Detection of infectious canine hepatitis virus by TaqMan real-time PCR method

TaqMan 실시간 PCR법에 의한 개 전염성 간염 바이러스의 검출

  • Wang, Hye-young (Bio-Safety Research Institute and College of Veterinary Medicine, Chonbuk National University) ;
  • Choi, Jae-yong (Bio-Safety Research Institute and College of Veterinary Medicine, Chonbuk National University) ;
  • Lee, Mi-jin (Bio-Safety Research Institute and College of Veterinary Medicine, Chonbuk National University) ;
  • Park, Jin-ho (Bio-Safety Research Institute and College of Veterinary Medicine, Chonbuk National University) ;
  • Cho, Mae-Rim (Bio-Safety Research Institute and College of Veterinary Medicine, Chonbuk National University) ;
  • Han, Jae-cheol (Bio-Safety Research Institute and College of Veterinary Medicine, Chonbuk National University) ;
  • Choi, Kyoung-seong (Bio-Safety Research Institute and College of Veterinary Medicine, Chonbuk National University) ;
  • Chae, Joon-seok (Bio-Safety Research Institute and College of Veterinary Medicine, Chonbuk National University)
  • 왕혜영 (전북대학교 수의과대학 생체안전성 연구소) ;
  • 최재용 (전북대학교 수의과대학 생체안전성 연구소) ;
  • 이미진 (전북대학교 수의과대학 생체안전성 연구소) ;
  • 박진호 (전북대학교 수의과대학 생체안전성 연구소) ;
  • 조매림 (전북대학교 수의과대학 생체안전성 연구소) ;
  • 한재철 (전북대학교 수의과대학 생체안전성 연구소) ;
  • 최경성 (전북대학교 수의과대학 생체안전성 연구소) ;
  • 채준석 (전북대학교 수의과대학 생체안전성 연구소)
  • Accepted : 2004.07.20
  • Published : 2004.12.30


The aim of this work was the validation of a rapid real-time PCR assay based on TaqMan technology for the unequivocal identification of infectious canine hepatitis (ICH) virus, to be used directly on DNA purified from blood specimens. A real-time PCR system targeting at the E3 ORFA gene sequence of canine adenovirus type 1 was optimized and validated through comparative analysis of samples using conventional PCR system. The real-time PCR assay based on TaqMan technology could disclose 23 (37.7%) out of 61 samples as PCR positive. In contrast, 18 (29.5%) samples were found PCR positive when conventional PCR was applied on these samples. The use of the ABI Prism 7700 sequence detection system allowed the efficient determination of the amplified product accumulation through a fluorogenic probe. The entire real-time TaqMan PCR assay, including DNA extraction, amplification, and detection could be completed within 3 hours. The detection method of real-time TaqMan PCR assay was 1,000 times more sensitive than conventional PCR. Real-time TaqMan probe and primer set developed and optimized in this study is a sensitive, rapid and accurate method for detection of ICH virus and can be effective screening tool for the detection of ICH in a diagnostic laboratory routines.


Supported by : 전북대학교 생체안전성연구소


  1. Appel, M. J. Canine adenovirus type 1(infectious canine hepatitis virus). In: Horzinek, M. C.(series ed.), Virus infectious of vertebrates, Vol. I. Virus infections of carnivores, p. 29-43, Elsevier Science Publishers, Amsterdam, 1987
  2. Cabasso, V. J. Canine viruses. II. Infectious canine hepatitis and rabies control. Southwest. Vet. 1953, 6, 137-141
  3. Cabasso, V. J. Infectious canine hepatitis virus. Ann. N. Y. Acad. Sci. 1962, 101, 498-514
  4. Chen, W., Martinez, G. and Mulchandani, A. Molecular Beacons: A real-time polymerase chain reaction assay for detecting Salmonella. Anal. Biochem. 2000, 280, 166-172
  5. Chouinard, L., Martineau, D., Forget, C. and Girard, C. Use of polymerase chain reaction and immunohistochemistry for detection of canine adenovirus type 1 in formalin-fixed, paraffin-embedded liver of dogs with chronic hepatitis or cirrhosis. J. Vet. Diagn. Invest. 1998, 10, 320-325
  6. Cooney, M. K. Adenoviruses. pp. 135-146, In E. H. Lennette, ed., Laboratory diagnosis of viral infectious. Marcel Dekker, New York. 1985
  7. Corless, C. E., Guiver, M., Borrow, R., Jones, V. E., Kaczmarski, E. B. and Fox, A. J. Contamination and sensitivity issues with a real-time universal 16S rRNA PCR. J. Clin. Microbiol. 2000, 38, 1747-1752
  8. Corpet, F. Multiple sequence alignment with hierarchical clustering. Nucleic Acids Res. 1988, 16, 10881-10890
  9. Craig, E. G. Infectious canine hepatitis and canine acidophilic cell hepatitis. In: Craig. E. G. (Edied.), Infectious Diseases of the Dog and Cat. p. 242-250. Saunders, Philadelphia, 1990
  10. Darai, G., Rosen, A., Delius, H. and Flugel, R. M. Characterization of the DNA of canine adenovirus by restriction enzyme analysis. Intervirology. 1985, 23, 23-28
  11. Desjardin, L. E., Chen, Y., Perkins, M. D., Teixeira, L., Cave, M. D. and Eisenach, K. D. Comparison of the ABI 7700 system (TaqMan) and competitive PCR for quantification of IS6110 DNA in sputum during treatment of tuberculosis. J. Clin. Microbiol. 1998, 36, 1964-1968
  12. Dragulev, B. P., Sira, S., Abouhaidar, M. G. and Campbell, J. B. Sequence analysis of putative E3 and fiber genomic regions of two strains of canine adenovirus type 1. Virology. 1991, 183, 298-305
  13. Green, C. E. Infectious canine hepatitis. In: Green, C. E. (ed.), Infectious Diseases of the Dog, and Cat. pp. 242-251. Saunders, Philadelphia, 1990
  14. Horwitz, M. S. Adenoviral diseases. In B. N. Fields, D. M. Knipe, R. M. Chanock, J. L. Melnick, B. Roizman, and R. E. Shope. eds., Virology. pp. 477-495. Raven Press, New York. 1985
  15. Hervas, J., Gomez-Villamandos, J. C., Perez, J., Carrasco, L. and Sierra, M. A. Focal mesangialsclerosing glomerulonephritis and acute-spontaneous infectious canine hepatitis: structural, immunohistochenical and subcellular studies. Vet. Immunol. Immunopathol. 1997, 57, 25-32
  16. Hu, R. L., Huang, G., Qiu, W., Zhong, Z. H., Xia, X. Z. and Yin, Z. Detection and differentiation of CAV-1 and CAV-2 by Polymerase Chain Reaction. Vet. Res. Commun. 2001, 25, 77-84
  17. Kiss, I., Matiz, K., Bajmoci, E., Rusvai, M. and Harrach, B. Infectious canine hepatitis: detection of canine adenovirus type 1 by polymerase chain reaction. Acta. Vet. Hung. 1996, 44, 253-258
  18. Kobayashi, Y., Ochiai, K. and Itakura, K. Dual infection with canine distemper virus and infectious canine hepatitis virus (canine adenovorus type 1) in a dog. J. Vet. Med. Sci. 1993, 55, 699-701
  19. Lyon, W. J. TaqMan PCR for Detection of Vibrio cholerae O1, O139, Non-O1, and Non-O139 in Pure Cultures, Raw Iysters, and Synthetic Seawater. Appl. Environ. Microbiol. 2001, 67, 4685-4693
  20. Ma, L., Bluyssen, H. A. and De Raeymaeker, M. Rapid determination of adenoviral vector titers by quantitative real-time PCR. J. Virol. Methods. 2001, 93,181-188
  21. Morrison, M. D., Onions, D. E. and Nicolson, L. Complete DNA sequence of canine adenovirus type 1. J. Gen. Virol. 1997, 78, 873-878
  22. Nlesters, H. G. M., Park, D. S., Lee, Y. J., Cho, J. H., Lappalainen, M. and Chae, J. S. Hepatitis B virus DNA assay using TaqMan PCR system. Korean J. Lab. Med. 2002, 22, 246-252
  23. Nogva, H. K., Rudi, K., Naterstad, K., Holck, A. and Lillehaug, D. Applcation of 5'-nuclease PCR assay for quantitative detection of Listeria monocytogenes in pure cultures, water, skim milk, and unpasteurized whole milk. Appl. Environ. Microbiol. 2000, 66, 4266-4271
  24. Pratelli, A., Martella, V., Elia, G., Tempesta, M., Guarda, F., Capucchio, M. T., Carmichael, L. E. and Buonavoglia, C. Severe Enteric Disease in an AnimalShelter Associated with Dual Infections by Canine Adenovirus Type 1 and Canine Coronavirus. J. Vet. Med B. Infect. Dis. Vet. Public. Health 2001, 48, 385-392
  25. Pusterla, N., Chang, C. C., Chomel, B. B., Chae, J. S., Foley, J. E., DeRock, E., Kramer, V. L., Lutz, H. and Madigan, J. E. Serologic and molecular evidenceof Ehrlichia spp. in coyotes in California. J. Wildl. Dis. 2000, 36, 494-499
  26. Rubarth, S. An acute virus disease with liver lesions in dogs (hepatitis contagiosa canis). A pathologicoanatomical and aetiologic investigation. Acta. Pathol. Microbiol. Scand. 1947, 24, 1-222
  27. Sasaki, N., Nakai, M., Iwamoto, I., Konishi, S. and Ikegami, T. Studies on infectious hepatitis of dogs. II. The distribution of the disease in Japan, and itsimmunization. Jpn. J. Vet. Sci. 1956, 18, 113-118
  28. Vishnubhatla, A. D., Fung, Y. C., Oberst, R. D., Hays, M. P., Nagaraja, T. G. and Flood, S. J. A. Rapid 5' Nuclease(TaqMan) Assay for Detection of Virulent Strains of Yersinia enterocolotica. Appl. Environ. Microbiol. 2000, 66, 4131-4135
  29. Whetstone, C. A., Draayer, H. and Collins, J. E. Characterization of canine adenovirus type 1 isolated from American black bears. Am. J. Vet. Res. 1988, 49,778-780
  30. Willis, A. M. Canine viral infectious. Vet. Clin. North. Am. Small. Anim. Pract. 2000, 30, 1119-1133