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© Borgis - Nowa Stomatologia 4/2003, s. 209-210
Jan Kowalski
DNA probe enhances periodontal diagnostics
from Zakład Chorób Błony Śluzowej i Przyzębia ISAM w Warszawie
Department´s Chief: prof. dr hab. n. med. Renata Górska



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.

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Piśmiennictwo
1. Herzberg M.C., Weyer M.W.: Dental plaque, platelets, and cardiovascular diseases. Ann Periodontol 1998, 3:151-60. 2. Wolff L. et al.: Bacteria as risk markers for periodontitis. J Periodontol 1994, 65:498-510. 3. Chuba P.J. et al.: Synthetic oligodeoxynucleotyde probes for the rapid detection of bacteria associated with human periodontitis. J Gen Microbiol 1988, 134:1931-8. 4. Liu L. et al.: Species-specific DNA probe for the detection of Porphyromonas gingivalis from adult Chinese periodontal patients and healthy subjects. J Periodontol 2003, 74:1000-6. 5. Komiya A. et al.: A rapid DNA probe method for detection of Porphyromonas gingivalis and Actinobacillus actinomycetemcomitans. J Periodontol 2000, 71:760-7. 6. Tanner A. et al.: Rapid chairside DNA probe assay of Bacteroides forsythus and Porphyromonas gingivalis. J Periodonal Res 1998, 33:105-17.
Nowa Stomatologia 4/2003
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