Korean Journal of Microbiology (미생물학회지)

The Microbiological Society of Korea (한국미생물학회)
권호 표지

Rapid Detection Method of Avian Influenza Subtype H5N1 using Quick Real-Time PCR

Quick Real-time PCR을 이용한 Avian Influenza Virus Subtype H5N1의 신속검출법

  • Kim, Eul-Hwan (Department of Life Science, College of Natural Science, Kyonggi University) ;
  • Lee, Dong-Woo (Department of Life Science, College of Natural Science, Kyonggi University) ;
  • Han, Sang-Hoon (Department of Life Science, College of Natural Science, Kyonggi University) ;
  • Kwon, Soon-Hwan (Chronic Inflammatory Disease Research Center, School of Medicine, Ajou University) ;
  • Yoon, Byoung-Su (Department of Life Science, College of Natural Science, Kyonggi University)
  • 김을환 (경기대학교 이과대학 생명과학과 분자생물학연구실) ;
  • 이동우 (경기대학교 이과대학 생명과학과 분자생물학연구실) ;
  • 한상훈 (경기대학교 이과대학 생명과학과 분자생물학연구실) ;
  • 권순환 (아주대학교 의과대학 만성염증질환센타) ;
  • 윤병수 (경기대학교 이과대학 생명과학과 분자생물학연구실)
  • Published : 2007.03.31
Download PDF
⟨ Previous Next ⟩

Abstract

The most rapid Real-time PCR based detection method for Avian influenza A virus (AIV) subtype H5N1 was developed. The target DNA sequence in this study was deduced from H5N1 subtype-specific 387 bp partial gene of hemagglutinin, and was synthesized by using PCR-based gene synthesis on the ground of safety. Real-Time PCR was performed by $GenSpector^{TM}$ using microchip-based, total $1{\mu}l$ of reaction mixture with extremely short time in each steps in PCR. The detection including PCR-amplication and analysis of melting temperature was totally completed within 13 min. The H5N1-specific 189 bp PCR product was correctly amplified until 2.4 molecules of hemagglutinin gene as minimum of templates. This kind of PCR was designated as Quick Real-Time PCR in this study and it could be applied to detect not only AIV H5N1, but also other pathogens using PCR-based detection.

조류 인플루엔자바이러스(AIV) H5N1 아형을 Real-time PCR법을 이용하여 가장 빠르게 진단할 수 있는 방법을 개발하였다. 검색 대상의 염기서열은 AIV H5N1 아형의 hemagglutinin 유전자 중 가장 상동성이 높은 387 bp의 부위를 선택하였고, 실험의 안전을 위하여 인공합섬의 방법으로 제작하였다. Microchip을 기반으로 한Real-time PCR법을 사용하였으며, 총PCR 반응액의 양을 $1{\mu}l$로, PCR 과정 중 각 단계, 즉 해리, 접합, 신장의 시간을 각1초, 1초, 3초로 하여 총 실험시간을 단축하였다. 진단을 위한 실험과정에서 PCR 및 융점분식에 소요된 최단 시간은 12분28초였으며, 민감도측정에서 최소2.4개의 hemaggutinin 유전자를 기질로 하여 목적한 특이 189 bp의 PCR 산물을 증폭할 수 있었기에, 본 연구에서는 이런 초고속 PCR 실험방식을 Quick Real-time PCR이라 명명하였다. 이 결과들은 가금류 및 사람에게 전파된 AIV H5N1아형의 진단에 적용될 수 있을 뿐 아니라, PCR이 사용되는 다른 신속검색법에도 널리 적용 될 수 있을 것으로 기대한다.

Keywords

References

  1. 남정구, 박근용, 심수경, 이영선, 이영희. 2005. 감염병실 험실진단; 질환별시험법 3판. 국립보건연구원 857-869
  2. 한상훈, 임윤규, 윤병수. 2006. 분자생물학 연구방법론 III. 경기대학교 출판부 82-84
  3. Alexander, D.J. 1995. The epidemiology and control of avian influenza and Newcastle disease. J. Comp. Pathol. 112, 105-126 https://doi.org/10.1016/S0021-9975(05)80054-4
  4. CDC. 2004. Outbreaks of avian influenza A (H5Nl) in Asia and interim recommendations for evaluation and reporting of suspected cases? United States. MMWR 13, 97-100
  5. Claas, E.C.J., A.D. Osterhaus, R. Van Beck, J.C. de Jong, G.F. Rimrnelzwaan, D.A. Senne, S. Krauss, K.F. Shortridge, and R.G. Webster. 1998. Human influenza A (H5Nl) virus related to highly pathogenic avian influenza virus. Lancet 351, 472-477 https://doi.org/10.1016/S0140-6736(97)11212-0
  6. Ellis, J.S., and M.C. Zambon. 2002. Molecular diagnosis of influenza. Rev. Med. Virol. 12, 375-389 https://doi.org/10.1002/rmv.370
  7. Fauci, A.S. Emerging and re-emerging infectious diseases: Influenza as a prototype of the host-pathogen balancing act. Cell 124, 665-670 https://doi.org/10.1016/j.cell.2006.02.010
  8. Fleming, D.M., P. Chakraverty, C. Sadler, and P. Litton. 1995. Combined clinical and virological surveillance of influenza in winters of 1992 and 1993-4. BMJ 29, 290-291
  9. Fouchier, R.A., V. Munster, A. Wallensten, T.M. Bestebroer S. Herfst, D. Smith, G.F. Rimmelzwaan, B. Olsen, and A.D. Osterhaus. 2005. Characterization of a Novel Influenza A Virus Hemagglutinin Subtype (H16) Obtained from Black-Headed Gulls. J. Virol. 79, 2814-2822 https://doi.org/10.1128/JVI.79.5.2814-2822.2005
  10. Gamblin, S.J., L.F. Haire, R.J. Russell, D.J. Stevens, B. Xiao, Y. Ha, N. Vasisht, D.A. Steinhauer, R.S. Daniels, A. Elliot, D.C. Wiley, and J.J. Skehel. 2004. The structure and receptor binding properties of the 1918 influenza hemagglutinin. Science 303, 1838-1842 https://doi.org/10.1126/science.1093155
  11. Guan, M.K., L.C. Hsueh, Y.K. Liang, T.J. Wen, L.C.J. Chulu, H.M. Liao, T.J. Chang, and H.J. Liu. 2006. Development of a quantitative Light Cycler real-time RT-PCR for detection of avian reovirus. J. Virol. Methods 133, 6-13 https://doi.org/10.1016/j.jviromet.2005.09.011
  12. Horimoto, T. and Y. Kawaoka. 2001. Pandemic threat posed by avian influenza A viruses. Clin. Microbiol. Rev. 14, 129-149 https://doi.org/10.1128/CMR.14.1.129-149.2001
  13. Lau, L.T., J. Banks, R. Aherne, I.H. Brown, N. Dillon, R.A. Collins, K.Y. Chan, Y. W. Fung, J. Xing, and A.C.H. Yu. 2003. Nucleic acid sequence-based amplification methods to detect avian influenza virus. Biochem. Biophys. Res. Commun. 313, 336-42
  14. Nicholson, K.G., J.M. Wood, and M. Zambon. 2003. Influenza. Lancet 362, 1733-1745 https://doi.org/10.1016/S0140-6736(03)14854-4
  15. Payungporn, S., P. Phakdeewirot, S. Chutinimitkul, A. Theamboonlers, J. Keawcharoen, K. Oraveerakul, A. Amonsin, and Y. Poovorawan. 2004. Single step multiplex reverse transcription polymerase chain reaction for Influenza A virus subtype H5Nl detection. J. Vir. lmmun. 17, 588-593
  16. Poddar, S.K. 2002. Influenza virus types and subtypes detection by single step single tube multiplex reverse transcription. polymerase chain reaction (RT-PCR) and agarose gel electrophoresis. J. Virol. Methods 99:63-70 https://doi.org/10.1016/S0166-0934(01)00380-9
  17. Trampuz, A., R.M. Prabhu, T.F. Smith, and L.M. Baddour. 2004. Avian influenza: a new pandemic threat? Mayo Clin. Proc. 79, 523-530
  18. Tran, T.H., T.L. Nguyen, T.D. Nguyen, T.S. Luong, P.M. Pham, V.C. Nguyen, T.S. Pham, C.D. Vo, T.Q. Le, T.T. Ngo, B.K. Dao, P.P. Le, T.T. Nguyen, T.L Hoang, V.T. Cao, T.G Le, D.T. Nguyen, H.N. Le, K.T. Nguyen, H.S. Le, et al. 2004. Avian influenza A (H5Nl) in 10 patients in Vietnam. N. Engl. J. Med. 350, 1179-1188 https://doi.org/10.1056/NEJMoa040419
  19. Yuen, K.Y, P.K.S. Chan, M. Peiris, D.N. Tsang, T.L. Que, K.F. Shortridge, P.T. Cheung, W.K. To, E.T. Ho, R. Sung, and A.F. Cheng. 1998. Clinical features and rapid viral diagnosis of human disease associated with avian influenza A H5Nl virus. Lancet 351, 467-471 https://doi.org/10.1016/S0140-6736(98)01182-9
  20. Xie, Z., Y.S. Pang, J. Liu, X. Deng, X. Tang, J. Sun, and M.I. Khan. 2006. A multiplex RT-PCR for detection of type A influenza virus and differentiation of avian H5, H7, and H9 hemagglutinin subtypes. Mol. Cell Probes. 20, 245-249 https://doi.org/10.1016/j.mcp.2006.01.003