| Autor: |
Takiya T; United Graduate School of Agricultural Science, Gifu University, Japan., Futo S, Tsuna M, Namimatsu T, Sakano T, Kawai K, Suzuki T |
| Jazyk: |
angličtina |
| Zdroj: |
Bioscience, biotechnology, and biochemistry [Biosci Biotechnol Biochem] 2004 Feb; Vol. 68 (2), pp. 360-8. |
| DOI: |
10.1271/bbb.68.360 |
| Abstrakt: |
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 degrees C and 45 degrees 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. |
| Databáze: |
MEDLINE |
| Externí odkaz: |
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