Polymerase Chain Reaction for the Detection of Aujeszky's Disease Virus

오제스키병 바이러스 검출을 위한 Polymerase Chain Reaction

  • Hwang, Dong-hee (Plasma Fractionation Center, Korean Red Cross) ;
  • Yeo, Sang-geon (College of Veterinary Medicine.Institute of Animal Medicine Gyeongsang National University)
  • 황동희 (대한적십자사 혈장분획센터) ;
  • 여상건 (경상대학교 수의과대학.동물의학연구소)
  • Accepted : 2003.03.06
  • Published : 2003.06.30


Polymerase chain reaction (PCR) was evaluated for the early detection of Aujeszky's disease virus (ADV) DNA from virus-infected cell cultures. For the purposes, the Korean ADV NYJ1-87 was propagated in swine kidney (SK) cells and subjected to the amplification of DNA (217 bp) by PCR using sense and antisense primers specific to gp50 gene of the ADV. In detection of cell-associated viral DNA, reliable PCR conditions were determined as 30 cycles of reaction consisting 1 minute each of denaturation at $94^{\circ}C$, annealing at $55^{\circ}C$ and polymerization at $72^{\circ}C$. The PCR encountered best results with reagent mixtures of $50{\mu}l$ containing $200{\mu}M$ dNTPs, $0.2{\mu}M$ each sense and antisense primers, 1 mM $MgCl_2$ and 10% (v/v) template DNA in the final concentrations. ADV-specific DNAs were detected as early as 6, 6, and 9 hours post-infection, respectively, from lysates of the SK cells infected with ADV of $10^3$, $10^2$ and $10^1\;TCID_{50}/ml$ by this condition. In culture supernatant, the DNAs were detected from ADV of as low infectivity as $10^ {-3}\;TCID_{50}/ml$ by the reduced reagent concentrations and 30 cycles of 1 minute each of denaturation at $94^{\circ}C$ and annealing at $55^{\circ}C$, and 2 minutes of polymerization at $72^{\circ}C$. The lowest amount of detectable ADV DNA was 1 fg. In conclusion, the PCR condition established in the present study was recognized as a feasible alternative to time-consuming procedures in isolation and characterization of the virus.



  1. 박정우, 전무형, 안수환. 국내분리 Aujeszky's disease virus의 실험적 감염자돈에 대한 바이러스학적 연구. 대한수의학회지. 1990, 30(2), 177-186
  2. 이중복, 안수환, 김병한, 송재영, 김용희, 설동섭. 돼지 오제스키병에 관한 연구. I. 감염자돈으로부터 원인체의 분리 및 동정. 대한수의학회지. 1988, 28(1), 99-103
  3. 전무형. 돼지 가성광견병의 실제와 대책. 대한수의사회지. 1987, 23(7), 433-443
  4. 전무형, 조성환, 안수환, 박성국, 윤석민, 하용공. 이환자돈으로부터 오제스키병 바이러스 분리와 생물학적성상. 대한수의사회지. 1988, 24(3), 163-171
  5. Allan, G. M., McNulty, M. S. and McCracken, R. M. Rapid diagnosis of Aujesky's disease in pig by immunofluorescence. Res. Vet. Sci. 1984, 36, 225-239
  6. Ben Porat, T. and Kaplan, A. S. The chemical Composition of herpes simplex and pseudorabies virus. Virology. 1962, 16, 261-266
  7. Brown, T. M., Osorio, F. A. and Rock, D. L. Detection of latent pseudorabies virus in swine using in situ hybridization. Vet. Microbiol. 1990, 24, 273-280
  8. Freshney, R. I. Culture of Animal Cells: A manual of basic technique. Alan R. Liss, Inc., New York, 1983. 119-128
  9. Hasebe, H., Wheeler, G. J. and Osorio, F. A. Gene specific assay to differentiate strains of pseudorabies virus. Vet. Microbiol. 1993, 34, 221-231
  10. Holmes, D. S. and Quigley, M. A rapid boiling method for the preparation of bacterial plasmids. Anal. Biochem 1981, 114, 193-195
  11. Hsing, G. D., Fong, C. K. Y. and Landry, M. L. Diagnostic Virology. Yale University Press, New haven, 1994. 238-249
  12. Jestin, A, Foulon, T, Pertuiset, B., Blanchard, P. and Labourdet, M. Rapid detection of pseudorabies virus genomic sequences in biological samples from infected pigs using polymerase chain reaction DNA amplification. Vet. Microbiol. 1990, 23, 317-328
  13. Johnson, M. E., Thawley, D. G. and Solorzano, R. F. Evaluation of the microimmunodiffusion test for the detection of antibody to pseudorabies virus. Am. J. Vet. Res. 1983, 44, 28-30
  14. MeFarlane, R G., Thawley, D. G and Solorzano, R. F. Detection of latent pseudorabies virus in porcine tissue, using a DNA hybridization dot-blot assay. Am. J. Vet. Res. 1986, 47, 2329-2336
  15. Murphy, F. A., Gibbs, E. P. J., Horzinek, M C and Studdert, M. J. Veterinary Virology. Acadanic Pcess, San Diego, 1999, 301-314
  16. Schoenbaum, N. A., Beran, G. W. and Murphy, D. P. A study comparing the immunologic responses of swine to pseudorabies viral antigens based on the ELISA, serum virus neutralization and latex agglutination tests. J. Vet. Diagn. Invest. 1990, 2, 29-34
  17. Straw, B. E., D'Allaire, S. D., Mengeling, W. L. and Taylor, D. I. (ed), Diseases of Swine, Iowa state University Press, Ames, 1999, 233-246
  18. Timoney, J. F., Gillespie, J. H., Scott, F. W. and Barlough, J. E. Hagan and Bruner's Microbiology and Infectious Diseases of Domestic Animals. Cornell Univeisity Pnass, Ithaca, 1988, 615-620
  19. Van Oirschot, J. T., Gielkens, A. L. T., Moormann, R. J. M. and Berns, A. J. M. Marker vaccines, virus protein specific antibody assays and the control of Aujeszky's disease. Vet. Microbiol. 1990, 23, 85-101
  20. Williams, J. F. Optimization strategies for the poly merase chain reaction. Biotechniques. 1989, 7, 762-768
  21. Wittman, G. and Hall, S. A. (ed), Aujeszky's disease. Martinus Nijhoff Publishers, The Hague, Netherlands. 1981, 51-54