Skip to main content
Journal of Clinical Microbiology logoLink to Journal of Clinical Microbiology
. 1991 Nov;29(11):2375–2379. doi: 10.1128/jcm.29.11.2375-2379.1991

Detection of Escherichia coli heat-stable enterotoxin genes in pig stool specimens by an immobilized, colorimetric, nested polymerase chain reaction.

E Hornes 1, Y Wasteson 1, O Olsvik 1
PMCID: PMC270341  PMID: 1774239

Abstract

A combination of selective enrichment by using immunomagnetic separation of F4 (K88)-positive Escherichia coli and a nested colorimetric polymerase chain reaction (PCR) was used on crude clinical and spiked samples for determination of genes encoding heat-stable enterotoxins (STs) Ia (ST Ia) and Ib (ST Ib). The combination increased the sensitivity of the nested PCR compared with that of application onto crude samples. Dead cells were also enriched by use of this technology, giving results that are not available by traditional cultivation as enrichment before PCR. The second step in the PCR was modified to be able to differentiate between ST Ia and ST Ib genes. The colorimetric PCR was performed in a microtiter format, making it useful for automation in clinical laboratories and for the screening of large numbers of samples.

Full text

PDF
2375

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Candrian U., Furrer B., Höfelein C., Meyer R., Jermini M., Lüthy J. Detection of Escherichia coli and identification of enterotoxigenic strains by primer-directed enzymatic amplification of specific DNA sequences. Int J Food Microbiol. 1991 Apr;12(4):339–351. doi: 10.1016/0168-1605(91)90148-i. [DOI] [PubMed] [Google Scholar]
  2. Dean A. G., Ching Y. C., Williams R. G., Harden L. B. Test for Escherichia coli enterotoxin using infant mice: application in a study of diarrhea in children in Honolulu. J Infect Dis. 1972 Apr;125(4):407–411. doi: 10.1093/infdis/125.4.407. [DOI] [PubMed] [Google Scholar]
  3. Echeverria P., Taylor D. N., Seriwatana J., Brown J. E., Lexomboon U. Examination of colonies and stool blots for detection of enteropathogens by DNA hybridization with eight DNA probes. J Clin Microbiol. 1989 Feb;27(2):331–334. doi: 10.1128/jcm.27.2.331-334.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Gouvea V., Glass R. I., Woods P., Taniguchi K., Clark H. F., Forrester B., Fang Z. Y. Polymerase chain reaction amplification and typing of rotavirus nucleic acid from stool specimens. J Clin Microbiol. 1990 Feb;28(2):276–282. doi: 10.1128/jcm.28.2.276-282.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Hill W. E., Payne W. L., Zon G., Moseley S. L. Synthetic oligodeoxyribonucleotide probes for detecting heat-stable enterotoxin-producing Escherichia coli by DNA colony hybridization. Appl Environ Microbiol. 1985 Nov;50(5):1187–1191. doi: 10.1128/aem.50.5.1187-1191.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Kemp D. J., Smith D. B., Foote S. J., Samaras N., Peterson M. G. Colorimetric detection of specific DNA segments amplified by polymerase chain reactions. Proc Natl Acad Sci U S A. 1989 Apr;86(7):2423–2427. doi: 10.1073/pnas.86.7.2423. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Klipstein F. A., Engert R. F., Houghten R. A. Immunological properties of purified Klebsiella pneumoniae heat-stable enterotoxin. Infect Immun. 1983 Nov;42(2):838–841. doi: 10.1128/iai.42.2.838-841.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Lund A., Hellemann A. L., Vartdal F. Rapid isolation of K88+ Escherichia coli by using immunomagnetic particles. J Clin Microbiol. 1988 Dec;26(12):2572–2575. doi: 10.1128/jcm.26.12.2572-2575.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Moseley S. L., Hardy J. W., Hug M. I., Echeverria P., Falkow S. Isolation and nucleotide sequence determination of a gene encoding a heat-stable enterotoxin of Escherichia coli. Infect Immun. 1983 Mar;39(3):1167–1174. doi: 10.1128/iai.39.3.1167-1174.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Nishibuchi M., Murakami A., Arita M., Jikuya H., Takano J., Honda T., Miwatani T. Detection with synthetic oligonucleotide probes of nucleotide sequence variations in the genes encoding enterotoxins of Escherichia coli. J Clin Microbiol. 1989 Oct;27(10):2272–2276. doi: 10.1128/jcm.27.10.2272-2276.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Olive D. M. Detection of enterotoxigenic Escherichia coli after polymerase chain reaction amplification with a thermostable DNA polymerase. J Clin Microbiol. 1989 Feb;27(2):261–265. doi: 10.1128/jcm.27.2.261-265.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Olsvik O., Fossum K. Enterotoxigenic Escherichia coli in the jejunum of piglets with neonatal diarrhea. Acta Vet Scand. 1984;25(1):21–28. doi: 10.1186/BF03547275. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Olsvik O., Solberg R., Bergan T. Characterization of enterotoxigenic Escherichia coli. Serotypes, enterotoxins, adhesion fimbriae, and the presence of plasmids. Acta Pathol Microbiol Immunol Scand B. 1985 Aug;93(4):255–262. [PubMed] [Google Scholar]
  14. Olsvik O., Wasteson Y., Lund A., Hornes E. Pathogenic Escherichia coli found in food. Int J Food Microbiol. 1991 Jan;12(1):103–113. doi: 10.1016/0168-1605(91)90051-p. [DOI] [PubMed] [Google Scholar]
  15. Pai C. H., Mors V. Production of enterotoxin by Yersinia enterocolitica. Infect Immun. 1978 Mar;19(3):908–911. doi: 10.1128/iai.19.3.908-911.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Rimstad E., Hornes E., Olsvik O., Hyllseth B. Identification of a double-stranded RNA virus by using polymerase chain reaction and magnetic separation of the synthesized DNA segments. J Clin Microbiol. 1990 Oct;28(10):2275–2278. doi: 10.1128/jcm.28.10.2275-2278.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Rönnberg B., Söderlind O., Wadström T. Evaluation of a competitive enzyme-linked immunosorbent assay for porcine Escherichia coli heat-stable enterotoxin. J Clin Microbiol. 1985 Dec;22(6):893–896. doi: 10.1128/jcm.22.6.893-896.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Saiki R. K., Gelfand D. H., Stoffel S., Scharf S. J., Higuchi R., Horn G. T., Mullis K. B., Erlich H. A. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science. 1988 Jan 29;239(4839):487–491. doi: 10.1126/science.2448875. [DOI] [PubMed] [Google Scholar]
  19. Skjerve E., Rørvik L. M., Olsvik O. Detection of Listeria monocytogenes in foods by immunomagnetic separation. Appl Environ Microbiol. 1990 Nov;56(11):3478–3481. doi: 10.1128/aem.56.11.3478-3481.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Sommerfelt H., Svennerholm A. M., Kalland K. H., Haukanes B. I., Bjorvatn B. Comparative study of colony hybridization with synthetic oligonucleotide probes and enzyme-linked immunosorbent assay for identification of enterotoxigenic Escherichia coli. J Clin Microbiol. 1988 Mar;26(3):530–534. doi: 10.1128/jcm.26.3.530-534.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Svennerholm A. M., Wikström M., Lindblad M., Holmgren J. Monoclonal antibodies against Escherichia coli heat-stable toxin (STa) and their use in a diagnostic ST ganglioside GM1-enzyme-linked immunosorbent assay. J Clin Microbiol. 1986 Oct;24(4):585–590. doi: 10.1128/jcm.24.4.585-590.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Wahlberg J., Lundeberg J., Hultman T., Uhlén M. General colorimetric method for DNA diagnostics allowing direct solid-phase genomic sequencing of the positive samples. Proc Natl Acad Sci U S A. 1990 Sep;87(17):6569–6573. doi: 10.1073/pnas.87.17.6569. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Journal of Clinical Microbiology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES