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

人类致病菌分为2级(350种)和3级(33种)。我们制备了针对大部分2级和3级病原菌的核糖体RNA芯片，以建立全面的病原菌鉴定和检测体系。这些核糖体DNA的聚合酶链式反应产物在玻片上定量斑点。将未知菌落的Cye3或Cye5标记的聚合酶链式反应产物与切片DNA杂交，用激光扫描仪对杂交DNA进行定量检测。核糖体RNA阵列不能区分一些具有相似核糖体RNA序列的强效病原体。因此，我们制备了致病因子基因芯片，将这两种不同来源的聚合酶链式反应产物固定起来，可以鉴定和检测大部分人类致病的2级和3级病原体。通用引物不适用于粪便、痰、尿涂片等污染标本的扩增。因此，我们准备了每个特定的引物，并将其混合在一个试管中，以简化扩增程序。这种方法成功地奏效了。我们混合了50-100多个不同的特异引物，但仍能成功地扩增出病原菌的DNA。

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.