The establishment of clinical examination system to detect periodontal microorganism using specific DNA probes

1992 Fiscal Year Final Research Report Summary

• Project/Area Number: 02454439
• Research Category: Grant-in-Aid for General Scientific Research (B)
• Allocation Type: Single-year Grants
• Research Field: Conservative dentistry
• Research Institution: KYUSHU UNIVERSITY
• Principal Investigator: HIROFUJI Takao Kyushu Univ., Fac. Dent., Assistant Professor, 歯学部, 講師 (10189897)
• Co-Investigator(Kenkyū-buntansha): KABASHIMA Hiroaki Kyushu Univ., Fac. Dent., Assistant, 歯学部, 助手 (20214504) ; HAMACHI Takafumi Kyushu Univ., Fac. Dent., Assistant, 歯学部, 助手 (80198811) ; MAEDA Katsumasa Kyushu Univ., Fac. Dent., Professor, 歯学部, 教授 (00117243)
• Project Period (FY): 1990 – 1992
• Keywords: Periodontitis / Porphyromonas gingivalis / Cysteine proteinase / DNA cloning / Actinobacillus actinomycetemcomitans / Synthetic oligonucleotide / PCR method

Research Abstract

Prophyromonas gingivalis (P.g.) has been implicated as a key organism in 2dult periodontitis. A cysteine proteinase was purified from the culture supernatant of P.g. to a homogeneity by several continuous chromatographic methods. The molecular size of this enzyme was estimated to be 50KDa and the pl of this factor was between pH 4.5 to 5.1. This proteinase was found to induce the dysfunction of polymorphonuclear leukocytes (PMNs), as judged from the suppression of their chemiluminescence response. These results indicate that P.g. release the unique cysteine proteinase having a damaging effect on the host defense system. We are now considering the possibility that this factor has been developed to aid in the diagnosis and treatment of human periodontal disease using DNA technology. Actinobacillus actinomycetemcomitans (A.a.) is generally recognized as the main infectious organism in localized juvenile periodontitis and also found in adult periodontitis patients. We prepared for several synthetic olygonucleotides derived from IktA gene in A.a., then, used as primers of polymerase chain reaction method (PCR method). One of these primers reacted specifically all genomes in A.a. we tested by PCR method and amplified only one specific band on DNA gel electrophoresis. When this primer was reacted with samples from subgingival plaques of twenty one patients with periodontal periodontitis, samples from six-teen patients with periodontitis were positive by PCR method and these reactions were specific.
These results indicate that this PCR method using specific synthetic oligonucleotide as a primer is very useful to detect A.a. in subgingival site in patient with periodontitis.