A fluorescence polarization assay using oligonucleotide probes for the rapid detection of verotoxin-producing Escherichia coli.
A hybridization assay using fluorescence polarization was combined with the asymmetric polymerase chain reaction (PCR) in a method for the detection of the verotoxin type 2 gene of verotoxin-producing Escherichia coli. Six oligonucleotide probes labeled with FITC were designed and evaluated. One of these gave a detection limit of 10(3) colony forming units per assay, and assay results could be obtained within 5 min after PCR. It appears that the detection limit was restricted mainly by the extent and fidelity of PCR amplification, rather than by the sensitivity of the fluorescence polarization technique, indicating that good probe design facilitates the rapid detection of the PCR product. The fluorescence polarization assay, in conjunction with DNA amplification by PCR, is a powerful and widely applicable method for the rapid and sensitive detection of oligonucleotide sequences.
Ohiso
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I
Diversified Products Division, Nishikawa Rubber Co., Ltd., 2-2-8 Misasa-cho, Nishi-ku, 733-8510, Hiroshima, Japan. oiso@nishikawa-rbr.co.jp
Tsuruoka
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M
Iida
T
T
Honda
T
T
Karube
I
I
D001427:Bacterial Toxins / Q000032:analysis* / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D005454:Fluorescence Polarization / Q000379:methods*; D017403:In Situ Hybridization; D015345:Oligonucleotide Probes / Q000737:chemistry / Q000235:genetics*; D016133:Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D022622:Shiga Toxin 1
D001427:Bacterial Toxins; D015345:Oligonucleotide Probes; D022622:Shiga Toxin 1