Development of an enzyme-labeled oligonucleotide probe for the cholera toxin gene.
An alkaline phosphatase-conjugated 30-mer oligonucleotide probe was developed to detect the cholera toxin gene (ctx) in Vibrio cholerae O1. For rapid identification, V. cholerae O1 was grown on selective agar (thiosulfate-citrate-bile salts agar) or in alkaline peptone water and organisms were transferred directly to nylon membranes. Lysis of cells, denaturation of DNA, neutralization, and hybridization were carried out on the membrane. These procedures required only 3 h for completion. The results of the hybridization test with 88 clinical and 20 environmental isolates agreed almost exactly with the results of the immunological tests (anti-cholera toxin antibody-sensitized latex agglutination tests). The specificity of the probe was also tested with strains of enterotoxigenic Escherichia coli, V. cholerae non-O1, and Vibrio mimicus.
Yoh
M
M
Research Institute for Microbial Diseases, Osaka University, Japan.
Miyagi
K
K
Matsumoto
Y
Y
Hayashi
K
K
Takarada
Y
Y
Yamamoto
K
K
Honda
T
T
D000469:Alkaline Phosphatase; D001483:Base Sequence; D002771:Cholera / Q000382:microbiology; D002772:Cholera Toxin / Q000235:genetics* / Q000302:isolation & purification; D004269:DNA, Bacterial / Q000235:genetics; D006801:Humans; D007841:Latex Fixation Tests; D015336:Molecular Probe Techniques / Q000706:statistics & numerical data; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D012680:Sensitivity and Specificity; D014734:Vibrio cholerae / Q000235:genetics*
D004269:DNA, Bacterial; D015345:Oligonucleotide Probes; D002772:Cholera Toxin; D000469:Alkaline Phosphatase