Abstract
A rapid method based on previously described DNA extraction procedures was developed for the isolation of DNA from dental plaque samples. The isolated DNA is suitable for use in the PCR. Freeze-thawing, cell wall-degrading enzymes, and guanidine isothiocyanate were used to lyse cells and release DNA. The released DNA was adsorbed onto diatomaceous earth and purified by washing with guanidine isothiocyanate, ethanol, and acetone. The purified DNA was released from the diatomaceous earth into an aqueous buffer and analyzed by PCR with 16S rDNA primers (rDNA is DNA coding for rRNA). As judged from studies with pure cultures of a number of bacterial species, gram-negative and gram-positive organisms were lysed equally well by this procedure. The amount of PCR product was proportional to the number of cells analyzed over the range tested, 500 to 50,000 cells. On the basis of studies with plaque samples that were spiked with known quantities of the oral bacterium Treponema denticola, the DNA prepared from plaque was free of substances inhibitory to PCR. This method should have utility in molecular genetic studies of bacterial populations not only in uncultured plaque samples but also in other complex bacterial assemblages.
Full Text
The Full Text of this article is available as a PDF (286.9 KB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Boom R., Sol C. J., Salimans M. M., Jansen C. L., Wertheim-van Dillen P. M., van der Noordaa J. Rapid and simple method for purification of nucleic acids. J Clin Microbiol. 1990 Mar;28(3):495–503. doi: 10.1128/jcm.28.3.495-503.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Dix K., Watanabe S. M., McArdle S., Lee D. I., Randolph C., Moncla B., Schwartz D. E. Species-specific oligodeoxynucleotide probes for the identification of periodontal bacteria. J Clin Microbiol. 1990 Feb;28(2):319–323. doi: 10.1128/jcm.28.2.319-323.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Loesche W. J., Lopatin D. E., Stoll J., van Poperin N., Hujoel P. P. Comparison of various detection methods for periodontopathic bacteria: can culture be considered the primary reference standard? J Clin Microbiol. 1992 Feb;30(2):418–426. doi: 10.1128/jcm.30.2.418-426.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Moncla B. J., Braham P. H., Persson G. R., Page R. C., Weinberg A. Direct detection of Porphyromonas gingivalis in Macaca fascicularis dental plaque samples using an oligonucleotide probe. J Periodontol. 1994 May;65(5):398–403. doi: 10.1902/jop.1994.65.5.398. [DOI] [PubMed] [Google Scholar]
- Moore W. E., Holdeman L. V., Cato E. P., Smibert R. M., Burmeister J. A., Palcanis K. G., Ranney R. R. Comparative bacteriology of juvenile periodontitis. Infect Immun. 1985 May;48(2):507–519. doi: 10.1128/iai.48.2.507-519.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Nguyen A. M., Engstrand L., Genta R. M., Graham D. Y., el-Zaatari F. A. Detection of Helicobacter pylori in dental plaque by reverse transcription-polymerase chain reaction. J Clin Microbiol. 1993 Apr;31(4):783–787. doi: 10.1128/jcm.31.4.783-787.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Porteous L. A., Armstrong J. L., Seidler R. J., Watrud L. S. An effective method to extract DNA from environmental samples for polymerase chain reaction amplification and DNA fingerprint analysis. Curr Microbiol. 1994 Nov;29(5):301–307. doi: 10.1007/BF01577445. [DOI] [PubMed] [Google Scholar]
- Reysenbach A. L., Giver L. J., Wickham G. S., Pace N. R. Differential amplification of rRNA genes by polymerase chain reaction. Appl Environ Microbiol. 1992 Oct;58(10):3417–3418. doi: 10.1128/aem.58.10.3417-3418.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Savitt E. D., Strzempko M. N., Vaccaro K. K., Peros W. J., French C. K. Comparison of cultural methods and DNA probe analyses for the detection of Actinobacillus actinomycetemcomitans, Bacteroides gingivalis, and Bacteroides intermedius in subgingival plaque samples. J Periodontol. 1988 Jul;59(7):431–438. doi: 10.1902/jop.1988.59.7.431. [DOI] [PubMed] [Google Scholar]
- Smith G. L., Sansone C., Socransky S. S. Comparison of two methods for the small-scale extraction of DNA from subgingival microorganisms. Oral Microbiol Immunol. 1989 Sep;4(3):135–140. doi: 10.1111/j.1399-302x.1989.tb00240.x. [DOI] [PubMed] [Google Scholar]
- Smith G. L., Socransky S. S., Smith C. M. Rapid method for the purification of DNA from subgingival microorganisms. Oral Microbiol Immunol. 1989 Mar;4(1):47–51. doi: 10.1111/j.1399-302x.1989.tb00406.x. [DOI] [PubMed] [Google Scholar]
- Tay F., Liu Y. B., Flynn M. J., Slots J. Evaluation of a non-radioactive DNA probe for detecting Porphyromonas gingivalis in subgingival specimens. Oral Microbiol Immunol. 1992 Dec;7(6):344–348. doi: 10.1111/j.1399-302x.1992.tb00634.x. [DOI] [PubMed] [Google Scholar]
- Watanabe K., Frommel T. O. Detection of Porphyromonas gingivalis in oral plaque samples by use of the polymerase chain reaction. J Dent Res. 1993 Jun;72(6):1040–1044. doi: 10.1177/00220345930720060801. [DOI] [PubMed] [Google Scholar]
- Weber F. H., Canale-Parola E. Pectinolytic enzymes of oral spirochetes from humans. Appl Environ Microbiol. 1984 Jul;48(1):61–67. doi: 10.1128/aem.48.1.61-67.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Weisburg W. G., Barns S. M., Pelletier D. A., Lane D. J. 16S ribosomal DNA amplification for phylogenetic study. J Bacteriol. 1991 Jan;173(2):697–703. doi: 10.1128/jb.173.2.697-703.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]