Use of checkerboard DNA–DNA hybridization to study complex microbial ecosystems

It has been difficult to conduct large scale studies of microbiologically complex ecosystems using conventional microbiological techniques. Molecular identification techniques in new probe‐target formats, such as checkerboard DNA–DNA hybridization, permit enumeration of large numbers of species in very large numbers of samples. Digoxigenin‐labeled whole genomic probes to 40 common subgingival species were tested in a checkerboard hydridization format. Chemifluorescent signals resulting from the hybridization reactions were quantified using a Fluorimager and used to evaluate sensitivity and specificity of the probes. Sensitivity of the DNA probes was adjusted to detect 10⁴ cells. In all, 93.5% of potential cross‐reactions to 80 cultivable species exhibited signals <5% of that detected for the homologous probe signal. Competitive hybridization and probes prepared by subtraction hybridization and polymerase chain reaction were effective in minimizing cross‐reactions for closely related taxa. To demonstrate utility, the technique was used to evaluate 8887 subgingival plaque samples from 79 periodontally healthy and 272 chronic periodontitis subjects and 8126 samples from 166 subjects taken prior to and after periodontal therapy. Significant differences were detected for many taxa for mean counts, proportion of total sample, and percentage of sites colonized between samples from periodontally healthy and periodontitis subjects. Further, significant reductions were observed post therapy for many subgingival species including periodontal pathogens. DNA probes used in the checkerboard DNA–DNA format provide a useful tool for the enumeration of bacterial species in microbiologically complex systems.     

Use of checkerboard DNA-DNA hybridization to study complex microbial ecosystems

It has been difficult to conduct large scale studies of microbiologically complex ecosystems using conventional microbiological techniques. Molecular identification techniques in new probe-target formats, such as checkerboard DNA-DNA hybridization, permit enumeration of large numbers of species in very large numbers of samples. Digoxigenin-labeled whole genomic probes to 40 common subgingival species were tested in a checkerboard hydridization format. Chemifluorescent signals resulting from the hybridization reactions were quantified using a Fluorimager and used to evaluate sensitivity and specificity of the probes. Sensitivity of the DNA probes was adjusted to detect 10(4) cells. In all, 93.5% of potential cross-reactions to 80 cultivable species exhibited signals <5% of that detected for the homologous probe signal. Competitive hybridization and probes prepared by subtraction hybridization and polymerase chain reaction were effective in minimizing cross-reactions for closely related taxa. To demonstrate utility, the technique was used to evaluate 8887 subgingival plaque samples from 79 periodontally healthy and 272 chronic periodontitis subjects and 8126 samples from 166 subjects taken prior to and after periodontal therapy. Significant differences were detected for many taxa for mean counts, proportion of total sample, and percentage of sites colonized between samples from periodontally healthy and periodontitis subjects. Further, significant reductions were observed post therapy for many subgingival species including periodontal pathogens. DNA probes used in the checkerboard DNA-DNA format provide a useful tool for the enumeration of bacterial species in microbiologically complex systems.     

Multiple displacement amplification as an aid in checkerboard DNA–DNA hybridization

Objective: The study aimed to determine if multiple displacement amplification could be used to provide abundant target DNA and DNA probes for checkerboard DNA–DNA hybridization. Methods: Multiple displacement amplification was used to amplify 1 and 10 ng DNA from 16 individual bacterial species, DNA from single colonies, from a mixture of 20 bacterial species and oral biofilm samples, such as supragingival plaque, subgingival plaque, buccal swab and root canal samples. Samples in reaction buffer were heat‐denatured at 95°C for 3 min and cooled to 4°C. Φ29 DNA polymerase was added and the mixture was incubated at 30°C for 16–18 h. The quantity of the product was evaluated by the Picogreen assay. The amplified material was labeled with digoxigenin. The probes were compared with probes obtained from unamplified DNA using checkerboard DNA–DNA hybridization. Both amplified DNA and unamplified DNA were used as targets on the membrane. Amplified oral biofilm samples were compared to unamplified samples using checkerboard DNA–DNA hybridization. Results: The DNA yield ranged from 4 to 11 μg. DNA–DNA hybridization showed that the amplified genome of each species used either as target or as probe provided signals equivalent to controls and that amplification of a mixture of species provided signals comparable to those provided by the unamplified source mixture. Amplified oral biofilm samples exhibited comparable proportions of bacterial DNA when compared to the original unamplified samples. Conclusions: The multiple displacement amplification technique is a simple and reliable method to uniformly amplify DNA for use in checkerboard DNA–DNA hybridization. It is also a useful tool in the amplification of clinical samples.     

Non-isotopic DNA probes for the identification of subgingival microorganisms

The purpose of the present investigation was to examine the potential of non-isotopic DNA probes to identify pure cultures in predominant cultivable microbiota studies. Non-isotopic DNA probes to 7 subgingival species were prepared by 2 methods. In the first, biotin-labelled probes were prepared by nick translation. In the second, single-stranded DNA was covalently linked to horseradish peroxidase via polyethyleneimine. The relative sensitivities and specificities of these probes were tested against pure cultures of a range of subgingival species. Aliquots of broth cultures were standardized by optical densities, placed on nitrocellulose or Whatman 541 filters and then treated to lyse the cells, denature and fix DNA to the filter. Using a streptavidin-alkaline phosphatase detection system, 10(4)-10(5) cells were detected by homologous biotin-labelled probes. Horseradish peroxidase-labelled probes were approximately one order of magnitude less sensitive. Non-specific reactions with unrelated species, displayed by both biotin- and horseradish peroxidase-labelled probes, were eliminated by treatment of filters with proteinase K and organic solvents. Cross-reactions between closely related species could be discriminated by comparing reaction intensities of the test strains with probes to the each of the cross-reacting species involved. Thus, nonisotopic DNA probes could be used for the rapid identification of subgingival isolates. The technique could also be used for the recognition and grouping of strains of unknown species. A probe made to the strain of an unknown species may be used to rapidly screen hundred of unknown isolates for related strains.     

Comparison of two methods for the small-scale extraction of DNA from subgingival microorganisms

Two methods were compared for the extraction of DNA from small numbers of bacterial cells. The first method involved lysis of cells with SDS in the presence of proteinase K, treatment with hexadecyltrimethyl ammonium bromide (CTAB) and precipitation of DNA with isopropanol. In the second method, DNA was extracted by treatment of the cells with guanidine hydrochloride (GHCl) and precipitated with ethanol. Thirty strains of representative gram positive and gram negative species were included in the study. Preparations derived from confluent growth on one-quarter of the surface of agar plates and from 10(8) cells were subjected to each extraction procedure and analyzed for their content of DNA, RNA and protein. The suitabilities of the resultant DNA for restriction enzyme digestion and biotin-labelling by a random primer technique were also assessed. In general, the CTAB method yielded greater amounts of DNA than the GHCl procedure. RNA was present in most preparations of both types, but in amounts detectable only by agarose gel electrophoresis. The latter technique also revealed that DNA was not excessively sheared by either procedure. Protein was detected in some CTAB and GHCl preparations, but was not consistently associated with one or the other method. DNA obtained by both methods could be digested by the restriction enzyme EcoR I. In addition, biotin-labelled DNA probes prepared from CTAB and GHCl preparations were capable of hybridizing with homologous target DNA fixed to nitrocellulose. Since the CTAB method was consistently successful in recovering DNA from preparations containing 10(8) cells, it may be more suitable for the direct treatment of single colonies taken from primary isolation plates or plaque samples.     

Multiple displacement amplification as an aid in checkerboard DNA-DNA hybridization

OBJECTIVE: The study aimed to determine if multiple displacement amplification could be used to provide abundant target DNA and DNA probes for checkerboard DNA-DNA hybridization. METHODS: Multiple displacement amplification was used to amplify 1 and 10 ng DNA from 16 individual bacterial species, DNA from single colonies, from a mixture of 20 bacterial species and oral biofilm samples, such as supragingival plaque, subgingival plaque, buccal swab and root canal samples. Samples in reaction buffer were heat-denatured at 95 degrees C for 3 min and cooled to 4 degrees C. Phi29 DNA polymerase was added and the mixture was incubated at 30 degrees C for 16-18 h. The quantity of the product was evaluated by the Picogreen assay. The amplified material was labeled with digoxigenin. The probes were compared with probes obtained from unamplified DNA using checkerboard DNA-DNA hybridization. Both amplified DNA and unamplified DNA were used as targets on the membrane. Amplified oral biofilm samples were compared to unamplified samples using checkerboard DNA-DNA hybridization. RESULTS: The DNA yield ranged from 4 to 11 microg. DNA-DNA hybridization showed that the amplified genome of each species used either as target or as probe provided signals equivalent to controls and that amplification of a mixture of species provided signals comparable to those provided by the unamplified source mixture. Amplified oral biofilm samples exhibited comparable proportions of bacterial DNA when compared to the original unamplified samples. CONCLUSIONS: The multiple displacement amplification technique is a simple and reliable method to uniformly amplify DNA for use in checkerboard DNA-DNA hybridization. It is also a useful tool in the amplification of clinical samples.     

Comparison of the microbiota of supra- and subgingival plaque in health and periodontitis

BACKGROUND, AIMS: The purpose of the present investigation was to compare the microbial composition of supra and subgingival plaque in 22 periodontally healthy (mean age 32+/-16 years) and 23 adult periodontitis subjects (mean age 51+/-14 years). METHODS: A total of 2358 supra and separately subgingival plaque samples were collected from the mesial aspect of all teeth excluding 3rd molars in each subject. Samples were examined for the presence and levels of 40 bacterial taxa using whole genomic DNA probes and checkerboard DNA-DNA hybridization. Clinical assessments including dichotomous measures of gingival redness, bleeding on probing, plaque accumulation and suppuration, as well as duplicate measures of pocket depth and attachment level, were made at 6 sites per tooth. Mean counts (x10(5), % DNA probe count and % sites colonized for each species were determined separately for supra and subgingival samples in each subject and then averaged across subjects in the 2 clinical groups. Significance of differences between healthy and periodontitis subjects was determined using the Mann-Whitney test and adjusted for multiple comparisons. RESULTS: Mean total DNA probe counts (x10(5), +/-SEM) for healthy and periodontitis subjects in supragingival plaque were 72.1+/-11 and 132+/-17.5, respectively (p<0.01), and in subgingival plaque 22.1+/-6.6 and 100.3+/-18.4, (p<0.001). Porphyromonas gingivalis, Bacteroides forsythus and Treponema denticola could be detected in supragingival plaque samples of both healthy and periodontitis subjects. Actinomyces species were the dominant taxa in both supra- and subgingival plaque from healthy and periodontitis subjects. 4 Actinomyces species accounted for 63.2%, of supragingival and 47.2% of subgingival plaque in healthy subjects and 48.% and 37.8% in periodontitis subjects respectively. Increased proportions of P. gingivalis, B. forsythus, and species of Prevotella, Fusobacterium, Campylobacter and Treponema were detected subgingivally in the periodontitis subjects. P. gingivalis, B. forsythus and T. denticola were significantly more prevalent in both supra- and subgingival plaque samples from periodontitis subjects. CONCLUSIONS: The main differences between supra and subgingival plaque as well as between health and disease were in the proportions and to some extent levels of Actinomyces, "orange" and "red" complex species.     

Rapid method for the purification of DNA from subgingival microorganisms

A method is described which facilitates the rapid purification of high molecular weight chromosomal DNA from gram positive and gram negative bacteria grown on solid media. A total of 32 reference strains and fresh isolates were examined in this study. The purification procedure involved lysis of cells with SDS in the presence of proteinase K, followed by removal of cellular polysaccharides and proteins with hexadecyltrimethyl ammonium bromide (CTAB) and phenol:chloroform:isoamyl alcohol. Preparations were incubated with RNase and, after removal of the enzyme, DNA was precipitated with ethanol. Several hundred micrograms of DNA could be prepared within 5 h from cells grown on 1-2 agar plates. None of the final preparations contained RNA; protein was detected in 12/32 preparations. The resultant DNA proved suitable for restriction enzyme digestion and biotin-labelling by a random primer technique. DNA probes constructed from these preparations were capable of detecting 100 pg of homologous target DNA fixed to nitrocellulose. Cross reactions between closely related species displayed weaker signal intensities than, and, thus, were easily distinguished from, true positive reactions between homologous species. DNA obtained by this procedure may also be suitable for DNA-DNA homology studies, recombinant DNA experiments and molecular fingerprinting.     

Microbial composition of supra- and subgingival plaque in subjects with adult periodontitis

Background, aims: The purpose of the present study was to compare and relate the microbial composition of supra and subgingival plaque in 23 adult periodontitis subjects (mean age 51±14 years). Methods: A total of 1,170 samples of supra and subgingival plaque were collected from the mesial aspect of every tooth (up to 28 supra and 28 subgingival samples) from each subject and evaluated for the presence and levels of 40 bacterial taxa using whole genomic DNA probes and checkerboard DNA‐DNA hybridization. Clinical assessments including dichotomous measures of gingival redness, bleeding on probing, plaque accumulation and suppuration, as well as duplicate measures of pocket depth and attachment level, were made at 6 sites per tooth. The counts (levels), % DNA probe count (proportion) and % of sites colonized (prevalence) of each species in supra and separately in subgingival plaque were computed for each subject. Significance of differences between supra and subgingival plaque for each species was sought using the Wilcoxon signed ranks test and adjusted for multiple comparisons. Results: All 40 taxa were detected in both supra and subgingival plaque. Actinomyces species were the most prevalent taxa in both habitats. 75 to 100% of supra and 62 to 100% of subgingival sites were colonized by at least one of the 5 Actinomyces species. Supragingival samples exhibited significantly higher counts of Actinomyces naeslundii genospecies 1, Actinomyces israelii, Actinomyces odontolyticus, Neisseria mucosa, Streptococcus gordonii, Capnocytophaga ochracea and Capnocytophaga sputigena when compared with mean counts in subgingival samples taken from the same tooth surfaces. Subgingival plaque samples presented significantly higher counts of Prevotella nigrescens, Prevotella intermedia, Bacteroides forsythus and Porphyromonas gingivalis. Subgingival samples exhibited a significantly higher proportion of “red” and “orange complex” species, while supragingival plaque exhibited higher proportions of “green” and “purple” complex species as well as Actinomyces species. Suspected periodontal pathogens could be detected in supragingival plaque from sites where subgingival samples were negative for the same species. Conclusions: The data indicate that supragingival plaque can harbor putative periodontal pathogens, suggesting a possible rôle of this environment as a reservoir of such species for the spread or reinfection of subgingival sites.     

Absence of Helicobacter pylori in subgingival samples determined by polymerase chain reaction

The polymerase chain reaction was used for the detection of Helicobacter pylori from subgingival plaque in 336 periodontitis patients. A pair of primers derived from the H. pylori urease gene A served to amplify a targeted 411-bp fragment of genomic DNA. This technique permitted the detection of as few as 60 H. pylori cells. Paper point samples from 3 deep periodontal pockets per patient were immersed in 1 ml of phosphate-buffered saline or distilled water, DNA was solubilized by detergent/protease method, 3.7 μl or 37 μl of lysate supernatant was used as template, and the amplification product was analyzed in 1% agarose gel containing ethidium bromide. Each experiment included purified DNA and cell lysate of H. pylori as positive controls. The presence of bacteria in the sample was verified by a primer pair common to prokaryote 16S rRNA. The present study did not reveal the specific polymerase chain reaction amplification product characteristic of H. pylori. We conclude that periodontal pockets do not constitute a natural reservoir for H. pylori.     

"Reverse" DMA hybridization method for the rapid identification of subgingival microorganisms

A "reverse" hybridization method is described, in which whole chromosomal DNA was extracted from 10-20 colonies of "unknown" strains in pure culture and labelled with digoxigenin by a random primer technique. DNA probes were prepared from a total of 23 strains and hybridized with targets containing 100 ng purified, denatured DNA from 38 reference strains fixed to nitrocellulose. 21/23 digoxigenin-labelled DNA probes successfully detected all members of the homologous species present on filters. Probes to Fusobacterium nucleatum strains 364 and MG detected 3/4 and 1/4 members of this species, respectively; 13/23 probes were 100% specific, but cross reactions between 10 probes and DNA targets from closely related, heterologous species occurred in 15/834 possible instances. False-positive reactions that occurred between closely related species were, however, easily distinguished and did not prevent the accurate identification of probe strains. Digoxigenin-labelled probes were capable of detecting 100 pg of homologous DNA. The reverse hybridization procedure allows identification or grouping of a large number of isolates within 3 days and provides a more economical means of characterizing subgingival isolates than predominant cultivable techniques and conventional phenotypic testing. This method could be adapted for the direct identification of microorganisms in subgingival plaque samples.     

Evaluation of the microbiota of primary endodontic infections using checkerboard DNA–DNA hybridization

Background/aims: The aim of this study was to evaluate the composition of the microbiota of primary endodontic infections in 111 selected cases of single‐rooted teeth with necrotic pulp. Methods: Samples were collected from the root canals using #15 Hedströen‐type files and two sterile paper points, which were introduced 1 mm short of the apical foramen. The presence, levels, and proportions of 40 different bacterial species in each sample were determined using DNA probes and checkerboard DNA–DNA hybridization techniques. Results: The mean number of species per sample was 22. Enterococcus faecalis (89.3%), Campylobacter gracilis (89.3%), Leptotrichia buccalis (89.3%), Neisseria mucosa (87.5%), Prevotella melaninogenica (86.6%), Fusobacterium nucleatum ssp. vincentii (85.7%), Eubacterium saburreum (75.9%), Streptococcus anginosus (75%), and Veillonella parvula (74.1%) were the most prevalent species. The species found in highest mean counts (over 10⁵) were F. nucleatum ssp. vincentii (13.14 × 10⁵), E. saburreum (5.67 × 10⁵), E. faecalis (5.38 × 10⁵), N. mucosa (4.19 × 10⁵), V. parvula (3.63 × 10⁵), C. gracilis (3.46 × 10⁵), Treponema socranskii (3.34 × 10⁵), Porphyromonas endodontalis (2.96 × 10⁵), Porphyromonas gingivalis (2.85 × 10⁵), Micromonas micros (2.81 × 10⁵), Prevotella nigrescens (2.68 × 10⁵) and Fusobacterium nucleatum ssp. nucleatum (2.64 × 10⁵). Most of these species were also found in high proportions. Conclusions: Our results suggest that several bacterial species considered to be oral pathogens seem to be implicated in the etiology of primary endodontic infections.     

“Reverse” DMA hybridization method for the rapid identification of subgingival microorganisms

A “reverse” hybridization method is described, in which whole chromosomal DNA was extracted from 10-20 colonies of “unknown” strains in pure culture and labelled with digoxigenin by a random primer technique. DNA probes were prepared from a total of 23 strains and hybridized with targets containing 100 ng purified, denatured DNA from 38 reference strains fixed to nitrocellulose. 21/23 digoxigenin-labelled DNA probes successfully detected all members of the homologous species present on filters. Probes to Fusobacterium nucleatum strains 364 and MG detected 3/4 and 1/4 members of this species, respectively; 13/23 probes were 100% specific, but cross reactions between 10 probes and DNA targets from closely related, heterologous species occurred in 15/834 possible instances. False-positive reactions that occurred between closely related species were, however, easily distinguished and did not prevent the accurate identification of probe strains. Digoxigenin-labelled probes were capable of detecting 100 pg of homologous DNA. The reverse hybridization procedure allows identification or grouping of a large number of isolates within 3 days and provides a more economical means of characterizing subgingival isolates than predominant cultivable techniques and conventional phenotypic testing. This method could be adapted for the direct identification of microorganisms in subgingival plaque samples     

Comparison of the subgingival microbiota of periodontally healthy and diseased adults in Northern Cameroon

Abstract Our study is the 1st report on subgingival microbiota in adult Cameronians. The aim was to investigate, using the checkerboard DNA-DNA hybridization technique, the prevalence of 18 oral species in subgingival plaque samples obtained from sex- and age-matched Cameronian adults with and without periodontal destruction. We also compared cultivation and the Affirm^TM DP test with the checkerboard technique in their capability to detect some selected species among the 18, 21 adult periodontitis patients and 21 periodontally healthy subjects were examined and the results were compared statistically. Each periodontitis patient had at least 4 pockets of ≥ 6 mm depth, while the healthy subjects had no sites more than 3 mm deep. Results of the checkerboard analysis showed that significantly (p<0.05) more periodontitis patients tested positive for most of the 18 bacterial species. The Gram-positive species Actinomyces naeslundii, Streptococcus mitis and Streptococcus sanguis, known as microbiota of healthy sites, were detected significantly more frequently in the healthy group. Cultivation demonstrated P. gingivalis, B. forsythus, A. actinomycetemcomitans, P. intermedia and F. nucleatum in significantly lower %s of patients as compared to the checkerboard technique. Furthermore, the Affirm^TM DP test detected P. gingivalis and B. forsythus in significantly fewer patients than did the checkerboard technique. A. actinomycetemcomitans was detected in 52.3% of the patients by the latter technique while the Affirm^TM DP test failed to detect the bacterium in any of the samples. Overall, the results of the present study confirm the importance of the screening method and indicate that the prevalences of the investigated putative periodontal pathogens and beneficial species in the healthy and diseased adult Cameronians were similar to those reported for adults in the West and in some developing countries.     

Comparative analysis of endodontic pathogens using checkerboard hybridization in relation to culture

Background/Aim:  The purpose of this study was to detect bacterial species and to quantify the total number of bacteria from samples of infected root canals before (S1) and after chemo-mechanical preparation using 2% chlorhexidine (CHX) gel as auxiliary chemical substance (S2) and after 7 days of intracanal dressing (S3) to compare microbial changes.
Method:  Twenty-four teeth were selected for this study. Chemo-mechanical preparation was performed using 2% CHX gel, then three different intracanal medicaments [M1: Ca(OH)₂ paste; M2: 2% CHX gel; and M3: Ca(OH)₂ paste plus 2% CHX gel] were used for 7 days. Checkerboard DNA–DNA hybridization was performed to detect 40 bacterial species. Aerobic and anaerobic culture techniques were used to determine the bacterial community by counting the colony-forming units (CFU).
Results:  The species most frequently identified by checkerboard in S1 were: Fusobacterium nucleatum ssp . polymorphum , Treponema socranskii ssp. socranskii , Parvimonas micra and Enterococcus faecalis. In S2 and S3 a total of eight different species were identified; and only one of them was gram-positive ( E. faecalis ). Microorganisms were not identified after use of M2 for 7 days. The quantification obtained on agar plates ranged from 4 × 10⁵ to 2.6 × 10⁶ CFU/ml in S1, mean CFU was reduced by 99.96% in S2, and there was no statistical difference between the CFU in S2 and S3.
Conclusion:  The antibacterial effect of the mechanical preparation supplemented by the use of an antibacterial auxiliary substance greatly reduced the microorganisms in the main root canal.     

Evaluation of the microbiota of primary endodontic infections using checkerboard DNA-DNA hybridization

BACKGROUND/AIMS: The aim of this study was to evaluate the composition of the microbiota of primary endodontic infections in 111 selected cases of single-rooted teeth with necrotic pulp. METHODS: Samples were collected from the root canals using #15 Hedstr?en-type files and two sterile paper points, which were introduced 1 mm short of the apical foramen. The presence, levels, and proportions of 40 different bacterial species in each sample were determined using DNA probes and checkerboard DNA-DNA hybridization techniques. RESULTS: The mean number of species per sample was 22. Enterococcus faecalis (89.3%), Campylobacter gracilis (89.3%), Leptotrichia buccalis (89.3%), Neisseria mucosa (87.5%), Prevotella melaninogenica (86.6%), Fusobacterium nucleatum ssp. vincentii (85.7%), Eubacterium saburreum (75.9%), Streptococcus anginosus (75%), and Veillonella parvula (74.1%) were the most prevalent species. The species found in highest mean counts (over 10(5)) were F. nucleatum ssp. vincentii (13.14 x 10(5)), E. saburreum (5.67 x 10(5)), E. faecalis (5.38 x 10(5)), N. mucosa (4.19 x 10(5)), V. parvula (3.63 x 10(5)), C. gracilis (3.46 x 10(5)), Treponema socranskii (3.34 x 10(5)), Porphyromonas endodontalis (2.96 x 10(5)), Porphyromonas gingivalis (2.85 x 10(5)), Micromonas micros (2.81 x 10(5)), Prevotella nigrescens (2.68 x 10(5)) and Fusobacterium nucleatum ssp. nucleatum (2.64 x 10(5)). Most of these species were also found in high proportions. CONCLUSIONS: Our results suggest that several bacterial species considered to be oral pathogens seem to be implicated in the etiology of primary endodontic infections.     

Detection of Prevotella intermedia in subgingival plaque of adult periodontitis patients by polymerase chain reaction

A PCR assay was developed that could specifically amplify DNA from the periodontal pathogen Prevotella intermedia. A pair of primers was selected from regions of the 16S rRNA gene of P. intermedia that were both divergent in sequence at their 3' ends with respect to the corresponding regions of the 16S rRNA gene of P. nigreseens , its most closely related species, and used in the PCR assay. Positivity was indicated by amplification of an 855 bp product. Using purified genomic DNA from these 2 species, assay conditions were determined under which only P. intermedia DNA and not P. nigrescens DNA was amplifiable. Absolute specificity of the assay was confirmed by the fact that no amplification products were obtained when using DNA from several other important periodontal organisms. The optimized PCR assay was used to identify P. intermedia in subgingival plaque samples of patients with adult periodontitis. Confirmation of amplification of P. intermedia DNA was achieved by digestion of PCR products with the restriction endonuclease R sal, which gives different restriction patterns for P. intermedia and P. nigrescens. Of the 97 samples analysed, 38 (39%) were positive for P. intermedia. The results obtained confirm P. intermedia as a possible aetiological agent of adult periodontitis. Additionally, PCR primers targeting the corresponding region of the 16S rRNA gene of P. nigrescens were shown to be specific for the organism when used in a PCR assay, although P. nigrescens was not detectable in any of the subgingival plaques analysed.     

Detection of Prevotella intermedia in subgingival plaque of adult periodontitis patients by polymerase chain reaction

A PCR assay was developed that could specifically amplify DNA from the periodontal pathogen Prevotella intermedia. A pair of primers was selected from regions of the 16S rRNA gene of P. intermedia that were both divergent in sequence at their 3′ ends with respect to the corresponding regions of the 16S rRNA gene of P. nigrescens, its most closely related species, and used in the PCR assay. Positivity was indicated by amplification of an 855 bp product. Using purified genomic DNA from these 2 species, assay conditions were determined under which only P. intermedia DNA and not P. nigrescens DNA was amplifiable. Absolute specificity of the assay was confirmed by the fact that no amplification products were obtained when using DNA from several other important periodontal organisms. The optimized PCR assay was used to identify P. intermedia in subgingival plaque samples of patients with adult periodontitis. Confirmation of amplification of P. intermedia DNA was achieved by digestion of PCR products with the restriction endonuclease Rsal, which gives different restriction patterns for P. intermedia and P. nigrescens. Of the 97 samples analysed, 38 (39%) were positive for P. intermedia. The results obtained confirm P. intermedia as a possible aetiological agent of adult periodontitis. Additionally, PCR primers targeting the corresponding region of the 16S rRNA gene of P. nigrescens were shown to be specific for the organism when used in a PCR assay, although P. nigrescens was not detectable in any of the subgingival plaques analysed.     

Proportional distribution of the red complex and its individual pathogens after sample storage using the checkerboard DNA-DNA hybridization technique

BACKGROUND: Information on the impact of sample storage prior to analysis by DNA methods is limited. AIMS: To investigate the effect of microbial sample storage on bacterial detection and proportional distribution of the red complex and its individual pathogens. MATERIAL AND METHODS: Subgingival plaque samples were analysed by (1) immediate processing, (2) after storage at +4 degrees C for 6 weeks, (3) after storage at -20 degrees C for 6 months or (4) after storage at -20 degrees C for 12 months using the checkerboard DNA-DNA hybridization. RESULTS: Proportional distribution of the red complex did not differ between the first three protocols. However, the total bacterial DNA for pathogens studied decreased significantly in protocols 3 and 4. Relative amounts of Tannerella forsythensis, Porphyromonas gingivalis and Treponema denticola remained stable in the second protocols and changed in an unpredictable way if stored for 6 or 12 months. CONCLUSIONS: Results from samples stored for maximum 6 months at -20 degrees C with high proportional amounts of the red complex and T. denticola may be used as an indicator of persistence. All bacterial samples for DNA extraction should be processed following a standardized storage protocol (i.e. samples stored at +4 degrees C for maximum 6 weeks) in order to get comparable qualitative and quantitative results for total DNA, bacterial complexes and individual pathogens. Most representative results are yielded if processing and hybridization could be performed immediately after sampling.