DNA probe enhances periodontal diagnostics

It is well known that one of the major challenges of periodontologist in treatment planning is to identify factors causing or facilitating the course of periodontal disease. Gram negative anaerobic rods are main species responsible for the advancement of moderate and severe stages of this inflammatory process. They do not only destroy the integrity of tooth supporting tissues, but also can be detected in the peripheral blood, and may be the cause of various systemic disorders and diseases (1). The nature of these organisms makes them – while well prepared for the environment of periodontal pocket – very susceptible to oxygen. This may be with success exploited in the elimination of subgingival bacteria, but makes it difficult to specifically identify the bacteria responsible for the disease. Culturing the anaerobic bacteria is of course possible, but costly, time-taking and unreliable, since one can´t be sure not to kill some very sensitive species during probing. This fact was narrowing the microbiological identification to scientific centers or patients who were able to afford such an expensive method. That also forced clinicians to use chemiotherapeutics with wide spectrum of activity, such as tetracyclines or metronidazole and derivatives. Though used with success, those agents have numerous side effects if used systemically.

Last decade brought the major breakthroughs in molecular genetics. From the periodontologist´s point of view DNA hybridization was the most important. Both allowed to develop DNA probing – synthesis of a short, single DNA chain, usually labelled isotopically or chemically, used to detect a complementary DNA sequence.

Techniques developed during Human Genome Project allowed to approach the problem from a different side: instead of difficult, expensive and time-taking to extract and identify bacteria via culturing, extracting and identifying of their DNA takes place. After finding a DNA fragment specific for certain species, complementary DNA sequence is assembled. After labelling (see above) that may serve as a probe or a sort of "assay”. After extracting another sample from periodontal tissues, bacteria are lysed and DNA is extracted. The next process taking place is DNA hybridization, which involves the denaturation of double-stranded DNA chain into single strands. Those nucleotide sequences are exposed to DNA obtained earlier. If it finds complementary fragment, it specifically binds to it. After washing the excess of the specimen, labelled fragments signal the presence of complementary, i.e. specific for the DNA fragment of certain species. Such probes are commercially available in sets including a number of bacterial pathogens. Also there are banks containing information of numerous DNA fragments specific for bacterial species, such as Pasteur Institute in Paris (www.pasteur.fr).

That method is much less expensive than microbiological culturing. It is also reliable, since specimens obtained from periodontal pocket are bacteria-rich. There is no need to preserve the bacteria in the sample alive, since DNA does not require any sophisticated transporting or culturing regime.

It is obvious that monitoring of the presence or absence of periodontal pathogens makes the prognosis of incidence (or recurrence) and aggressiveness of periodontal disease much more accurate than recording of standard clinical parameters (as pocket probing depth, clinical attachment loss, bleeding and plaque indices, mobility, furcation involvement etc.) (2).

The DNA probes have been used in periodontology since early 1990´s. In the beginning the genomic probes were used. Those were purified DNA fragments labelled with phosphorium isotope. Later on, oligonucleotyde probes were introduced – since shorter, are much easier to obtain. Nowadays they can be produced synthetically. They are also more stable. First use of oligonucleotyde probes in periodontal practice took place in 1988 (3). Chuba et al. used sequences of 16S rRNA to detect P. gingivalis, P. intermedia and A. actinomycetemcomitans. 16S rRNA is a part of subunit of bacterial rybosome, species-specific sequence, which makes it an ideal target for DNA probe.

The specificity of DNA probe reaches 100% in pure cultures of bacteria, but may be lower in complex environments, such dental plaque. The sensitivity of bacteria is about 100-1000 cells per specimen, depending on labelling method. Two most frequently used tests in Europe are IAI Pado system, using radioactive agents as labels, and LCL Biokey, using chemiluminescents. Latest research is focusing on the novel design of DNA probes for increasing number of microorganisms. Liu et al. studied own-constructed DNA probe for P. gingivalis, targeted on fimriae subunit protein, labelled with phosphorium isotope (4). This probe detected P. gingivalis in all strains containing this bacteria, and in none of the specimens in which the bacteria was absent. This, according to authors, proved usefulness of this probe in detecting P.gingivalis.

The future of bacteria identification surely belongs to DNA probes, because of their exquisite specificity and sensitivity. Moreover, recently so called rapid DNA probes were introduced. They may be used chairside, and it takes only half an hour (5) or two hours (6) from specimen obtaining to bacteria identification. Such quickness makes the DNA probe a useful tool in everyday clinical practice.