Abstract
Alkaline phosphatase-conjugated synthetic oligodeoxyribonucleic acid probes were used to detect enterotoxigenicEscherichia coli strains containing either the heat stable or heat labile toxin genes. Both of the synthetic probes detected as little as 5 ng of purified plasmid DNA bearing the appropriate toxin gene. In addition, both probes could detect 5 × 106 toxigenic bacteria by colony hybridisation. No cross reactivity was observed between probes. When 197 clinical isolates ofEscherichia coli were examined for toxigenicity using bioassays, 13 heat stable and 17 heat labile toxin strains were identified. Of the 13 heat stable toxin strains, 12 were positive using the heat stable toxin synthetic probe (sensitivity, 92 %; specificity, 98 %) while 16 of 17 bioassay heat labile toxin positive samples were identified using the heat labile toxin synthetic probe (sensitivity, 94 %; specificity, 97 %). Alkaline phosphatase-conjugated synthetic probes with high sensitivity and specificity should provide a rapid means of identifying toxigenicEscherichia coli.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Moseley, S. L., Huq, I., Alim, A. L., So, M., Samadpour-Motalebi, M., Falkow, S. Detection of enterotoxigenicEscherichia coli by DNA colony hybridization. Journal of Infectious Disease 1980, 142: 892–898.
Boileau, C. R., d'Hauteville, H. M., Sansonetti, P. J. DNA hybridization technique to detectShigella species and enteroinvasiveEscherichia coli. Journal of Clinical Microbiology 1984, 20: 959–961.
Fitts, R., Diamond, M., Hamilton, C., Neri, M. DNA-DNA hybridization assay for detection ofSalmonella species in foods. Applied and Environmental Microbiology 1983, 46: 1146–1151.
Horn, J. E., Quinn, T., Hammer, M., Palmer, L., Falkow, S. Use of nucleic acid probes for the detection of sexually transmitted infectious agents. Diagnostic Microbiology and Infectious Disease 1986, 4: S101-S109.
Tompkins, L. S., Krajden, M. Approaches to the detection of enteric pathogens, includingCampylobacter, using nucleic acid hybridization. Diagnostic Microbiology and Infectious Disease 1986, 4: S78-S88.
Hill, W. E., Payne, W. L., Zon, G., Moseley, S. L. Synthetic oligodeoxyribonucleotide probes for detecting heat-stable enterotoxin-producingEscherichia coli by DNA colony hybridzation. Applied and Environmental Microbiology 1985, 50: 1187–1191.
Nishibuchi, M., Ishibashi, M., Takeda, Y., Kaper, J. Detection of the thermostable direct hemolysin gene and related DNA sequences inVibrio parahaemolyticus and otherVibrio species by the DNA colony hybridization test. Infection and Immunity 1985, 49: 481–486.
Langer, P. R., Waldrop, A. A., Ward, D. C. Enzymatic synthesis of biotin-labeled polynucleotides: Novel nucleic acid affinity probes. Proceedings of the National Academy of Science of the USA 1981, 78: 6633–6637.
Sethabutr, O., Hanchalay, S., Echeverria, P., Taylor, D. N., Leksomboon, U. A nonradioactive DNA probe to identifyShigella and enterotoxigenicEscherichia coli in stools of children with diarrhoca. Lancet 1985, ii: 1095–1097.
Bialkowska-Hobrzanska, H. Detection of enterotoxigenicEscherichia coli by dot blot hybridization with biotinylated DNA probes. Journal of Clinical Microbiology 1987, 25: 338–343.
Beck, S. Colorimetric-detected DNA sequencing. Analytical Biochemistry 1987, 164: 514–520.
Jablonski, E., Moomaw, E. W., Tullis, R. H., Ruth, J. L. Preparation of oligodeoxynucleotide-alkaline phosphatase conjugates and their use as hybridization probes. Nucleic Acids Research 1986, 14: 6115–6128.
Dallas, W. S., Gill, P. M., Falkow, S. Cistrons encodingEscherichia coli heat-labile toxin. Journal of Bacteriology 1979, 139: 850–858.
Lathe, R., Hirth, P., DeWilde, M., Lecocq, J. P. Cell-free synthesis of enterotoxin ofEscherichia coli from a cloned gene. Nature 1980, 284: 473–474.
Messing, J., Vieira, J. A new pair of M13 vectors for selecting either DNA strand of double-digest restriction fragments. Gene 1982, 19: 2969–2976.
Maniatis, T., Fritsch, E. F., Sambrook, J. Molecular Cloning: A laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1982, p. 88–94.
Sack, D. A., Sack, R. B. Test for enterotoxigenicEscherichia coli using Y-1 adrenal cells in miniculture. Infection and Immunity 1975, 11: 334–336.
Dean, A. G., Ching, Y.-C., Williams, R. G., Harden, L. B. Test forEscherichia coli enterotoxin using infant mice: application in a study of diarrhea in children in Honolulu. Journal of Infectious Disease 1972, 125: 407–411.
Black, R. E., Meyerson, M. H., Huq, I., Alim, A. R. M. A., Yunus, M. Incidence and severity of rotavirus andEscherichia coli diarrhea in rural Bangladesh. Lancet 1981, i: 141–143.
Wallace, R. B., Shaeffer, J., Murphy, R. F., Bonner, J., Hirose, T., Itakura, K. Hybridization of synthetic oligodeoxyribonucleotide to OX174 DNA: the effect of single base pair mismatch. Nucleic Acids Research 1979, 6: 3543–3557.
Seriwatana, J., Escheverria, P., Taylor, D. N., Sakuldaipeara, T., Changchawalit, S., Chivoratanond, O. Identification of enterotoxigenicEscherichia coli with synthetic alkaline phosphatase-conjugated oligonucleotide DNA probes. Journal of Clinical Microbiology 1987, 25: 1438–1441.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Olive, D.M., Khalik, D.A. & Sethi, S.K. Identification of enterotoxigenicEscherichia coli using alkaline phosphatase-labeled synthetic oligodeoxyribonucleotide probes. Eur. J. Clin. Microbiol. Infect. Dis. 7, 167–171 (1988). https://doi.org/10.1007/BF01963071
Issue Date:
DOI: https://doi.org/10.1007/BF01963071