Detection of two variant Vero toxin genes in Escherichia coli by capillary electrophoresis.
Three methods [capillary electrophoresis (CE)-allele-specific PCR, CE-single-strand conformation polymorphism (SSCP) and CE-cleavase fragment length polymorphism (CFLP)] were developed in order to effect rapid and specific analysis of the vero toxin (VT)1 and VT2 genes of O157. The allele-specific polymerase chain reaction (PCR) method, which utilized specific duplex PCR with specific primers for VT1 and VT2, showed that VT1 and VT2 consisted of 174 and 128 bp, respectively. Subsequent CE analysis was carried out. Separation time was 4 min. SSCP, which utilized one primer set which reacted with both VT1 and VT2 in the PCR method, was followed by CE analysis of secondary structure of single-strand DNA. Two genes could be analyzed in approximately 18 min. CFLP, like SSCP, is a method for detecting mutation-induced changes in secondary structure of single-stranded DNA. The endonuclease cleavase I recognizes and cleaves the 5' side of hairpin loops in self-annealed single-strand DNA of PCR product 169 bp obtained from VT1 and VT2. The produced DNA fragments are analyzed by CE and the electrophelogram reveals a sequence-specific CFLP. Separation time was 6 min. These techniques are suitable for the detection and the identification of O157.
Arakawa
Hidetoshi
H
School of Pharmaceutical Sciences, Showa University, Shinagawa-ku, Tokyo 142-8555, Japan.
Watanabe
Kazuyuki
K
Kashiwazaki
Hiroyuki
H
Maeda
Masako
M
D000483:Alleles; D001483:Base Sequence; D017931:DNA Primers; D019075:Electrophoresis, Capillary / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics*; D007834:Lasers; D016133:Polymerase Chain Reaction; D018807:Polymorphism, Single-Stranded Conformational; D020033:Protein Isoforms / Q000235:genetics*; D022603:Shiga Toxins / Q000235:genetics*; D013050:Spectrometry, Fluorescence / Q000379:methods
D017931:DNA Primers; D020033:Protein Isoforms; D022603:Shiga Toxins