Detection of a heat-labile enterotoxin gene in enterotoxigenicEscherichia coli by densitometric evaluation using highly specific enzyme-linked oligonucleotide probes

  • S. Tamatsukuri
  • K. Yamamoto
  • S. Shibata
  • F. Leaño
  • T. Honda
  • T. Miwatani


Two alkaline phosphatase-conjugated 24-mer oligonucleotide probes were developed to detect the heat-labile enterotoxin gene in enterotoxigenicEscherichia coli. Probes were antisense codon sequences, which are transcribed into mRNA, of the heat-labile enterotoxin gene of enterotoxigenicEscherichia coli of human origin. Using dot-blot hybridization, probes were tested with 100 clinical isolates and evaluated by a reflectance-type densitometer. Results agreed very well with those of an immunological test, the Biken test, and a32P-labelled recombinant DNA probe. The oligonucleotide probes did not react with nucleic acids prepared from other diarrhoeagenic bacterial pathogens. Thus, the alkaline phosphatase-conjugated oligonucleotide probes seem to be highly sensitive and specific for detection of heat-labile enterotoxin-producing enterotoxigenicEscherichia coli. Moreover, the results indicate a potential usefulness for densitometric evaluation of DNA hybridization.


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Levine MM Escherichia coli that cause diarrhea: enterotoxigenic, enteropathogenic, enteroinvasive, enterohemorrhagic, and enteroadherent. Journal of Infectious Diseases 1987, 155: 377–389.PubMedGoogle Scholar
  2. 2.
    Dean AG, Ching YC, Williams RG, Harden LB Test forEscherichia coli enterotoxin using infant mice: application in a study of diarrhea in children in Honolulu. Journal of Infectious Diseases 1972, 125: 407–411.PubMedGoogle Scholar
  3. 3.
    Sack DA, Sack RB Test for enterotoxigenicEscherichia coli using Y1 adrenal cells in miniculture. Infection and Immunity 1975, 11: 334–336.PubMedGoogle Scholar
  4. 4.
    Honda T, Akhtar Q, Glass RI, Kibriya AKMG A simple assay to detectEscherichia coli producing heat-labile enterotoxin: results of a field study of the Biken test in Bangladesh. Lancet 1981, ii; 609–610.Google Scholar
  5. 5.
    Honda T, Arita M, Takeda Y, Miwatani T Further evaluation of the Biken test (modified Elek test) for detection of enterotoxigenicEscherichia coli producing heat-labile enterotoxin. Journal of Clinical Microbiology 1982, 16: 60–62.PubMedGoogle Scholar
  6. 6.
    Honda T, Sato M, Miwatani T Differential detection of cholera enterotoxin andEscherichia coli heat-labile enterotoxin by enzyme-linked immunosorbent assays with antibodies specific to the two toxins. Journal of Clinical Microbiology 1984, 20: 664–667.PubMedGoogle Scholar
  7. 7.
    Honda T, Yoh M, Wang Y, Tsuji T, Miwatani T Quantitative analysis by reversed Mancini test of cross-reacting antibodies in rabbit antisera against porcine and humanEscherichia coli heat-labile enterotoxins and cholera toxin. FEMS Microbiology Letters 1986, 33: 267–270.Google Scholar
  8. 8.
    Echeverria P, Seriwatana J, Leksomboon U, Tirapat C, Chaicumpa W, Rowe B Identification by DNA hybridization of enterotoxigenicEscherichia coli in homes of children with diarrhoea. Lancet 1984, i: 65–66.Google Scholar
  9. 9.
    Echeverria P, Seriwatana J, Taylor DN, Tirapat C, Chaicumpa W, Lowe B Identification by DNA hybridization of enterotoxigenicEscherichia coli in a longitudinal study of villages in Thailand. Journal of Infectious Diseases 1987, 151: 124–130.Google Scholar
  10. 10.
    Echeverria P, Taylor DN, Seriwatana J, Chatkaeomorakot A, Khungvalert V, Sakuldaipera T, Smith RD A comparative study of enterotoxin gene probes and tests for toxin production to detect enterotoxigenicEscherichia coli. Journal of Infectious Diseases 1986, 153: 255–260.PubMedGoogle Scholar
  11. 11.
    Moseley SL, Echeverria P, Seriwatana J, Tirapat C, Chaicumpa W, Sakuldaipeara T, Falkow S Identification of enterotoxigenicEscherichia coli by colony hybridization using three enterotoxin gene probes. Journal of Infectious Diseases 1982, 145: 863–869.PubMedGoogle Scholar
  12. 12.
    Moseley SL, Hardy JW, Hug MI, Echeverria P, Falkow S Isolation and nucleotide sequence determination of a gene encoding a heat-stable enterotoxin ofEscherichia coli. Infection and Immunity 1983, 39: 1167–1174.PubMedGoogle Scholar
  13. 13.
    Moseley SL, Huq I, Alim ARMA, So M, Samadpour-Motalebi M, Falkow S Detection of enterotoxigenicEscherichia coli by DNA hybridization. Journal of Infectious Diseases 1980, 142: 892–899.PubMedGoogle Scholar
  14. 14.
    So M, McCarthy BJ Nucleotide sequence of the bacterial transposon Tn1681 encoding a heat-stable (ST) toxin and its identification in enterotoxigenicEscherichia coli strains. Proceedings of National Academy of Sciences of the USA 1980, 77: 4011–4015.Google Scholar
  15. 15.
    Echeverria P, Taylor DN, Seriwatana J, Moe C Comparative study of synthetic oligonucleotide and cloned polynucleotide enterotoxin gene probes to identify enterotoxigenicEscherichia coli by DNA colony hybridization. Applied and Environmental Microbiology 1987, 25: 106–109.Google Scholar
  16. 16.
    Hill WE, Payne WL, Zon G, Moseley SL Synthetic oligodeoxyribonucleotide probes for detecting heat-stable enterotoxin-producingEscherichia coli by DNA colony hybridization. Applied and Environmental Microbiology 1985, 50: 1187–1191.PubMedGoogle Scholar
  17. 17.
    Murray BE, Mathewson JJ, DuPont HL, Hill WE Utility of oligodeoxyribonucleotide probes for detecting enterotoxigenicEscherichia coli. Journal of Infectious Diseases 1987, 155: 809–811.PubMedGoogle Scholar
  18. 18.
    Nishibuchi M, Murakami A, Arita M, Jikuya H, Tanaka J, Honda T, Miwatani T Detection with synthetic oligonucleotide probes of nucleotide sequence variations in the genes encoding enterotoxins ofEscherichia coli. Journal of Clinical Microbiology 1989, 27: 2272–2276.PubMedGoogle Scholar
  19. 19.
    Seriwatana J, Echeverria P, Escamilla J, Glass R, Huq I, Rockhill R, Stoll BJ Identification of enterotoxigenicEscherichia coli in patients with diarrhea in Asia with three enterotoxin gene probes. Infection and Immunity 1983, 42: 152–155.PubMedGoogle Scholar
  20. 20.
    Sommerfelt H, Grewal HMS, Bahm MK Simplified and accurate nonradioactive polynucleotide gene probe assay for identification of enterotoxigenicEscherichia coli. Journal of Clinical Microbiology 1990, 28: 49–54.PubMedGoogle Scholar
  21. 21.
    Sommerfelt H, Kalland KH, Raj P, Moseley SL, Bhan MK, Bjorvatn B Cloned polynucleotide and synthetic oligonucleotide probes used in colony hybridization are equally efficient in the identification of enterotoxigenicEscherichia coli. Journal of Clinical Microbiology 1988, 26: 2275–2278.PubMedGoogle Scholar
  22. 22.
    Sommerfelt H, Svennerholm AM, Kalland KH, Haukanes BI, Bjorvatn B Comparative study of colony hybridization with synthetic oligonucleotide probes and enzyme-linked immunosorbent assay for identification of enterotoxigenicEscherichia coli. Journal of Clinical Microbiology 1988, 26: 530–534.PubMedGoogle Scholar
  23. 23.
    Alvarado-Urbina G, Sathe GM, Liu W, Gillen MF, Duck PD, Bender R, Ogilvie KK Automated synthesis of gene fragments. Science 1981, 214: 270–274.PubMedGoogle Scholar
  24. 24.
    Bialkowska-Hobrzanska H Detection of enterotoxigenicEscherichia coli by dot blot hybridization with biotinylated DNA probes. Journal of Clinical Microbiology 1987, 25: 338–343.PubMedGoogle Scholar
  25. 25.
    Kirii Y, Danbara H, Komase K, Arita H, Yoshikawa M Detection of enterotoxigenicEscherichia coli by colony hybridization with biotinylated enterotoxin probes. Journal of Clinical Microbiology 1987, 25: 1962–1965.PubMedGoogle Scholar
  26. 26.
    Longer PR, Waldrop AA, Ward CC Enzymatic synthesis of biotin-labeled polynucleotides: novel nucleic acid affinity probes. Proceedings of the National Academy of Sciences of the USA 1981, 78: 6633–6637.PubMedGoogle Scholar
  27. 27.
    Jablonski E, Moomaw EW, Tullis RH, Ruth JL Preparation of oligodeoxynucleotide-alkaline phosphatase conjugates and their use as hybridization probes. Nucleic Acids Research 1986, 14: 6115–6128.PubMedGoogle Scholar
  28. 28.
    Li P, Medon PP, Skingle DC, Lanser JA, Symons RH Enzyme-linked synthetic oligonucleotide probes non-radioactive detection of enterotoxigenicEscherichia coli in faecal specimens. Nucleic Acids Research 1987, 15: 5275–5287.PubMedGoogle Scholar
  29. 29.
    Medon PP, Lanser JA, Monckton PR, Li P, Symons RH Identification of enterotoxigenicEscherichia coli isolated with enzyme-labeled synthetic oligonucleotides probes. Journal of Clinical Microbiology 1988, 26: 2173–2176.PubMedGoogle Scholar
  30. 30.
    Nishibuchi M, Arita M, Honda T, Miwatani T Evaluation of a nonisotopically labeled oligonucleotide probe to detect the heat-stable enterotoxin gene ofEscherichia coli by the DNA colony hybridization test. Journal of Clinical Microbiology 1988, 26: 784–786.PubMedGoogle Scholar
  31. 31.
    Oprandy JJ, Thornton SA, Gardiner CH, Burr D, Batchelor R, Bourgeois L Alkaline phosphatase-conjugated oligonucleotide probes for enterotoxigenicEscherichia coli in travelers to South America and West Africa. Journal of Clinical Microbiology 1988, 26: 92–95.PubMedGoogle Scholar
  32. 32.
    Seriwatana J, Echeverria P, Taylor DN, 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.PubMedGoogle Scholar
  33. 33.
    Sambrook J, Fritsch EF, Maniatis T Molecular cloning. A laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989.Google Scholar
  34. 34.
    Spicer EK, Noble JA Escherichia coli heat-labile enterotoxin. Nucleotide sequence of the A subunit gene. Journal of Biological Chemistry 1982, 257: 5716–5721.PubMedGoogle Scholar
  35. 35.
    Dallas WS, Falkow S Amino acid sequence homology between cholera toxin andEscherichia coli heat-labile toxin. Nature 1980, 288: 499–501.PubMedGoogle Scholar
  36. 36.
    Yamamoto T, Nakazawa T, Miyata T, Kaji A, Yokota T Evolution and structure of two ADP-ribosylation enterotoxins,Escherichia coli heat-labile toxin and cholera toxin. FEBS Letters 1984, 169: 241–246.PubMedGoogle Scholar
  37. 37.
    Yamamoto T, Tamura T, Yokota T Primary structure of heat-labile enterotoxin produced byEscherichia coli pathogenic for humans. Journal of Biological Chemistry 1984, 259: 5037–5044.PubMedGoogle Scholar
  38. 38.
    Takeshita S, Sato M, Toba M, Masahashi W, Hashimoto-Gotoh T High-copy-number and low-copy-number plasmid vectors forlacZ-complementation and chloramphenicol-or kanamycin-resistance selection. Gene 1987, 61: 63–74.PubMedGoogle Scholar
  39. 39.
    Lockman HA, Galen JE, Kaper JB Vibrio cholerae enterotoxin genes: nucleotide sequence analysis of DNA encoding ADP-ribosyltransferase. Journal of Bacteriology 1984, 159: 1086–1089.PubMedGoogle Scholar
  40. 40.
    Lockman H, Kaper JB Nucleotide sequence analysis of the A2 and B subunits ofV. cholerae enterotoxin. Journal of Biological Chemistry 1983, 258: 13722–13726.PubMedGoogle Scholar
  41. 41.
    Mekalanos JJ, Swarts DJ, Pearson GDN, Harford N, Gyoyne F, Wilde M Cholera toxin gene: nucleotide sequence, deletion analysis and vaccine development. Nature 1983, 308: 551–557.Google Scholar
  42. 42.
    Redfield DC, Richman DD, Albanil S, Oxman MN, Wahl GM Detection of herpes simplex virus in clinical specimens by DNA hybridization. Diagnosis of Microbial Infectious Diseases 1983, 1: 117–128.Google Scholar
  43. 43.
    Sethabutr O, Hanchalay S, Echeverria P, Taylor DN, Leksomboon U A non-radioactive DNA probe to identifyShigella and enteroinvasiveEscherichia coli in stools of children with diarrhoea. Lancet 1985, ii: 1095.Google Scholar
  44. 44.
    Wetmur JG Acceleration of DNA renaturation rate. Biopolymers 1975, 14: 2517–2524.Google Scholar
  45. 45.
    Matteucci MD, Caruthers MH Synthesis of deoxyoligonucleotides on a polymer support. Journal of the American Chemical Society 1981, 103: 3185–3191.Google Scholar
  46. 46.
    Endo Y, Iwata T, Nagasaki H, Miyai K Quantification of DNA hybrids on discs of nitrocellulose filter. Clinical Chemistry 1988, 34: 1523.Google Scholar
  47. 47.
    Wolf SF, Haines L, Fisch J, Kremsky JN, Dougherty JP, Jacob K Rapid hybridization kinetics of DNA attached to submicron latex particles. Nucleic Acids Research 1987, 15: 2911–2926.PubMedGoogle Scholar
  48. 48.
    Nagata Y, Yokota H, Kosuda O Quantification of picogram levels of specific DNA immobilized in microtiter wells. FEBS Letters 1985, 183: 379–382.PubMedGoogle Scholar

Copyright information

© Friedr. Vieweg & Sohn Verlagsgesellschaft mbH 1991

Authors and Affiliations

  • S. Tamatsukuri
    • 1
    • 2
  • K. Yamamoto
    • 1
  • S. Shibata
    • 2
  • F. Leaño
    • 1
  • T. Honda
    • 1
  • T. Miwatani
    • 1
  1. 1.Department of Bacteriology and Serology, Research Institute for Microbial DiseasesOsaka UniversitySuita, OsakaJapan
  2. 2.Toyobo Research CenterOhtsu, ShigaJapan

Personalised recommendations