Rapid Detection for Shiga Toxin Type 1 (Stxl) by Using Two-Step Ultra-Rapid Real-Time (URRT) PCR

초고속 이단계 PCR에 의한 Shiga 독소 타입 1의 신속 검출법

  • Kim, Il-Wook (Department of Biology, Kyonggi University) ;
  • Kang, Min-Hee (Department of Biology, Kyonggi University) ;
  • Kwon, Soon-Hwan (Chronic Inflammatory Disease Research Center, School of Medicine, Ajou University) ;
  • Cho, Seung-Hak (Team of Enterobacteria, Korean Centers for Disease Control and Prevention) ;
  • Yoon, Byoung-Su (Department of Biology, Kyonggi University)
  • 김일욱 (경기대학교 자연과학대학 생명과학과) ;
  • 강민희 (경기대학교 자연과학대학 생명과학과) ;
  • 권순환 (아주대학교 의과대학 만성염증질환센타) ;
  • 조성학 (질병관리본부 장내세균팀) ;
  • 윤병수 (경기대학교 자연과학대학 생명과학과)
  • Published : 2008.09.30

Abstract

Rapid detection-method for Shiga toxin type 1 that was produced from Shiga toxin-producing Escherichia coli (STEC) was developed by two-step ultra-rapid real-time (URRT) PCR. The specific primers were deduced from 80 bp stable region of stx type 1 (stxl) gene among various informations of STEC strains. URRT PCR is a microchip-based real-time PCR using 6 ${\mu}l$ of reaction volume with extremely short denaturation step and annealing/extension step (1 sec, 3 sec, respectively) in each cycle of PCR. Using the stx1-specific URRT PCR, 35 cycled PCR were finished in time of 6 min and 38 see, also measured 7 min and 28 see including melting temperature (Tm) analysis. The detection-limit of stxl-specific URRT-PCR was estimated until 3 colony forming units / PCR with products with stable Tm at $81.42{\pm}0.34^{\circ}C$. In the applications to various STEC strains and contaminated genomic DNAs, stx1-specific URRT-PCR were tested and shown that it would be expected an useful method for the rapid detection of stx1-coded STEC strains.

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