생명과학회지 (Journal of Life Science)

  • 제18권3호
  • /
  • Pages.374-380
  • /
  • 2008
  • /
  • 1225-9918(pISSN)
  • /
  • 2287-3406(eISSN)
한국생명과학회 (Korean Society of Life Science)
권호 표지
DOI QR코드

DOI QR Code

엔테로바이러스 검출을 위한 real-time nucleic acid sequence-based amplification (NASBA), reverse transcription-PCR (RT-PCR) 및 바이러스 배양법의 비교

Comparison of the Real-Time Nucleic Acid Sequence-Based Amplification (NASBA) Assay, Reverse Transcription-PCR (RT-PCR) and Virus Isolation for the Detection of Enterovirus RNA.

  • 나영란 (부산광역시보건환경연구원) ;
  • 조현철 (부산광역시보건환경연구원) ;
  • 이영숙 (부산광역시보건환경연구원) ;
  • 빈재훈 (부산광역시보건환경연구원) ;
  • 최홍식 (부산광역시보건환경연구원) ;
  • 민상기 (부산광역시보건환경연구원)
  • Na, Young-Ran (Division of Epidemiology, Busan Institute of Health and Environment) ;
  • Joe, Hyeon-Cheol (Division of Epidemiology, Busan Institute of Health and Environment) ;
  • Lee, Young-Suk (Division of Epidemiology, Busan Institute of Health and Environment) ;
  • Bin, Jae-Hun (Division of Epidemiology, Busan Institute of Health and Environment) ;
  • Cheigh, Hong-Sik (Division of Epidemiology, Busan Institute of Health and Environment) ;
  • Min, Sang-Kee (Division of Epidemiology, Busan Institute of Health and Environment)
  • 발행 : 2008.03.31
PDF 다운로드
⟨ 이전 논문 다음 논문 ⟩

초록

본 연구는 무균성수막염 의심환자의 다양한 검체로부터 enterovirus의 진단을 위하여 real-time NASBA, 2 step RT-PCR 시험과 세포배양 시험을 각각 실시하여 각 시험법의 검출율, 특이도, 사용자 편리성, 시험소요 시간, 교차오염의 가능성 등을 비교 검토하였다. 비교시험 결과 전체 292건의 검체로부터 real-time NASBA에서 145건, 세포배양에서 101건, 2 step RT-PCR에서 86건이 양성으로 나타나 real-time NASBA가 가장 검출율이 높은 시험법임을 알 수 있었다. Enterovirus 외의 무균성수막염 원인바이러스에 대한 특이도 비교 시험결과 2 step RT-PCR 시험에서 rhinovirus 10건 중 1건이 위양성 반응을 나타내어 다른 시험법에 비해 특이도가 떨어지는 것으로 나타났다. Real-time NASBA는 하나의 튜브에서 증폭과 검출이 동시에 일어나 다른 시험과 비교하여 교차오염의 가능성이 낮으며 또한 시험 소요시간이 5시간 정도로 세포배양(5-14일 소요) 및 2 step RT-PCR(9시간소요) 에 비하여 신속하게 진단할 수 있어 일선병원이나 실험실에서 enterovirus를 검출을 위하여 적용할 수 있을 것으로 사료된다.

Rapid detection of enterovirus (EVs) is important in the management of aseptic meningitis. We examined the relative efficiency and specificity of the real-time nucleic acid sequence-based amplification (NASBA) comparing with the established reverse transcription polymerase chain reaction (RT-PCR) and viral culture method which were used for the detection of enterovirus RNA in clinical specimens. Of the total 292 samples, 145 were found to be positive to enterovirus RNA by real-time NASBA, 101 were positive by viral culture, and 86 were positive by RT-PCR. 147 samples and 46 samples were determined to be negative and positive by all methods respectively, but 4 samples were positive only by real-time NASBA. To compare the specificity of each method, various clinical samples which were diagnosed for herpes simplex virus (HSV)-1, HSV-2, adenovirus, mumps, and rhinovirus were applied. Except one rhinovirus sample which was false positive to enterovirus RNA by RT-PCR, the other different samples were negative to all three methods. The real-time NASBA procedure can be completed within 5 hours in contrast with 9 hours for the RT-PCR and 3-14 days for the viral culture. From this study, it was suggested that the real-time NASBA assay could be a standardized, rapid, specific, and sensitive procedure for the detection of enterovirus RNA.

키워드

참고문헌

  1. Capaul, S. E. and M. H. Gorgioevski. 2005. Detection of enterovirus RNA in cerebrospinal fluid (CSF) using NucliSens EasyQ enterovirus assay. J. Clin. Virol. 32, 236-240. https://doi.org/10.1016/j.jcv.2004.08.006
  2. Compton, J. 1991. Nucleic acid sequence based amplification. Nature 350, 91. https://doi.org/10.1038/350091a0
  3. Fox, J. D., S. Han, A. Samuelson, Y. Zhang, M. L. Neale and D. Westmoreland. 2002. Development and evaluation of a nucleic acid sequence based amplification (NASBA) for diagnosis of enterovirus infections using the NucliSens Basic kit. J. Clinical Virol. 24, 117-120. https://doi.org/10.1016/S1386-6532(01)00241-4
  4. Ginocchion, C., F. Zhang, A. Malhotra, R. Manji, P. Sillekens, F. Helma, M. Overdyk and M. Peeters. 2005. Development technical performance and clinical evaluation of a nucli sens basic kit application for detection of enterovirus RNA cerebrospinal fluid. J. Clin. Microbiol. 2616-2623.
  5. Glimarker, M., B. Johasson, P. Olcen, A. Ehrnst and M. Forsgren. 1993. Detection of enterovirus RNA by polymeric chain reaction in cerebrospinal fluid from patients with aseptic meningitis. Scan. J. Infect. Dis. 25, 547-557. https://doi.org/10.3109/00365549309008542
  6. Gorgioevski-Hrisoho, M., J. D. Schmacher, N. Vilimonovic, D. Germann and L. Matter. 1998. Detection by PCR of enteroviruses in cerebrospinal fluid during a summer outbreak of aseptic meningitis in Switzerland. J. Clin. Microbiol. 36, 2408-2412.
  7. Grandien, M., M. Forsgren and A. Ehrnst. 1995. Enteroviruses diagnostic procedures for viral, rickettsial and chlamydial infection. pp. 279-297, In Lennette E. H. and E. T. Lennette, 7th eds. American Public Health Association, Washington DC.
  8. Hellen, C. U. and E. Wimmer. 1995. Enterovirus structure and assembly. pp. 155-174, Human enterovirus infection, ASM press, Washington DC.
  9. Heim, A. and J. Schumann. 2002. Development and evaluation of a nucleic acid sequence based amplification (NASBA) protocol for the detection of enterovirus RNA in cerebrospinal fluid samples. J. Virological Methods 103, 101-107. https://doi.org/10.1016/S0166-0934(01)00454-2
  10. Hsiung, G. D. 1994. Picornaviridae. pp. 119, 4th eds. Hsiung's diagnostic virology, New Haven, Yale University press.
  11. Jean, J., B. Blais, A. Darveau and I. Fliss. 2001. Detection of hepatitis a virus by the nucleic acid sequence-based amplification technique and comparison with reverse transcription- PCR. Appl. Environ. Microbiol. 67, 5593-5600. https://doi.org/10.1128/AEM.67.12.5593-5600.2001
  12. Lai, K. K. Y., L. Cook, S. Wendt, L. Conrey and K. R. Jerome. 2003. Evaluation of realtime PCR versus PCR with liquid phase hybridization for detection of enterovirus RNA in cerebrospinal fluid. J. Clin. Microbiol. 41, 3133-3141. https://doi.org/10.1128/JCM.41.7.3133-3141.2003
  13. Landry, M. L., R. Garner and D. Ferguson. 2003. Comparison of the NuceiSens basic kit (nucleic acid sequence based amplification) and Argene Biosoft enterovirus consensus reverse transcription PCR assay for rapid enterovirus RNA in clinical specimens. J. Clin. Microbiol. 41, 5006-5010. https://doi.org/10.1128/JCM.41.11.5006-5010.2003
  14. Landry, M. L., R. Garner and D. Ferguson. 2003. Rapid enterovirus RNA detection in clinical specimens by using nucleic acid sequence based amplification. J. Clin. Microbiol. 41, 346-350. https://doi.org/10.1128/JCM.41.1.346-350.2003
  15. Loens, K., D. Ursi, M. Ieven, A. P. Van, P. Sillekens, P. Oudshoorn and H. Goossens. 2002. Detection of Mycoplasma pneumoniae in spiked clinical samples by Nucleic Acid Sequence-Based Amplification. J. Clin. Microbiol. 40, 1339-1345. https://doi.org/10.1128/JCM.40.4.1339-1345.2002
  16. Min, J. and A. J. Baeumer. 2002. Highly sensitive and specific detection of viable Escherichia coli in drinking water. Anal. Biochem. 303, 186-193. https://doi.org/10.1006/abio.2002.5593
  17. Oberste, M. S., K. Maher, D. R. Kilpatrick and A. Pallansch. 1999. Molecular evolution of the human enteroviruses: Correlation of serotype with VP1 sequence and application to picornavirus classification. J. Virol. 73, 1941-1948.
  18. Oberste, M. S., K. Maher, D. R. Kilpatrick, M. R. Flemister, B. A. Brown and M. A. Pallansch. 1999. Typing of human enteroviruses by partial sequencing of VP1. J. Clin. Microbiol. 37, 1288-1293.
  19. Rotbart, H. A. 1990. Enzymatic RNA amplification of enteroviruses. J. Clin. Microbiol. 28, 438-442.
  20. Wacharapluesadee, S. and T. L. Hemachudha. 2001. Nucleic-acid sequence based amplification in the rapid diagnosis of rabies. J. Clin. Microbiol. 358, 892-893.
  21. Zoll, G. J., W. J. Melcher, H. Kopecka, G. Jambros and H. J. Vanderpoel. 1992. General primer mediated polymeric chain reaction for detection of enteroviruses; application for diagnostic routine and persistant infections. J. Clin. Microbiol. 30, 160.