Molecular Biotechnology

, Volume 7, Issue 3, pp 207–216

Decontamination of polymerase chain reaction reagents for detection of low concentrations of 16S rRNA genes

  • Farida Hilali
  • Patrick Saulnier
  • Elisabeth Chachaty
  • Antoine Andremont
Research

Abstract

We describe a polymerase chain reaction (PCR) that allowed detection of rRNA consensus sequences from the DNA extracted from a wide range of bacterial species in amounts as low as 10 fg. To avoid false-positive results with universal primers for 16S rRNA PCR, contaminating DNA had to be eliminated from the polymerase preparations. Decontamination was undertaken before PCR to optimize treatment with DNase I and was followed by DNase inactivation at 94°C for 50 min, which eliminated contaminating DNA at concentrations of up to 100 pg. After optimization of PCR conditions for each polymerase, Deep-Vent Exo-®polymerase (New England Biolabs, Beverly, MA), and super-Taq® polymerase (HT Biotechnology, Cambridge, UK) were more effective than Ampli-Taq® polymerase (Perkin-Elmer Cetus, Norwalk, CT), Ampli-Taq LD® polymerase (Perkin-Elmer Cetus) or Deep-vent® polymerase (New England Biolabs). The technique described in this article might prove to be a universal method for PCR detection of small numbers of unidentified bacteria in usually sterile clinical sites, such as blood and cerebrospinal fluids, in which a broad spectrum of pathogens can be expected.

Index Entries

PCR polymerase DNase 16S rRNA sequences bacteria 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. 1.
    Medlin, L. H., Elwood, J., Stickel, S., and Sogin, M. L. (1988) The characterization of enzymatically amplified eukaryotic 16S-like rRNA-coding regions.Gene 71, 491–499.PubMedCrossRefGoogle Scholar
  2. 2.
    Woese, C. R. (1987) Bacterial evolution.Microbiol. Rev. 51, 221–271.PubMedGoogle Scholar
  3. 3.
    Lane, D. J., Pace, B., Olsen, G. J., Stahl, D. A., Sogin, M. L., and Pace, N. R. (1985) Rapid determination of 16S ribosomal RNA sequences for phylogenetic analyses.Proc. Natl. Acad. Sci. USA 82, 6955–6959.PubMedCrossRefGoogle Scholar
  4. 4.
    Böttger, E. C. (1989) Rapid determination of bacterial ribosomal RNA sequences by direct sequencing of enzymatically amplified DNA.FEMS Microbiol. Lett. 65, 171–176.CrossRefGoogle Scholar
  5. 5.
    Weisburg, W. G., Barns, S. M., Pelletier, D. A., and Lane, D. J. (1991) 16S ribosomal DNA amplification for phylogenetic study.J. Bacteriol. 173, 697–703.PubMedGoogle Scholar
  6. 6.
    Wilson, K. H., Blitchington, R., and Greene, R. C. (1990) Amplification of bacterial 16S ribosomal DNA with polymerase chain reaction.J. Clin. Microbiol. 28, 1942–1946.PubMedGoogle Scholar
  7. 7.
    Relman, D. A., Loutit, J. S., Schmidt, T. M., Falkow, S., and Tompkins, L. S. (1990) The agent of bacillary angiomatosis. An approach to the identification of uncultured pathogens.N. Engl. J. Med. 323, 1573–1583.PubMedCrossRefGoogle Scholar
  8. 8.
    Relman, D. A., Schmidt, T. M., MacDermott, R. P., and Falkow, S. (1992) Identification of uncultured Bacillus of Whipple's Disease.N. Engl. J. Med. 323, 293–301.CrossRefGoogle Scholar
  9. 9.
    Kwok, S. and Higuchi, R. (1989). Avoiding false positives with PCR.Nature (Lond.) 339, 237, 238.CrossRefGoogle Scholar
  10. 10.
    Böttger, E. C. (1990) Frequent contamination of Taq polymerase with DNA.Clin. Chem. 36, 1258, 1259.PubMedGoogle Scholar
  11. 11.
    Schmidt, T. M., Pace, B., and Pace, N. R. (1991) Detection of DNA contamination in Taq polymerase.BioTechniques 11, 176,177.PubMedGoogle Scholar
  12. 12.
    Meier, A., Persing, D. H., Finken, M., and Böttger, E. C. (1993) Elimination of contaminating DNA within polymerase chain reaction reagents: implications for a general approach to detection of uncultured pathogens.J. Clin. Microbiol. 31, 646–652.PubMedGoogle Scholar
  13. 13.
    Rys, P. N. and Persing, D. H. (1993) Preventing false positives: quantitative evaluation of three protocols for inactivation of polymerase chain reaction amplification products.J. Clin. Microbiol. 31, 2356–2360.PubMedGoogle Scholar
  14. 14.
    Widjojoatmodjo, M. N., Fluit, A. D. C., and Verhoef, J. (1994) Rapid identification of bacteria by PCR-single-strand conformation polymorphism.J. Clin. Microbiol. 32, 3002–3007.PubMedGoogle Scholar
  15. 15.
    Widjojoatmodjo, M. N., Fluit, A. D. C., and Verhoef, J. (1995) Molecular identification of bacteria by fluorescent-based PCR-single-strand conformation polymorphism analysis of the 16S rRNA gene.J. Clin. Microbiol. 33, 2601–2606.PubMedGoogle Scholar
  16. 16.
    Sahn, D. F., Kissinger, J., Gilmore, M. S., Murray, P. R., Mulder, R., Solliday, J., and Clarke, B. (1989) In vitro susceptibility studies of vancomycin-resistantEnterococcus faecalis.Antimicrob. Agents Chemother. 33, 1588–1591.Google Scholar
  17. 17.
    Dutka-Malen, S., Leclercq, R., Coutant, V., Duval, J., and Courvalin, P. (1990) Phenotypic and genotypic heterogeneity of glycopeptide resistance determinants in Gram-positive bacteria.Antimicrob. Agents Chemother. 34, 1875–1879.PubMedGoogle Scholar
  18. 18.
    Raleigh, E. A., Murray, N. E., Revel, H., Blumenthal, R. M., Westaway, D., Reith, A. D., Rigby, P. W. J., Ethai, J., and Hanahan, D. (1987) Mcr A and Mcr B restriction phenotypes of someE. coli strains and implications for gene cloning.Nucleic Acids Res. 16, 1563–1575.CrossRefGoogle Scholar
  19. 19.
    Böddinghaus, B., Rogall, T., Flohr, T., Blöcker, H., and Böttger, E. C. (1990) Detection and identification of mycobacteria by amplification of rRNA.J. Clin. Microbiol. 28, 1751–1759.PubMedGoogle Scholar
  20. 20.
    Boom, R., Sol, C. J. A., Salimans, M. M. M., Jansen, C. L., Wertheim-Van Dillen, P. M. E., and Van der Noordaa, J. (1990) Rapid and simple method for purification of nucleic acids.J. Clin. Microbiol. 28, 495–503.PubMedGoogle Scholar
  21. 21.
    Manchester, K. L. (1996) Use of UV methods for measurement of protein and nucleic acid concentrations.Biotechniques 20, 968–970.PubMedGoogle Scholar
  22. 22.
    Edwards, U., Rogall, T., Blöcker, H., Emde, M., and Böttger, E. C. (1989) Isolation and direct complete nucleotide determination of entire genes. Characterization of a gene coding for 16S ribosomal RNA.Nucleic Acids Res. 17, 7843–7853.PubMedCrossRefGoogle Scholar
  23. 23.
    Brosius, J., Palmer, M. L., Kennedy, P. J., and Noller, H. F. (1978) Complete nucleotide sequence of a 16S Ribosomal RNA gene fromEscherichia coli.Proc. Natl. Acad. Sci. USA 75, 4801–4805.PubMedCrossRefGoogle Scholar
  24. 24.
    Erlich, H. A. (ed.) (1989)PCR Technology, Principles and Applications for DNA amplification. Stockton, England.Google Scholar
  25. 25.
    Chevet, E., Lemaître, G., and Katinka, M. D. (1995) Low concentrations of tetramethylammonium chloride increase yield and specificity of PCR.Nucleic Acids Res. 23, 3343, 3344.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc 1997

Authors and Affiliations

  • Farida Hilali
    • 1
  • Patrick Saulnier
    • 1
  • Elisabeth Chachaty
    • 1
  • Antoine Andremont
    • 2
  1. 1.Laboratoire de Microbiologie MédicaleInstitut Gustave-RoussyVillejuifFrance
  2. 2.Laboratoire de BactériologieGroupe Hospitalier Bichat-Claude BernardParis CEDEX 18France

Personalised recommendations