Identification of periodontal pathogen by DNA probe-biolobiclal and chemical luminescence system

DNA探针-生物化学发光系统鉴定牙周病原菌

• 批准号: 05557086
• 负责人: ABIKO Yoshimitsu
• 金额: $ 5.44万
• 依托单位: NIHON UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Developmental Scientific Research (B)
• 财政年份: 1993
• 资助国家: 日本
• 起止时间: 1993 至 1995
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-05557086/
• 关键词: periodontal disease; bacterial examination; P.gingivalis; A.actinomycetemcomitans; A.viscosus.; DNA probe; riboprobe; genetic diagnosis; 生物化学発光; DNA診断; Porphyromonas gingivalis; Campylobacter rectus

One approach to the development of a rapid, sensitive and specific assay for the detection of the pathogens is molecular hybridization by using DNA or RNA probe. We focused to detection and identification of periodontal pathogens, P.gingivalis and A.actinomycetemcomitans as disease-active site and A.viscosus as inactive site during the progression of periodontal disease. We cloned genes ; outer membrane protein and glycylprolyl amonopeptidase from P.gingivalis, leukotoxin from A.actinomycetemcomitans and sialidase from A.viscosus. Then non-radiolabeled riboprobes were developed using in vitro transcription RNA synthesizing system. All probes were highly specific for identification of each pathogen.We also examined the usefullness of alkaline phosphatase/lumigen PPD detection system using anti-DNA-RNA complex antibody. Result showed a good corelation between optical density and number of molecules until 10^4 molecules but poor corelation was found over 10^5 molecules. These unfortunate results may occur by contamination of RNA or DNA-RNA hybrid in the sample. Then we amplified the specific region from chromosomal DNA and measured by the same procedure, this experiment produced a good corelation for detection of pathogens.

开发快速、灵敏且特异的病原体检测方法的一种方法是使用 DNA 或 RNA 探针进行分子杂交。我们重点检测和鉴定牙周病原菌，在牙周病进展过程中，P.gingivalis和A.actinomycetemcomitans作为疾病活性位点，A.viscosus作为非活性位点。我们克隆了基因；来自 P.gingivalis 的外膜蛋白和甘氨酰氨肽酶、来自 A.actinomycetemcomitans 的白细胞毒素和来自 A.viscosus 的唾液酸酶。然后利用体外转录RNA合成系统开发非放射性标记的核糖探针。所有探针对于每种病原体的鉴定都具有高度特异性。我们还使用抗 DNA-RNA 复合物抗体检查了碱性磷酸酶/lumigen PPD 检测系统的有用性。结果显示，直到 10^4 个分子为止，光密度与分子数之间存在良好的相关性，但在 10^5 个分子以上，发现相关性较差。这些不幸的结果可能是由于样品中 RNA 或 DNA-RNA 杂交体的污染而导致的。然后我们从染色体DNA中扩增特定区域并通过相同的程序进行测量，该实验为病原体的检测产生了良好的相关性。

项目成果

玉舎秀規: "Porphyromonas gingivalis外膜タンパク質遺伝子合成オリゴヌクレオチドDNAプローブ" 日大口腔科学. 20. 406-413 (1994)

Hidenori Tamasha：“牙龈卟啉单胞菌外膜蛋白基因合成寡核苷酸DNA探针”日本大学口腔医学。 20. 406-413 (1994)

小板橋誠: "Porphyromonas gingivalis Glycylprolyl Aminopeptidase遺伝子リボプローブの開発" 日大口腔科学. 20. 499-507 (1994)

Makoto Koitabashi：“牙龈卟啉单胞菌甘氨酰氨基肽酶基因核糖核酸探针的开发”日本大学口腔医学院 20. 499-507 (1994)。

Katsukata SHIMIZU,Mitsuo HAYAKAWA and Hisashi TAKIGUCHI: "Construction of the Gene Clone Highly Expressing Porphyromonas gingivalis Glycylprolyl Diaminopeptidase" Nihon University Journal of Oral Science. Vol.22. 36-44 (1996)

Katsukata SHIMIZU、Mitsuo HAYAKAWA 和 Hisashi TAKIGUCHI：“高表达牙龈卟啉单胞菌甘氨酰脯氨酰二氨基肽酶的基因克隆的构建”日本大学口腔科学杂志。

清水克貴: "Porphyromonas gingivalis glycylprolyl diaminopeptidase高産生性遺伝子クローンの作成" 日大口腔科学. 22. 36-44 (1996)

Katsutaka Shimizu：“牙龈卟啉单胞菌甘氨酰二氨基肽酶的高产基因克隆的创建”日本大学口腔医学院22. 36-44（1996）。

Kanasugi N.: "Detection of Porphyromonas gingivalis by DNA-RNA hybridization with non-radioactivity labeled riboprobe" 日大口腔科学. 22. (1996)

Kanasugi N.：“通过 DNA-RNA 杂交与非放射性标记的核糖核酸探针检测牙龈卟啉单胞菌”，日本大学口腔医学杂志 22。（1996）