1371\/journal.pone.0213295"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"001843","DOI":"10.1099\/jgv.0.001843","article-title":"ICTV virus taxonomy profile: Coronaviridae 2023","volume":"104","author":"Woo","year":"2023","journal-title":"J. Gen. Virol."},{"key":"ref_9","doi-asserted-by":"crossref","unstructured":"Buonavoglia, A., Pellegrini, F., Decaro, N., Galgano, M., and Pratelli, A. (2023). A one health perspective on canine coronavirus: A wolf in sheep\u2019s clothing?. Microorganisms, 11.","DOI":"10.3390\/microorganisms11040921"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"216","DOI":"10.1016\/S0042-6822(03)00160-0","article-title":"Detection of a group 2 coronavirus in dogs with canine infectious respiratory disease","volume":"310","author":"Erles","year":"2003","journal-title":"Virology"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"78","DOI":"10.1016\/j.virusres.2006.10.004","article-title":"Isolation and sequence analysis of canine respiratory coronavirus","volume":"124","author":"Erles","year":"2007","journal-title":"Virus Res."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"57","DOI":"10.1016\/j.tvjl.2017.12.009","article-title":"New viruses associated with canine gastroenteritis","volume":"232","author":"Caddy","year":"2018","journal-title":"Vet. J."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"1306","DOI":"10.3201\/eid1408.080062","article-title":"Detection and molecular characterization of a canine norovirus","volume":"14","author":"Martella","year":"2008","journal-title":"Emerg. Infect. Dis."},{"key":"ref_14","doi-asserted-by":"crossref","unstructured":"Zhu, Y., Xu, J., Lian, S., Zhang, R., Hou, J., Wang, M., and Yan, X. (2022). Difference analysis between canine adenovirus types 1 and 2. Front. Cell. Infect. Microbiol., 12.","DOI":"10.3389\/fcimb.2022.854876"},{"key":"ref_15","first-page":"238","article-title":"Association of canine adenovirus (Toronto A 26\/61) with an outbreak of laryngotracheitis (\u201ckennel cough\u201d): A preliminary report","volume":"3","author":"Ditchfield","year":"1962","journal-title":"Can. Vet. J."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"355","DOI":"10.1051\/vetres:2006058","article-title":"Canine respiratory viruses","volume":"38","author":"Buonavoglia","year":"2007","journal-title":"Vet. Res."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"467","DOI":"10.1177\/0300985820926485","article-title":"Canine respiratory coronavirus: A naturally occurring model of COVID-19?","volume":"57","author":"Priestnall","year":"2020","journal-title":"Vet. Pathol."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"587024","DOI":"10.1155\/2013\/587024","article-title":"The serological and virological investigation of canine adenovirus infection on the dogs","volume":"2013","author":"Bulut","year":"2013","journal-title":"Sci. World J."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"365","DOI":"10.1016\/j.tvjl.2014.04.019","article-title":"Detection of respiratory viruses and Bordetella bronchiseptica in dogs with acute respiratory tract infections","volume":"201","author":"Schulz","year":"2014","journal-title":"Vet. J."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"572","DOI":"10.1111\/jsap.12389","article-title":"Prevalence of canine infectious respiratory pathogens in asymptomatic dogs presented at US animal shelters","volume":"56","author":"Lavan","year":"2015","journal-title":"J. Small Anim. Pract."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"75","DOI":"10.1016\/j.meegid.2017.04.020","article-title":"Identification of a novel canine norovirus","volume":"52","author":"Bodnar","year":"2017","journal-title":"Infect. Genet. Evol."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"898","DOI":"10.1128\/CVI.00048-14","article-title":"Seroprevalence of canine norovirus in 14 European countries","volume":"21","author":"Mesquita","year":"2014","journal-title":"Clin. Vaccine Immunol."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"225","DOI":"10.1016\/j.vetmic.2006.12.001","article-title":"Serological and molecular evidence that canine respiratory coronavirus is circulating in Italy","volume":"121","author":"Decaro","year":"2007","journal-title":"Vet. Microbiol."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"2615","DOI":"10.1128\/JCM.02397-05","article-title":"Survey of dogs in Japan for group 2 canine coronavirus infection","volume":"44","author":"Yachi","year":"2006","journal-title":"J. Clin. Microbiol."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"125","DOI":"10.1007\/978-1-4939-6730-8_8","article-title":"Multiplex analyses using real-time quantitative PCR","volume":"1546","author":"Hawkins","year":"2017","journal-title":"Methods Mol. Biol."},{"key":"ref_26","doi-asserted-by":"crossref","unstructured":"Kralik, P., and Ricchi, M. (2017). A basic guide to real time PCR in microbial diagnostics: Definitions, parameters, and everything. Front. Microbiol., 8.","DOI":"10.3389\/fmicb.2017.00108"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"8","DOI":"10.1016\/j.mcp.2020.101618","article-title":"One-step multiplex TaqMan probe-based method for real-time PCR detection of four canine diarrhea viruses","volume":"53","author":"Wang","year":"2020","journal-title":"Mol. Cell. Probes"},{"key":"ref_28","doi-asserted-by":"crossref","unstructured":"Thieulent, C.J., Carossino, M., Peak, L., Wolfson, W., and Balasuriya, U.B.R. (2023). Multiplex one-step RT-qPCR assays for simultaneous detection of SARS-CoV-2 and other enteric viruses of dogs and cats. Viruses, 15.","DOI":"10.3390\/v15091890"},{"key":"ref_29","doi-asserted-by":"crossref","unstructured":"Thieulent, C.J., Carossino, M., Peak, L., Strother, K., Wolfson, W., and Balasuriya, U.B.R. (2023). Development and validation of a panel of one-step four-plex qPCR\/RT-qPCR assays for simultaneous detection of SARS-CoV-2 and other pathogens associated with canine infectious respiratory disease complex. Viruses, 15.","DOI":"10.3390\/v15091881"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"8","DOI":"10.1016\/j.mex.2023.102476","article-title":"Validation of a real-time PCR panel for detection and quantification of nine pathogens commonly associated with canine infectious respiratory disease","volume":"11","author":"Dong","year":"2023","journal-title":"MethodsX"},{"key":"ref_31","doi-asserted-by":"crossref","unstructured":"Dong, J.S., Tsui, W.N.T., Leng, X., Fu, J.P., Lohman, M., Anderson, J., Hamill, V., Lu, N.Y., Porter, E.P., and Gray, M. (2022). Development of a three-panel multiplex real-time PCR assay for simultaneous detection of nine canine respiratory pathogens. J. Microbiol. Methods, 199.","DOI":"10.1016\/j.mimet.2022.106528"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1016\/j.jviromet.2016.03.011","article-title":"A duplex real-time PCR assay based on TaqMan technology for simultaneous detection and differentiation of canine adenovirus types 1 and 2","volume":"234","author":"Dowgier","year":"2016","journal-title":"J. Virol. Methods"},{"key":"ref_33","doi-asserted-by":"crossref","unstructured":"Caddy, S., Emmott, E., El-Attar, L., Mitchell, J., de Rougemont, A., Brownlie, J., and Goodfellow, I. (2013). Serological evidence for multiple strains of canine norovirus in the UK dog population. PLoS ONE, 8.","DOI":"10.1371\/journal.pone.0081596"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"746","DOI":"10.3390\/microbiolres15020049","article-title":"A quadruplex reverse transcription quantitative polymerase chain reaction for detecting canine coronavirus, canine rotavirus, canine parvovirus, and canine distemper virus","volume":"15","author":"Shi","year":"2024","journal-title":"Microbiol. Res."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"104879","DOI":"10.1016\/j.meegid.2021.104879","article-title":"First detection of canine norovirus in dogs and a complete GVI.2 genome in mainland China","volume":"92","author":"Ma","year":"2021","journal-title":"Infect. Genet. Evol."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"1171","DOI":"10.1016\/j.cvsm.2011.08.002","article-title":"Canine noroviruses","volume":"41","author":"Martella","year":"2011","journal-title":"Vet. Clin. N. Am. Small Anim. Pract."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"350","DOI":"10.3201\/eid2602.191151","article-title":"Human norovirus infection in dogs, Thailand","volume":"26","author":"Charoenkul","year":"2020","journal-title":"Emerg. Infect. Dis."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"2839","DOI":"10.1007\/s00705-015-2571-3","article-title":"Sewage surveillance reveals the presence of canine GVII norovirus and canine astrovirus in Uruguay","volume":"160","author":"Lizasoain","year":"2015","journal-title":"Arch. Virol."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"155","DOI":"10.1186\/s12985-024-02425-8","article-title":"A systematic review and meta-analysis of canine enteric coronavirus prevalence in dogs of mainland China","volume":"21","author":"Chen","year":"2024","journal-title":"Virol. J."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"105289","DOI":"10.1016\/j.rvsc.2024.105289","article-title":"Epidemiological investigation of enteric canine coronaviruses in domestic dogs: A systematic review and meta-analysis","volume":"174","author":"Odigie","year":"2024","journal-title":"Res. Vet. Sci."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"446","DOI":"10.1093\/cid\/ciab456","article-title":"Novel canine coronavirus isolated from a hospitalized patient with pneumonia in east Malaysia","volume":"74","author":"Vlasova","year":"2022","journal-title":"Clin. Infect. Dis."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"E1184","DOI":"10.1093\/cid\/ciab924","article-title":"Isolation of a novel recombinant canine coronavirus from a visitor to Haiti: Further evidence of transmission of coronaviruses of zoonotic origin to humans","volume":"75","author":"Lednicky","year":"2022","journal-title":"Clin. Infect. Dis."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"699","DOI":"10.1080\/22221751.2022.2040341","article-title":"Animal alphacoronaviruses found in human patients with acute respiratory illness in different countries","volume":"11","author":"Vlasova","year":"2022","journal-title":"Emerg. Microbes Infect."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"789","DOI":"10.2460\/javma.23.03.0148","article-title":"Continuous surveillance and viral discovery in animals and humans are a core component of a one-health approach to address recent viral reverse zoonoses","volume":"261","author":"Kibenge","year":"2023","journal-title":"J. Am. Vet. Med. Assoc."}],"container-title":["Pathogens"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2076-0817\/13\/12\/1054\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T16:43:03Z","timestamp":1760114583000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2076-0817\/13\/12\/1054"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2024,11,29]]},"references-count":44,"journal-issue":{"issue":"12","published-online":{"date-parts":[[2024,12]]}},"alternative-id":["pathogens13121054"],"URL":"https:\/\/doi.org\/10.3390\/pathogens13121054","relation":{},"ISSN":["2076-0817"],"issn-type":[{"value":"2076-0817","type":"electronic"}],"subject":[],"published":{"date-parts":[[2024,11,29]]}}}