Identification of Single Base-pair Mutation on uidA Gene of Escherichia coli O157:H7 by Peptide Nucleic Acids (PNA) Mediated PCR Clamping
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概要
- 論文の詳細を見る
Peptide Nucleic Acids (PNA) is a new type of DNA analogue with a peptide backbone. We developed a rapid identification system of Escherichia. coli O157:H7 using PNA mediated PCR clamping. Firstly, we confirmed a single nucleotide alteration in the uidA gene (T93G), which is specific to E. coli O157: H7. We designed forward mutant DNA primer, wild type PNA, and a reverse DNA primer corresponding to the uidA sequence. PCR cycle consisted of four steps including dual annealing temperatures, 57°C and 45°C. Among 20 E. coli strains with various serotypes and 4 neighboring strains, the amplified bands (517 bp) were detected only in E. coli O157:H7 strains. PNA has specifically inhibited the PCR amplification from a wild type uidA gene. We successfully developed a multiplex PCR system, which detects both shigatoxin (stx) and uidA genes at once, to get reliable results by easier and rapid operation. We also analyzed kinetic parameters of PNA/DNA association using surface plasmon resonance and melting temperature using fluorescence resonance energy transfer (FRET). We discussed a selection mechanism of PCR clamping from these results.
- 2004-02-23
著者
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TAKIYA Toshiyuki
United Graduate School of Agricultural Science, Gifu University
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FUTO Satoshi
Nippon Flour Mills Co., Ltd.
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TSUNA Mika
Nippon Flour Mills Co., Ltd.
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NAMIMATSU Takanori
Zen-noh Institute of Animal Health
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SAKANO Tetsuya
Zen-noh Institute of Animal Health
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KAWAI Keiichi
United Graduate School of Agricultural Science, Gifu University
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SUZUKI Tohru
United Graduate School of Agricultural Science, Gifu University
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Kawai K
Faculty Of Applied Biological Sciences Gifu University
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Tsuna M
Nippon Flour Mills Co. Ltd.
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Futo Satoshi
Nippon Flour Mills Co. Ltd.
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Futo Satoshi
Fasmac Co. Ltd.
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Takiya Toshiyuki
United Graduate School Of Agricultural Science Gifu University
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Suzuki T
Ihara & Company Ltd.
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Kawai K
Fac. Appl. Biol. Sci. Gifu Univ.
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Kawai Keiichi
School Of Health Sciences Faculty Of Medicine Kanazawa University
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Tsuna Mika
Nippon Flour Mills Co. Ltd.
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Suzuki Tohru
United Graduate School Of Agricultural Sci. Gifu Univ.
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Kawai Keiichi
Fac. Appl. Biol. Sci. Gifu Univ.
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Suzuki Tohru
United Grad. Sch. Agr. Sci. Gifu Univ.:fac. Appl. Biol. Sci. Gifu Univ.
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Kamimura Kiyohisa
Department Of Radiology Fujimoto Hayasuzu Hospital
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