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

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)
Publication Information
Korean Journal of Microbiology / v.44, no.3, 2008 , pp. 203-211 More about this Journal
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.
Keywords
rapid diagnosis; Shiga toxin-producing Escherichia coli; Shiga toxin type 1; stx1; ultra-rapid real-time PCR;
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