Preparation of DNA chip of level 2 and 3 pathogenic bacteria and development of diagnostic sutem of infectious diseases

2、3级病原菌DNA芯片的制备及传染病诊断系统的开发

• 批准号: 12557238
• 负责人: EZAKI Takayuki
• 金额: $ 7.17万
• 依托单位: Gifu University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2000
• 资助国家: 日本
• 起止时间: 2000 至 2001
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-12557238/
• 关键词: Lebel 2; Lebel 3; detection; DNA chip; Identification; Microarray; Multiplex PR; Diagnosis; 感染症診断; 病原細菌; 感染症; 診断

Human pathogenic bacteria are classified as level 2 ( 350 species ) and level 3 ( 33 species ). We prepared ribosomal RNA microarray for most of these level 2 and 3 pathogens to establish comprehensive identification and detection system of pathogenic bacteria. PCR products of ribosomal DNA of these were quantitatively Spotted on glass slide. Cye3 or Cye5 labeled PCR products from unidentified bacterial colonies were hybridized On DNAs of slides and hybridized DNAs were quantitatively measured by a laser scanner. Ribosomal RNA array could not differentiate some potent pathogens which shared similar ribosomal RNA sequences. Therefore, we prepared pathogenic factprs DNA microarray for them.Immobilizing these two different sources of PCR products, we could identify and detect most of human pathogenic level 2 and level 3 pathogens. Universal primers were not applicable to amplify contaminated sample such as stool, sputum and urinary smears. So we prepared each specific primer and mixed in a single tube to simplify amplification procedure. This method successfully worked. We mixed more than 50-100 different specific primers but could successfully amplify DNA of pathogens.

人类致病菌分为2级（350种）和3级（33种）。我们针对大部分2级和3级病原体制备了核糖体RNA微阵列，以建立全面的病原菌鉴定和检测体系。将这些核糖体DNA的PCR产物定量点在载玻片上。来自未鉴定细菌菌落的 Cye3 或 Cye5 标记的 PCR 产物与载玻片的 DNA 杂交，并通过激光扫描仪定量测量杂交的 DNA。核糖体RNA阵列无法区分一些共享相似核糖体RNA序列的强效病原体。因此，我们为它们制备了致病因子DNA微阵列。固定这两种不同来源的PCR产物，我们可以识别和检测大多数人类致病2级和3级病原体。通用引物不适用于放大粪便、痰液和尿涂片等污染样本。因此，我们准备了每种特异性引物并在单管中混合以简化扩增程序。这个方法成功了。我们混合了50-100多个不同的特异性引物，但可以成功扩增病原体的DNA。

项目成果

江崎孝行: "病原体の危険度レベルに応じた細菌検査のあり方"日本臨床微生物学会誌. 10・4. 34-36 (2000)

Takayuki Ezaki：“根据病原体风险水平进行细菌学检测”日本临床微生物学会杂志10/4（2000）。

Ezaki,T., Kawamura,Y., Li,N., Li,Z-Y., Zhao,L., and Shu,S.: "Propo sal of genera Anaerococcus, gen. nov., Peptoniphilus, gen. nov., and Catonia, gen. nov. for members of Genus Peptostreptococcus."Int. J. Syst. Evol Microbiol. 1528 (2001)

Ezaki,T.、Kawamura,Y.、Li,N.、Li,Z-Y.、Zhao,L. 和 Shu,S.：“厌氧球菌属，第 1 代，嗜胃杆菌属，第 1 代的提案，

"Borrelia sinica sp. nov., a Lyme disease-related Borrelia species isolated in China"Int.J.Syst.Evol.Microbiol.. 51. 1817-1824 (2001)

“Borrelia sinica sp. nov.，在中国分离的一种与莱姆病相关的疏螺旋体种”Int.J.Syst.Evol.Microbiol.. 51. 1817-1824 (2001)

江崎 孝行: "Relationship between bath enrironment and Mycobacteria in the public-bath water"環境感染. 16. 279-2284 (2001)

Takayuki Ezaki：“浴室环境与公共浴室水中分枝杆菌的关系”环境感染。 16. 279-2284 (2001)

Xu.H., et al.: "A rapid method to determine the G+C"Int. J. Syst. Evol. Microbiol. 50. 1463-1469 (2000)

Xu.H. 等人：“确定 G C 的快速方法”Int。