Abstract
Contamination of polymerase chain reaction (PCR) reagents continues to be a major problem when consensus primers are used for detection of low concentrations of bacterial DNA. We designed a real-time polymerase chain reaction (PCR) for quantification of bacterial DNA by using consensus primers that bind specifically to the 16S region of bacterial DNA. We have tested four different methods of decontamination of PCR reagents in a project aimed at detecting bacterial DNA at low concentrations: deoxyribonuclease (DNAse) treatment, restriction endonuclease digestion, UV irradiation, and 8-methoxypsoralen in combination with long-wave UV light to intercalate contaminating DNA into double-stranded DNA. All four methods result in inhibition of the PCR reaction, and most of the decontamination procedures failed to eliminate the contaminating bacterial DNA. Only the DNAse decontamination proved to be efficient in eliminating contaminating DNA while conserving PCR efficiency. All four decontamination methods are time consuming and have the possibility of carrying new contamination into the reaction mixture. However, decontamination with DNAse may help, together with the use of highly purified PCR reagents, in detecting small amounts of bacterial DNA in clinical specimens.
Similar content being viewed by others
References
Medlin, L., Elwood, H. J., Stickel, S., and Sogin, M. L. (1988) The characterization of enzymatically amplified eukaryotic 16S-like rRNA-coding regions. Gene 71, 491–499.
Gutell, R. R., Larsen, N., and Woese, C. R. (1994) Lessons from an evolving rRNA: 16S and 23S rRNA structures from a comparative perspective. Microbiol Rev 58, 10–26.
Woese, C. R. (1987) Bacterial evolution. Microbiol Rev 51, 221–271.
Bottger, E. C. (1989) Rapid determination of bacterial ribosomal RNA sequences by direct sequencing of enzymatically amplified DNA. FEMS Microbiol Lett 53, 171–176.
Chen, K., Neimark, H., Rumore, P., and Steinman, C. R. (1989) Broad range DNA probes for detecting and amplifying eubacterial nucleic acids. FEMS Microbiol Lett 48, 19–24.
Wilson, K. H., Blitchington, R. B., and Greene, R. C. (1990) Amplification of bacterial 16S ribosomal DNA with polymerase chain reaction. J Clin Microbiol 28, 1942–1946.
Barry, T., Powell, R. and Gannon, F. (1990) A general method to generate DNA probes for microorganisms. Biotechnology (N Y) 8, 233–236.
Greisen, K., Loeffelhoz, M., Purohit, A., & Leong, D. (1994) PCR primers and probes for the 16S rRNA gene of most species of pathogenic bacteria, including bacteria found in cerebrospinal fluid. J Clin Microbiol 32, 335–351.
Bottger, E. C. (1990) Frequent contamination of Taq polymerase with DNA. Clin Chem 36, 1258–1259.
Kwok, S. and Higuchi, R. (1989) Avoiding false positives with PCR. Nature 339, 237–238.
Rand, K. H. and Houck, H. (1990) Taq polymerase contains bacterial DNA of unknown origin. Mol Cell Probes 4, 445–450.
Schmidt, T. M., Pace, B., and Pace, N. R. (1991) Detection of DNA contamination in Taq polymerase. Biotechniques 11, 176–177.
Carroll, N. M., Adamson, P., and Okhravi, N. (1999) Elimination of bacterial DNA from Taq DNA polymerases by restriction endonuclease digestion. J Clin Microbiol 37, 3402–3404.
Corless, C. E., Guiver, M., Borrow, R., Edwards-Jones, V., Kaczmarski, E. B., and Fox, A. J. (2000) Contamination and sensitivity issues with a real-time universal 16S rRNA PCR. J Clin Microbiol 38, 1747–1752.
DeFilippes, F. M. (1991) Decontaminating the polymerase chain reaction. Biotechniques 10, 26, 28, 30.
Hilali, F., Saulnier, P., Chachaty, E., and Andremont, A. (1997) Decontamination of polymerase chain reaction reagents for detection of low concentrations of 16S rRNA genes. Mol Biotechnol. 7, 207–216.
Hughes, M. S., Beck, L. A., and Skuce, R. A. (1994) Identification and elimination of DNA sequences in Taq DNA polymerase. J Clin Microbiol 32, 2007–2008.
Jinno, Y., Yoshiura, K., and Niikawa, N. (1990) Use of psoralen as extinguisher of contaminated DNA in PCR. Nucleic. Acids. Res 18, 6739.
Klausegger, A., Hell, M., Berger, A., et al. (1999) Gram type-specific broad-range PCR amplification for rapid detection of 62 pathogenic bacteria. J Clin Microbiol 37, 464–466.
Meier, A., Persing, D. H., Finken, M., and Bottger, 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.
Ou, C. Y., Moore, J. L., and Schochetman, G. (1991) Use of UV irradiation to reduce false positivity in polymerase chain reaction. Biotechniques 10, 442, 444, 446.
Rochelle, P. A., Weightman, A. J., and Fry, J. C. (1992) DNase I treatment of Taq DNA polymerase for complete PCR decontamination. Biotechniques 13, 520.
Sarkar, G. and Sommer, S. S. (1990) Shedding light on PCR contamination. Nature 343, 27.
Ou, C. Y., McDonough, S. H., Cabanas, D., et al. (1990) Rapid and quantitative detection of enzymatically amplified HIV-1 DNA using chemiluminescent oligonucleotide probes. AIDS Res Hum Retroviruses 6, 1323–1329.
Widjojoatmodjo, M. N., Fluit, A. C., and Verhoef, J. (1994) Rapid identification of bacteria by PCR-single-strand conformation polymorphism. J Clin Microbiol 32, 3002–3007.
Sharma, J. K., Gopalkrishna, V., and Das, B. C. (1992) A simple method for elimination of unspecific amplifications in polymerase chain reaction. Nucleic Acids Res 20, 6117–6118.
Roizes, G., Nardeux, P. C., and Monier, R. (1979) A new specific endonuclease from Anabaena variabilis. FEBS Lett 104, 39–44.
Furrer, B., Candrian, U., Wieland, P., and Luthy, J. (1990) Improving PCR efficiency. Nature 346, 324.
Mariani, B. D., Martin, D. S., Levine, M. J., Booth, R. E. J., and Tuan, R. S. (1996) The Coventry Award. Polymerase chain reaction detection of bacterial infection in total knee arthroplasty. Clin Orthop 331, 11–22.
Widjojoatmodjo, M. N., Fluit, A. C., and Verhoef, J. (1995) Molecular identification of bacteria by fluorescence-based PCR-single-strand conformation polymorphism analysis of the 16S rRNA gene. J Clin Microbiol 33, 2601–2606.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Klaschik, S., Lehmann, L.E., Raadts, A. et al. Comparison of different decontamination methods for reagents to detect low concentrations of bacterial 16S DNA by real-time-PCR. Mol Biotechnol 22, 231–242 (2002). https://doi.org/10.1385/MB:22:3:231
Issue Date:
DOI: https://doi.org/10.1385/MB:22:3:231