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）。我们对大部分2级和3级病原菌制备了核糖体RNA芯片，建立了全面的病原菌鉴定检测体系。将这些核糖体DNA的PCR产物定量地标记在玻片上。将未识别菌落的Cye3或Cye5标记PCR产物杂交在载玻片的dna上，用激光扫描仪对杂交后的dna进行定量测定。核糖体RNA阵列不能区分某些具有相似核糖体RNA序列的强效病原体。为此，我们为其制备致病因子DNA芯片。固定这两种不同来源的PCR产物，我们可以鉴定和检测大多数人类致病性2级和3级病原体。通用引物不适用于扩增粪、痰、尿涂片等污染样品。因此，我们准备了每个特定的引物并混合在一个管中，以简化扩增程序。这个方法很成功。我们混合了50-100多种不同的特异性引物，但都能成功地扩增病原体的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.