Abstract
In the study, we established a hydrolysis probe-based real-time polymerase chain reaction (PCR) assay to rapidly detect Canine circovirus (CanineCV) DNA in faecal samples. We designed a pair of specific primers and one probe targeting Rep in CanineCV, and sensitivity, specificity, and repeatability tests were performed to evaluate the efficacy of the assay. The assay showed high sensitivity and a minimum detection limit of 8.42 × 101 copies/μL, which is 1000-fold more sensitive compared to traditional PCR. The method was also highly specific, without cross-reaction with other common canine viruses. Moreover, the assay showed high repeatability, and the mean intra-assay and inter-assay coefficients of variation were 0.26 and 0.36%, respectively. The results of the detection of clinical samples showed that the positive detection rate of CanineCV was 14.04% (8/57). Notably, 8% of clinical samples were co-infected with other canine pathogens. In conclusion, the establishment of a hydrolysis probe-based real-time PCR method provides a fast, sensitive, specific, reliable, and repeatable method for CanineCV detection.
This is a preview of subscription content, log in via an institution to check access.
Explore related subjects
Discover the latest articles and news from researchers in related subjects, suggested using machine learning.References
Anderson A, Hartmann K, Leutenegger CM, Proksch AL, Mueller RS, Unterer S (2017) Role of canine circovirus in dogs with acute haemorrhagic diarrhoea. Vet Rec 180(22):542. https://doi.org/10.1136/vr.103926
Apte A, Daniel S (2009) PCR primer design. Cold Spring Harb Protoc 2009(3):ip65. https://doi.org/10.1101/pdb.ip65
Battilani M, Gallina L, Vaccari F, Morganti L (2007) Co-infection with multiple variants of canine parvovirus type 2 (CPV-2). Vet Res Commun 31(Suppl 1):209–212. https://doi.org/10.1007/s11259-007-0007-6
Breitbart M, Delwart E, Rosario K, Segales J, Varsani A, Ictv Report C (2017) ICTV virus taxonomy profile: Circoviridae. J Gen Virol 98(8):1997–1998. https://doi.org/10.1099/jgv.0.000871
Decaro N, Martella V, Desario C, Lanave G, Circella E, Cavalli A, Elia G, Camero M, Buonavoglia C (2014) Genomic characterization of a circovirus associated with fatal hemorrhagic enteritis in dog, Italy. PLoS ONE 9(8):e105909. https://doi.org/10.1371/journal.pone.0105909
Du H, Li W, Hao W, Liao X, Li M, Luo S (2013) Taqman real-time PCR assay based on ORFV024 gene for rapid detection of orf infection. Toxicol Mech Methods 23(5):308–314. https://doi.org/10.3109/15376516.2012.753968
Espy MJ, Uhl JR, Sloan LM, Buckwalter SP, Jones MF, Vetter EA, Yao JD, Wengenack NL, Rosenblatt JE, Cockerill FR 3rd, Smith TF (2006) Real-time PCR in clinical microbiology: applications for routine laboratory testing. Clin Microbiol Rev 19(1):165–256. https://doi.org/10.1128/CMR.19.1.165-256.2006
Hornbeck PV (2015) Enzyme-linked immunosorbent assays. Curr Protoc Immunol. https://doi.org/10.1002/0471142735.im0201s110
Hsu HS, Lin TH, Wu HY, Lin LS, Chung CS, Chiou MT, Lin CN (2016) High detection rate of dog circovirus in diarrheal dogs. BMC Vet Res 12(1):116. https://doi.org/10.1186/s12917-016-0722-8
Kapoor A, Dubovi EJ, Henriquez-Rivera JA, Lipkin WI (2012) Complete genome sequence of the first canine circovirus. J Virol 86(12):7018. https://doi.org/10.1128/JVI.00791-12
Kotsias F, Bucafusco D, Nunez DA, Lago Borisovsky LA, Rodriguez M, Bratanich AC (2019) Genomic characterization of canine circovirus associated with fatal disease in dogs in South America. PLoS ONE 14(6):e0218735. https://doi.org/10.1371/journal.pone.0218735
Li Y, Fan P, Zhou S, Zhang L (2017) Loop-mediated isothermal amplification (LAMP): a novel rapid detection platform for pathogens. Microb Pathog 107:54–61. https://doi.org/10.1016/j.micpath.2017.03.016
Li L, McGraw S, Zhu K, Leutenegger CM, Marks SL, Kubiski S, Gaffney P, Dela Cruz FN Jr, Wang C, Delwart E, Pesavento PA (2013) Circovirus in tissues of dogs with vasculitis and hemorrhage. Emerg Infect Dis 19(4):534–541. https://doi.org/10.3201/eid1904.121390
Mackay IM (2004) Real-time PCR in the microbiology laboratory. Clin Microbiol Infect 10(3):190–212. https://doi.org/10.1111/j.1198-743x.2004.00722.x
Niu L, Wang Z, Zhao L, Wang Y, Cui X, Shi Y, Chen H, Ge J (2020) Detection and molecular characterization of canine circovirus circulating in northeastern China during 2014–2016. Arch Virol 165(1):137–143. https://doi.org/10.1007/s00705-019-04433-4
Piewbang C, Jo WK, Puff C, van der Vries E, Kesdangsakonwut S, Rungsipipat A, Kruppa J, Jung K, Baumgartner W, Techangamsuwan S, Ludlow M, Osterhaus A (2018) Novel canine circovirus strains from Thailand: evidence for genetic recombination. Sci Rep 8(1):7524. https://doi.org/10.1038/s41598-018-25936-1
Silva Zatti M, Domingos Arantes T, Cordeiro Theodoro R (2020) Isothermal nucleic acid amplification techniques for detection and identification of pathogenic fungi: a review. Mycoses 63(10):1006–1020. https://doi.org/10.1111/myc.13140
Sun W, Wang W, Xin J, Cao L, Zhuang X, Zhang C, Zhu Y, Zhang H, Qin Y, Du Q, Han Z, Lu H, Zheng M, Jin N (2019a) An epidemiological investigation of porcine circovirus 3 infection in dogs in the Guangxi Province from 2015 to 2017, China. Virus Res 270:197663. https://doi.org/10.1016/j.virusres.2019.197663
Sun W, Zhang H, Zheng M, Cao H, Lu H, Zhao G, Xie C, Cao L, Wei X, Bi J, Yi C, Yin G, Jin N (2019b) The detection of canine circovirus in Guangxi, China. Virus Res 259:85–89. https://doi.org/10.1016/j.virusres.2018.10.021
Thaiwong T, Wise AG, Maes RK, Mullaney T, Kiupel M (2016) Canine circovirus 1 (CaCV-1) and Canine Parvovirus 2 (CPV-2): recurrent dual infections in a papillon breeding colony. Vet Pathol 53(6):1204–1209. https://doi.org/10.1177/0300985816646430
Tignon M, Gallardo C, Iscaro C, Hutet E, Van der Stede Y, Kolbasov D, De Mia GM, Le Potier MF, Bishop RP, Arias M, Koenen F (2011) Development and inter-laboratory validation study of an improved new real-time PCR assay with internal control for detection and laboratory diagnosis of African swine fever virus. J Virol Methods 178(1–2):161–170. https://doi.org/10.1016/j.jviromet.2011.09.007
Wan C, Chen C, Cheng L, Chen H, Fu Q, Shi S, Fu G, Liu R, Huang Y (2018) Specific detection of Muscovy duck parvovirus infection by TaqMan-based real-time PCR assay. BMC Vet Res 14(1):267. https://doi.org/10.1186/s12917-018-1600-3
Wang J, Luo Y, Liang L, Li J, Cui S (2018) A fast and simple one-step duplex PCR assay for canine distemper virus (CDV) and canine coronavirus (CCoV) detection. Arch Virol 163(12):3345–3349. https://doi.org/10.1007/s00705-018-3982-8
Wang J, Zhang Y, Wang J, Liu L, Pang X, Yuan W (2017a) Development of a TaqMan-based real-time PCR assay for the specific detection of porcine circovirus 3. J Virol Methods 248:177–180. https://doi.org/10.1016/j.jviromet.2017.07.007
Wang Y, Li Y, Cui Y, Jiang S, Liu G, Wang J, Li Y (2020) Establishment of a duplex SYBR green I-based real-time polymerase chain reaction assay for the rapid detection of canine circovirus and canine astrovirus. Mol Cell Probes 54:101666. https://doi.org/10.1016/j.mcp.2020.101666
Wang Y, Yang K, Bai C, Yin D, Li G, Qi K, Wang G, Li Y (2017b) Development of a SYBR Green I real-time PCR for the detection of the orf virus. AMB Express 7(1):21. https://doi.org/10.1186/s13568-016-0322-9
Weber MN, Cibulski SP, Olegario JC, da Silva MS, Puhl DE, Mosena ACS, Alves C, Paim WP, Baumbach LF, Mayer FQ, Fernandes ARF, Azevedo SS, Canal CW (2018) Characterization of dog serum virome from Northeastern Brazil. Virol 525:192–199. https://doi.org/10.1016/j.virol.2018.09.023
Zincke D, Norris MH, Cruz O, Kurmanov B, McGraw WS, Daegling DJ, Krigbaum J, Hoang TTH, Khanipov K, Golovko G, Hadfield T, Blackburn JK (2020) TaqMan assays for simultaneous detection of Bacillus anthracis and Bacillus cereus biovar anthracis. Pathogens. https://doi.org/10.3390/pathogens9121074
Acknowledgements
We would like to thank Editage (www.editage.cn) for English language editing.
Funding
This study was supported by the Ningbo Health Branding Subject Fund (No. ppxk2018-10).
Author information
Authors and Affiliations
Contributions
PS and YY conceived of the study, carried out the experiment and drafted the manuscript, contributed equally to this work. YeL and YC participated in the data collection and analysis. TZ and YoL participated in statistical analysis. YW conceived of the study, revising the manuscript critically. All authors have read and approved the final manuscript.
Corresponding author
Ethics declarations
Conflict of interest
The authors declare no conflict of interest.
Ethical approval
All experiments were compliant with the ethical standards of Anhui Agricultural University.
Supplementary Information
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Sun, P., Ye, Y., Li, Y. et al. Establishment of hydrolysis probe system real-time PCR assay for rapid detection of canine circovirus. 3 Biotech 11, 472 (2021). https://doi.org/10.1007/s13205-021-03031-z
Received:
Accepted:
Published:
DOI: https://doi.org/10.1007/s13205-021-03031-z