미생물학회지 (Korean Journal of Microbiology)

  • 제47권1호
  • /
  • Pages.30-37
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  • 2011
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  • 0440-2413(pISSN)
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  • 2383-9902(eISSN)
한국미생물학회 (The Microbiological Society of Korea)
권호 표지

Ultra Real-Time PCR을 활용한 Avian Influenza Virus Subtype의 조기진단법

Early Diagnostic Method of Avian Influenza Virus Subtype Using Ultra Real-Time PCR

  • 김상태 (이화여자대학교 생명약학부) ;
  • 김영균 (국민대학교 삼림대학 삼림생명공학과) ;
  • 김장수 (고려대학교 구로병원 진단검사의학과)
  • Kim, Sang-Tae (Divion of Biological and Phamaceutical, Ewha Womans University) ;
  • Kim, Young-Kyoon (Department of Forest Products, College of Forest Science, Kookmin University) ;
  • Kim, Jang-Su (Department of Laboratory Medicine, Korea University Guro Hospital)
  • 투고 : 2011.01.14
  • 심사 : 2011.01.26
  • 발행 : 2011.03.31
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초록

조류 인플루엔자 바이러스(AIV) 아형을 ultra-time PCR법(UPCR)을 이용하여 초스피드로 진단할 수 있는 방법을 고안하였다. 표적 대상의 프라이머는 AIV H5N1 아형의 hemagglutinin(HA) 유전자 중 가장 상보성이 높은 133 bp의 부위를 선택하였고, 실험의 안전을 위하여 인공합성의 방법으로 제작하였다. 압타머와 결합한 molecular beacon 기반 Mini-Opticon Q-PCR 기기를 사용한 UPCR법으로, 총 UPCR 반응액의 양을 10 ${\mu}l$으로, UPCR과 용융온도 분석시간을 15분 이내로 매우 짧게 단축시켰다. 민감도 측정에서 최소의 주형인 5분자의 HA 유전자만으로 정확히 AIV의 특이적 133 bp를 합성하였다. UPCR로 디자인된 이 PCR은 AIV 아형의 진단에 적용될 수 있을 뿐 아니라, UPCR이 기반되는 진단을 이용하여 다른 병원체에도 널리 적용 될 수 있을 것으로 기대된다.

This ultra real-time PCR (UPCR) based diagnosis system for avian influenza A virus (AIV) subtype was designed. The target primer in this study was derived from H5N1 subtype-specific 133 bp partial gene of hemagglutinin (HA), and was synthesized by using PCR-based gene synthesis on the ground of safety. UPCR was operated by Mini-Opticon Q-PCR Quantitative Thermal Cycler using aptamer-based molecular beacon, total 10 ${\mu}l$ of reaction mixture with extraordinarily short time in each steps in PCR. The detection including UPCR and analysis of melting temperature was totally operated within 15 min. The AIV-specific 133 bp PCR product was correctly amplified until 5 molecules of HA gene as minimum of templates. This kind of PCR was drafted as UPCR in this study and it could be used to detect not only AIV subtype, but also other pathogens using UPCR-based diagnosis.

키워드

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