실시간-염기순서기반증폭(Real-Time Nucleic Acid Sequence-based Amplification)을 통한 장바이러스 검출
Detection of Enterovirus using Real-Time Nucleic Acid Sequence-based Amplification
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배경: 장바이러스는 무균수막염의 가장 흔한 원인으로 바이러스에 의한 수막염 중 70%에서 90%를 차지한다. 임상 증상은 환자의 연령이나 면역능에 따라 매우 다양하게 나타나 진단에 어려움이 있다. 본 연구에서는 장바이러스 검출에 있어서 실시간-염기순서기반증폭(nucleic acid sequence-based amplification, NASBA)의 유용성을 실시간-이중-역전사중합효소연쇄반응과의 비교를 통해 검증하고자 하였다. 방법: 임상에서 무균수막염이 의심되어 장바이러스 검출을 의뢰한 96검체를 대상으로 NucliSENS EasyMAG (bioMérieux,Marcy l'Etoile, France)으로 RNA를 추출한 후 NucliSENS EasyQ Basic 2 (bioMérieux)와 NucliSens EasyQ Enterovirus (bioMérieux)를 사용하여 실시간-염기순서기반증폭을 시행하였다. 실시간-이중-역전사중합효소연쇄반응은 RNA를 QIAamp Viral RNA Mini (QIAGEN Inc., Valencia, CA, USA)로 추출한 후 검사실 자체의 방법에 의해 시행하였다. 결과: 실시간-염기순서기반증폭에 의한 장바이러스 양성률은 45.8% (44/96)였다. 일차 실시간-역전사중합효소연쇄반응에의한 양성률은 22.9% (22/96)였으며 이차 실시간-중합효소연쇄반응의 양성률은 57.3% (55/96)였다. 일차 실시간-역전사중합효소연쇄반응의 결과와 실시간-염기순서기반증폭과의 일치율은 75.0% (72/96)였으며, 이차 실시간-중합효소연쇄반응의 결과와 실시간-염기순서기반증폭과의 일치율은 86.5% (83/96)였다. 결론: 실시간-염기순서기반증폭을 통한 장바이러스 검출은 증폭산물 오염의 가능성이 없으며 역전사 과정이 없어 검사과정이 단순함으로 임상검체를 대상으로 하는 검사실에서 무균수막염의 신속한 감별에 유용하게 사용될 수 있을 것으로판단된다. [대한임상미생물학회지 2010;13:53-58]
Abstract
Background: Enteroviruses are the most frequent etiologic agents of aseptic meningitis and are estimated to be the cause of 70% to 90% of viral meningitis cases. Enterovirus diagnosis can be difficult because clinical features vary according to patient immunity and age. The purpose of this study was to evaluate the performance of the real-time nucleic acid sequence-based amplification (NASBA) assay compared to that of the real-time nested RT-PCR assay for enterovirus detection. Methods: This study was performed on 96 patients suspected of aseptic meningitis based on clinical features. RNA was extracted using NucliSENS EasyMAG and real-time NASBA assay was performed using NucliSENS EasyQ Enterovirus and NucliSENS EasyQ Basic 2. We also executed in-house real-time nested RT-PCR assay for RNA extracted via QIAamp Viral RNA Mini. Results: The positive rate of real-time NASBA assay was 45.8% for enterovirus detection. The positive rate of first real-time reverse transcription PCR was 22.9% and the second real-time PCR was 57.3%. The concordant rate of the real-time NASBA assay and first real-time reverse transcription PCR was 75.0%. The concordant rate of the real-time NASBA assay and second real-time PCR was 86.5%. Conclusion: The detection of enteroviruses using the real-time NASBA assay is less prone to cross-contamination and is simple, without the need for reverse transcription. We conclude that the NASBA assay is an effective method for the rapid diagnosis of aseptic meningitis. (Korean J Clin Microbiol 2010;13:53-58)
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