A Rapid Method for Extraction and Purification of DNA from Dental

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Nov. 1995, p. 4120–4123 Vol. 61, No. 11 0099-2240/95/$04.00ϩ0 Copyright ᭧ 1995, American Society for Microbiology

A Rapid Method for Extraction and Puriﬁcation of DNA from Dental Plaque

1,2 1 KENNETH D. PARRISH AND E. P. GREENBERG * Department of Microbiology, College of Medicine,1 and Department of Periodontics, College of Dentistry,2 University of Iowa, Iowa City, Iowa 52242

Received 22 May 1995/Accepted 1 September 1995

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.

Over 325 bacterial species have been cultured from dental a method for extracting nucleic acids from gram-negative bac- plaque (6). However, the use of culture techniques to study the teria in serum and urine (1), and the cell lysis is somewhat ecology of these species is problematic for several reasons: it is similar to a recently described method for lysis of bacteria in labor-intensive and time-consuming, plating efficiencies and soil samples (8). The procedure involves the thawing of frozen related problems bias the results, and not all species can be bacterial cells or plaque samples. Mutanolysin (100-␮g/ml final cultured routinely. Thus, considerable effort has been put into concentration) and lysozyme (1-mg/ml final concentration) the development of molecular techniques by which nucleic were added to each cell suspension (both enzymes were pur- acids of individual bacterial groups can be analyzed to study chased from Sigma Chemical Co., St. Louis, Mo.). After1hat the oral microflora (3–5, 11–14). The use of this molecular 37ЊC, 900 ␮l of a lysis buffer (which contained the following per genetic technology for ecological studies of oral microflora has 100 ml of 0.1 M Tris [pH 6.4]: 120 g of guanidine isothiocya- been hampered by the lack of a simple, rapid technique for nate, 22 ml of a 0.2 M EDTA solution adjusted to pH 8.0 with lysis of both gram-negative and gram-positive bacteria in sodium hydroxide, and 2.6 ml of Triton X-100) was added plaque. Such a technique should also remove substances in the together with diatomaceous earth (Celite; Sigma Chemical plaque that can interfere with further manipulations of the Co.) in a mixture prepared as described by Boom et al. (1). The DNA released upon bacterial cell lysis. We describe a tech- samples were mixed and then incubated at 23 to 25ЊC for 10 nique that appears to meet these criteria, and we present min. The samples were again mixed and then centrifuged at evidence in support of this conclusion. 12,000 ϫ g for 15 s. The DNA-containing pellet of diatoma- The bacterial species and strains used and the culture con- ceous earth was washed twice with 1 ml of guanidine isothio- ditions and media for their growth are described in Table 1. cyanate in Tris, twice with 1 ml of 70% ethanol, and once with The number of cells per milliliter in pure cultures of oral 1 ml of acetone, as described by Boom et al. (1). The pellets bacteria was determined by using a Petroff-Hausser counting were dried by incubation at 56ЊC for 10 min, and then the DNA chamber and a phase-contrast microscope. Cells were har- was eluted in 100 ␮l of TE by incubation at 56ЊC for 10 min. vested by microcentrifugation and resuspended in 100 lofTE ␮ The final solution of purified DNA was separated from the buffer (10 mM Tris and 1 mM EDTA at pH 8.0). These sam- diatomaceous earth by microcentrifugation at 12,000 ϫ g for 2 ples were frozen and stored at 20 C. Samples of dental Ϫ Њ min. To assess the utility of the DNA purification procedure, plaque were collected as follows. After patient consent was we subjected purified DNA to PCR and examined the PCR obtained, sites with probing depths of 5 mm were selected. Ͼ products as follows. For most experiments two universal 16S Supragingival plaque was removed with a sterile curette, and rDNA primers complementary to homologous sequences in subgingival plaque was then collected in a single pass with a the 16S rDNA of all eubacteria were employed (17). These are second sterile curette, starting at the most apical extent of the 5Ј-AGAGTTYGATYMTGGCT-3Ј (corresponding to nucle- periodontal pocket. The plaque samples were suspended in otides 8 to 24 in the Escherichia coli 16S rDNA), and 5Ј-AC 100 ␮l of TE and stored at Ϫ20ЊC. Subjects who had antibiotic GGHTACCTTGTTACGACTT-3Ј (corresponding to nucleo- treatment or periodontal therapy in the previous 6 months tides 1512 to 1492 in the E. coli 16S rDNA). For experiments were removed from the study. in which plaque samples were spiked with Treponema denti- The DNA purification procedure we developed is based on cola, we employed the following spirochete-specific primers: 5Ј-CACATTGGGACTGAGATAC-3Ј (nucleotides 312 to 330 in the E. coli numbering system), and 5Ј-TACCTGTTAGT * Corresponding author. Phone: (319) 335-7775. Fax: (319) 335- AACYGGCAGTAG-3Ј (spanning nucleotides 1138 to 1115 in 7949. Electronic mail address: [email protected]. the E. coli 16S rDNA) (7a). PCR (Gene-Amp kit; Perkin-

4120 VOL. 61, 1995 NOTES 4121

TABLE 1. Bacterial species and strains

Bacterial straina Description Source or referenceb Actinomyces viscosus ATCC 27044 Gram-positive, oral isolate ATCC Bacillus subtilis ATCC 168 Gram-positive, soil isolate 2 Eubacterium alactolyticum ATCC 23263 Gram-negative, oral isolate ATCC Fusobacterium nucleatum ATTC 49256 Gram-negative, oral isolate ATCC Lactobacillus casei ATTC 393 Gram-positive, oral isolate D. Drake Peptostreptococcus micros ATTC 33270 Gram-positive, oral isolate ATCC Selenomonas sputigena ATCC 35185 Gram-negative, oral isolate ATCC Staphylococcus aureus EV Gram-positive, skin isolate L. Caslavka Streptococcus mutans ATCC 25175 Gram-positive, oral isolate ATCC Treponema denticola ATCC 33521 Oral spirochete ATCC Treponema pectinovorum ATCC 33768 Oral spirochete ATCC Treponema socranskii N7A Oral spirochete P. Kolenbranderc Treponema socranskii N18B Oral spirochete P. Kolenbranderc Treponema socranskii ATCC 35536 Oral spirochete ATCC Treponema strain P2 Oral spirochete 16 Treponema strain P8 Oral spirochete 16 Treponema vincentii ATCC 35580 Oral spirochete ATCC

a With the exception of B. subtilis and S. aureus, which were grown aerobically at 37ЊC, all cultures were grown anaerobically in a Gas-Pak Plus system (BBL) at 37ЊC. Treponema species and strains were grown in GM-1 pectin medium (16); E. alactolyticum was grown in 3.7% brain heart infusion broth (Difco) plus 0.5% yeast extract (Difco) and 0.05% cysteine; P. micros was grown in 3.7% brain heart infusion broth plus 0.5% yeast extract, 0.1% Tween 80, 0.1% cellobiose, 0.1% maltose, and 0.1% starch; F. nucleatum and S. sputigena were grown in Schaedler broth (Difco); and all other bacteria were grown in a medium containing 3% Trypticase soy broth (BBL) plus 0.5% yeast extract. b ATCC, American Type Culture Collection. c The strain was from the VPI Anaerobe Laboratory culture collection.

Elmer Cetus, Norwalk, Conn.) were performed with 10 ␮lof some detail. From a PCR analysis similar to that described DNA in a total volume of 100 ␮l. Acetamide was added to a above, it was apparent that omission of enzyme treatment final concentration of 5% (9). Standard PCR techniques were greatly reduced DNA yield in all cases, while omission of employed with annealing at 42ЊC for 1 min. The PCR product freeze-thawing appreciably reduced the DNA yield from S. was subjected to agarose gel electrophoresis (2% NuSieve aga- mutans but not S. aureus or A. viscosus (Fig. 3). rose; FMC Bioproducts, Rockland, Maine). The gels were Does the procedure yield pure, high-quality DNA from den- stained with ethidium bromide (10) and examined by UV illu- tal plaque? It has been shown elsewhere that DNA extracted mination. from plaque by other procedures contains substances that can Does the procedure lyse and release DNA from both gram- interfere with PCR analysis (7). On the basis of the analysis of negative and gram-positive bacteria? We subjected a number DNA isolated from gram-negative bacteria in blood and urine of oral bacteria, including spirochetes, gram-negative bacteria, described by Boom et al. (1), we expected that DNA isolated and gram-positive bacteria, to the DNA extraction and purifi- from plaque by the procedure we describe would be free of cation procedure. In addition, we used two gram-positive bacteria that were not isolated from dental plaque (Table 1). In the first analysis, DNA was extracted and purified from 108 cells of each organism. A portion of the DNA was then am- plified by 25 cycles of PCR. Roughly equivalent amounts of PCR product were obtained regardless of species or strain (Fig. 1). This indicates that DNA was released and purified equally well regardless of the bacterial type. To more closely examine whether the procedure quantitatively released DNA from gram-positive cells, we compared the amount of PCR product obtained from different numbers of cells of Lactobacillus casei, a difficult-to-lyse gram-positive species, and T. denticola, a fragile, easily lysed organism (Fig. 2). For either organism, the amount of PCR product appeared to be proportional to the number of cells. Furthermore, for any given cell number, the amount of PCR product from L. casei was equivalent to the amount from T. denticola. This indicates FIG. 1. Ethidium bromide-stained agarose gel of PCR products obtained that the lysis and purification were equally effective with these with a variety of bacteria. DNA templates were purified from axenic bacterial two bacterial species. PCR product was detected at the lowest cultures. The PCR primers used were the eubacterial 16S rDNA primers. Lanes: cell density tested. DNA was purified from 500 cells, but only 1, negative control (no bacteria); 2, Peptostreptococcus micros;3,Treponema 10 ␮l of the 100 ␮l of purified DNA was used for the PCR. socranskii N7A; 4, Fusobacterium nucleatum;5;Treponema vincentii;6,Trepo- nema sp. strain P8; 7, A. viscosus;8,T. socranskii ATCC 35536; 9, Treponema sp. Therefore, the PCR product represented that obtained from strain P2; 10, Treponema pectinovorum; 11, Eubacterium alactolyticum; 12, Sel- 50 cells. We did not study the limits of detection with respect enomonas sputigena; 13, L. casei; 14, Bacillus subtilis; 15, S. mutans; 16, S. aureus; to bacterial numbers further. 17, T. socranskii N18B; 18, T. denticola; 19, DNA size standards (1-kb DNA We examined the effects of freeze-thawing and enzyme ladder; Bethesda Research Laboratories). The numbers on the right indicate the sizes of some of the DNA standards. For each organism, DNA was extracted and treatment on the quantity of DNA obtained from Staphylococ- purified from 108 cells. One-tenth of the total DNA purified was subjected to 25 cus aureus, Streptococcus mutans, and Actinomyces viscosus in cycles of PCR. 4122 NOTES APPL.ENVIRON.MICROBIOL.

FIG. 4. Agarose gel electrophoresis of PCR product obtained with DNA purified from spirochete-spiked and nonspiked subgingival plaque samples. Lanes: 1, 50,000 T. denticola cells from a pure culture; 2, 4, 6, 8, 10, and 12, different plaque samples without added T. denticola; 3, 5, 7, 9, 11, and 13, each of the plaque samples plus 50,000 T. denticola cells from the pure culture; 14, FIG. 2. Agarose gel electrophoresis of PCR products with DNA from differ- molecular size markers. PCR was done for 30 cycles with the spirochete 16S ent numbers of L. casei or T. denticola cells. Lanes: 1 to 3, 50,000, 5,000, and 500 rDNA primer set. The numbers on the right indicate the positions of the 500- and L. casei cells, respectively; 4 to 6, 50,000, 5,000, and 500 T. denticola cells, 1,000-bp DNA size markers. respectively; 7, negative control (no template DNA added to the PCR mixture); 8, molecular size markers. PCR was done with the eubacterial 16S rDNA primer set for 35 cycles. The numbers on the right indicate the sizes of some of the DNA standards. to be effective for purifying DNA from gram-negative bacteria in samples of blood and urine. The procedure described here adds to the array of molecular techniques for identifying oral bacteria (4, 5, 12, 13, 15). It is PCR inhibitors. To confirm this, we subjected DNA isolated now possible to obtain DNA which should be qualitatively and from plaque to PCR. Subgingival plaque samples were sus- quantitatively representative of the microflora present in an pended in 100 ␮l of TE buffer. The suspensions were divided uncultured plaque sample, and this DNA should be suitable T. denticola in half, 50,000 cells were added to one half, and no for subsequent molecular analyses. It should thus be possible addition was made to the other half. Each pair of samples was to apply molecular techniques to the study of population ecol- then subjected to the cell lysis and DNA purification proce- ogy of oral bacteria. This method for release and purification dure, and the purified DNA was used as a PCR template with of DNA from both gram-negative and gram-positive bacteria the spirochete-specific rDNA primers. Results for six plaque should have general applicability for the isolation of bacterial samples are shown in Fig. 4. We always observed a spirochete DNA from other complex bacterial assemblages, such as fecal rDNA PCR product, and the amount of product in the samples material and rumen fluid. containing 50,000 added T. denticola cells appeared to be equivalent to the amount of product from the cognate unadul- We thank Ercole Canale-Parola, Lynne Caslavka, David Drake, terated plaque samples plus the amount of product from DNA Paul Kolenbrander, and Donald Staley for providing cultures, and we purified from 50,000 cells from a pure culture of T. denticola thank Bruce Paster and Floyd Dewhirst for helpful advice and com- (Fig. 4). This is consistent with our expectation that the DNA ments. we purified from subgingival plaque is of high quality and free Support for this research was provided by a grant from the National of substances that interfere with PCR analysis. Institutes of Health (DE10730). K.D.P. was a Dental Scientist Trainee In summary, we have described a cell lysis and DNA puri- supported by U.S. Public Health Service Training Grant DE00175 fication procedure that appears to be suitable for obtaining from the National Institute of Dental Research. high-quality DNA from both gram-positive and gram-negative REFERENCES bacteria in uncultured dental plaque. The procedure is rapid 1. Boom, R., C. J. A. Sol, M. M. M. Salimans, C. L. Jansen, P. M. E. Wertheim- and convenient: multiple samples can be processed in under 2 van Dillen, and J. van der Noordaa. 1990. Rapid and simple method for h, and the complete lysis, extraction, and purification proce- purification of nucleic acids. J. Clin. 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