Identification of single base-pair mutation on uidA gene of Escherichia coli O157:H7 by Peptide Nucleic Acids (PNA) mediated PCR clamping.
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.
United Graduate School of Agricultural Science, Gifu University, Japan.
Toshiyuki
T
Takiya
Satoshi
S
Futo
Mika
M
Tsuna
Takanori
T
Namimatsu
Tetsuya
T
Sakano
Keiichi
K
Kawai
Tohru
T
Suzuki
D020137:Base Pair Mismatch / Q000235:genetics*; D004247:DNA / Q000235:genetics / Q000378:metabolism; D019453:Escherichia coli O157 / Q000235:genetics* / Q000378:metabolism*; D005798:Genes, Bacterial / Q000235:genetics*; D007700:Kinetics; D009154:Mutation / Q000235:genetics*; D020135:Peptide Nucleic Acids / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D020349:Surface Plasmon Resonance; D013696:Temperature
D020135:Peptide Nucleic Acids; D004247:DNA