Korean Journal of Microbiology (미생물학회지)

The Microbiological Society of Korea (한국미생물학회)
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Ultra-Rapid Real-Time PCR for the Detection of Human Immunodeficiency Virus (HIV)

Ultra Rapid Real-Time PCR에 의한 Human Immunodeficiency Virus (HIV)의 신속진단법

  • 이동우 (경기대학교 자연과학대학 생명과학과) ;
  • 김을환 (경기대학교 자연과학대학 생명과학과) ;
  • 유미선 (경기대학교 자연과학대학 생명과학과) ;
  • 한상훈 (경기대학교 자연과학대학 생명과학과) ;
  • 윤병수 (경기대학교 자연과학대학 생명과학과)
  • Published : 2007.06.30
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Abstract

For the detection of Human Immunodeficiency Virus (HIV), multiple and ultra-rapid real-time PCR methods were developed. The target DNA sequences were deduced from HIV-1 specific 495bp partial env gene (gi_1184090) and from HIV-2 specific 294 bp partial env gene (gi_1332355), and were synthesized by using PCR-based gene synthesis on the reason of safety. Ultra-rapid real-time PCR was performed by $Genspector^{TM}$ using microchip-based, $1\;{\mu}l$ of reaction volume with extremely short time in each 3 step in PCR. The detection including DNA-amplification and melting temperature analysis was completed inner 15 minutes. The HIV-1 specific 117 bp-long and HIV-2 specific 119 bp-long PCR products were successfully amplified from minimum of template,2.3 molecules of each env gene. This kind of real-time PCR was designated as ultra-rapid real-time PCR in this study and it could be applied not only an alternative detection method against HIV, but also other pathogens using PCR-based detection.

인간면역결핍바이러스(Human immunodeficiency virus; HIV) 진단을 위한 다중, 초고속실시간 PCR법을 개발하였다. 검출대상의 DNA 염기서열은 env 유전자를 기반으로 설계되었으며, 각기 HIV-1 특이 495염기(gi_1184090) 및 HIV-2 특이 294염기(gi_1332355)의 DNA를 안정상의 이유로 PCR을 이용한 유전자합성법으로 제작하여 사용하였다. 초고속 실시간 PCR은, PCR의 회전 중 각 단계별 설정시간을 극단적으로 축소하여, $1\;{\mu}l$의 PCR 용액용 microchip을 탑재할 수 있는 $Genspector^{TM}$을 사용하여 수행하였다. DNA 증폭과 융점분석을 포함한 총 PCR 검색 시간은 HIV_1 및 HIV-2 모두에서 15분 이내로 완료되었으며, 각기 최소 2.3개의 합성 env 유전자로부터도 HIV-1 특이 117염기와 HIV-2 특이 119염기의 PCR산물을 성공적으로 증폭시킬 수 있는 민감성을 보여주었다. 이런 형식의 실시간 PCR법을 본 연구에서 초고속실시간 PCR (Ultra-rapid real-time PCR)이라 명명하였다. 이는 본 연구의 대상인 HIV에 대한 보조적 진단방법일 뿐 아니라 PCR 검색법이 사용되고 있는 다른 병원체에 대하여도 적용될 수 있을 것이나, 우선 HIV 임상시료에 대한 본 검색법의 효용성 실험 등 추가 연구가 필요할 것으로 사료된다.

Keywords

References

  1. 김을환, 이동우, 유미선, 한상훈, 윤병수. 2007. ultrarapid Real-time PCR을 이용한 Avian Influenza Virus subtype H5N1의 신속검출법. 한국미생물학회지 43, 23-30
  2. 윤병수, 임윤규, 한상훈. 2005. 분자생물학 연구방법론 III (서적). p. 574. 경기대학교 연구지원팀
  3. Arens, M.Q, L.M. Mundy, D. Amsterdam, J.T. Barrett, D. Bigg, D. Bruckner, B. Hanna, H. Prince, T. Purington, T. Hanna, R. Hewitt, C. Kalinka, T. Koppes, S. Maxwell, A. Moe, M. Doymaz, M. Poulter, M.S. Tehrani, L. Simard, D.W. Carmody, J. Vidaver, C. Berger, A.H. Davis, and M.T. Alzona. 2005. Preclinical and clinical performance of the efoora test, a rapid test for detection of human immunodeficiency virus-specific antibodies. J. Clin. Microbiol. 43, 2399-2406 https://doi.org/10.1128/JCM.43.5.2399-2406.2005
  4. Benoit, K.M., P. Henrivaux, J. Ruelle, N. Delferriere, M. Bodeus, and P. Goubau. 2005. Human immunodeficiency virus type 1 (HIV-1) proviral DNA load in purified CD4+ cells by LightCycler Real-time PCR. BMC Infect. Dis. 5, 1471-1479
  5. Bergroth, T., A. Sonnerborg, and Z. Yun. 2005. Discrimination of lamivudine resistant minor HIV-1 variants by selective real-time PCR. J. Virol. Methods 127, 100-107 https://doi.org/10.1016/j.jviromet.2005.03.007
  6. Candotti, D., J. Temple, O.O. Shirley, and J.P. Allain. 2004. Multiplex real-time quantitative RT-PCR assay for hepatitis B virus, hepatitis C virus, and human immunodeficiency virus type 1. J. Virol. Methods 118, 39-47 https://doi.org/10.1016/j.jviromet.2004.01.017
  7. Cho, Y.K., J.T. Kim, Y.S. Lee, Y.A. Kim, K. Namkoong, H.K. Lim, K.W. Oh, S.H. Kim, J.I. Han, C.S. Park, Y.E. Pak, C.S. Ki, J.R. Chio, H.K. Myeong, and C. Ko. 2006. Clinical evaluation of micro-scale chip-based PCR system for rapid detection of hepatitis B virus. Biosens. Bioelectron. 21, 2161-2169 https://doi.org/10.1016/j.bios.2005.10.005
  8. Davide, G., V. Francesca, G. Vitone, G. Elisa, L.P. Michele, and C.R. Maria. 2004. Quantitative detection of human immunodeficiency virus type 1(HIV-1) viral load by SYBR green real-time RT-PCR technique in HIV-1 seropositive patients. J. Virol. Methods 115, 183-189 https://doi.org/10.1016/j.jviromet.2003.09.030
  9. Desire, N., A. Dehee, V. Schneider, C. Jacomet, C. Goujon, P.M. Girard, W. Rozenbaum, and J.C. Nicolas. 2001. Quantification of human immunodeficiency virus type 1 proviral load by a TaqMan real-time PCR assay. J. Clin. Microbiol. 39, 1303-1310 https://doi.org/10.1128/JCM.39.4.1303-1310.2001
  10. Ghosh, M.K., L. Kuhn, J. West, K. Semrau, D. Decker, D.M. Thea, and G.M. Aldrovandi. 2003. Quantitation of Human Immunodeficiency Virus Type 1 in Breast Milk. J. Clin. Microbiol. 41, 2465-2470 https://doi.org/10.1128/JCM.41.6.2465-2470.2003
  11. Guan, M.K., L.C. Hsueh, Y.K. Liang, T.J. Wen, L.C. Julius, H.L. Ming, T.J. Chang, and J.L. Hung. 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. Gupta, A. and V.K. Chaudhary. 2006. Bifunctional recombinant fusion proteins for rapid detection of antibodies to both HIV-1 and HIV-2 in whole blood. BMC Biotechnol. 6, 1472-1483
  13. Luo, W., H. Yang, K. Rathbun, C.P. Pau, and C.Y. Ou. 2005. Detection of human immunodeficiency virus type 1 DNA in dried blood spots by a duplex Real-time PCR assay. J. Clin. Microbiol. 43, 1851-1857 https://doi.org/10.1128/JCM.43.4.1851-1857.2005
  14. Mello, A.J. 2003. DNA amplification moves on. Nature 422, 28-29 https://doi.org/10.1038/422028a
  15. O'Shea, J., I. Chrystie, R. Cranston, J. Mullen, K. Corbett, G. Murphy, J.V. Parry, A.D. Ruiter, and J. Banatvala. 2000. Problems in the interpretation of HIV-1 viral load assays using commercial reagents. J. Med. Virol. 61, 187-194 https://doi.org/10.1002/(SICI)1096-9071(200006)61:2<187::AID-JMV3>3.0.CO;2-8
  16. Palmer, S., A.P. Wiegand, F. Maldarelli, H. Bazmi, J.M. Mican, M. Polis, R. L. Dewar, A. Planta, S. Liu, J.A. Metcalf, J.W. Mellors, and M. Coffin. 2003. New Real-Time Reverse Transcriptase-Initiated PCR Assay with Single-Copy Sensitivity for Human Immunodeficiency Virus Type 1 RNA in Plasma. J. Clin. Microbiol. 41, 4531-4536 https://doi.org/10.1128/JCM.41.10.4531-4536.2003
  17. Primer 3. http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www. cgi
  18. Rouet, F., D.K. Ekouevi, M.L. Chaix, M. Burgard, A. Inwoley, T.D. Tony, C. Danel, X. Anglaret, V. Leroy, P. Msellati, F. Dabis, and C. Rouzioux. 2005. Transfer and evaluation of an automated, low-cost Real-Time Reverse Transcription-PCR test for diagnosis and monitoring of human immunodeficiency virus type 1 infection in a west African resource-limited setting. J. Clin. Microbiol. 43, 2709-2717 https://doi.org/10.1128/JCM.43.6.2709-2717.2005
  19. Schutten, M., B. Van den Hoogen, M.E. van den Ende, R.A. Gruters, A.D. Osterhaus, and H.G. Niester. 2000. Development of a real-time quantitative RT-PCR for the detection of HIV-2 RNA in plasma. J. Virol. Methods 88, 81-87 https://doi.org/10.1016/S0166-0934(00)00177-4
  20. Simon, V., D.D. Ho, and Q.A. Karim. 2006. HIV/AIDS epidemiology, pathogenesis, prevention, and treatment. Lancet. 268, 489-504
  21. Singer, D.E., N. Kiwanuka, D. Serwadda, F. Nalugoda, L. Hird, J.B. Hoover, G. Kigozi, J.A. Malia, E.K. Calero, W. Sateren, M.L. Robb, F.W. Mangen, M. Wawer, R.H. Gray, N. Sewankambo, D.L. Birx, and N.L. Michael. 2005. Use of stored serum from uganda for development and evaluation of a human immunodeficiency virus type 1 testing algorithm involving multiple rapid immunoassays. J. Clin. Microbiol. 43, 5312-5315 https://doi.org/10.1128/JCM.43.10.5312-5315.2005
  22. Teo, I.A., J.W. Chio, J. Moriese, G. Tayler, and S. Shaunak. 2002. LightCycler qPCR optimisation for low copy number target DNA. J. Immunol. Methods 270, 119-133 https://doi.org/10.1016/S0022-1759(02)00218-1
  23. vPCR. http://grup.cribi.unipd.it/cgi-bin/mateo/vpcr2.cgi