Development of DNA microarry-based molecular diagnosis system for male urethritis

为了检测非衣原体非淋菌性尿道炎(NCNGU)的病原体，我们开发了一种分子诊断系统。首先，用支原体和解脲支原体通用引物从NCNGU患者的临床标本中扩增出16S rRNA基因，并对扩增产物进行测序。根据扩增出的16S rRNA基因序列对病原菌进行了鉴定。临床标本中仅检出生殖支原体、人型支原体、解脲支原体和细小支原体。其次，我们将生殖支原体、人型支原体、解脲支原体和解脲支原体的物种特异性寡核苷酸固定在微量平板上，并将PGR产物与微量平板上的每一种寡核苷酸杂交。用该方法在临床标本中只能检出生殖支原体、人型支原体、解脲支原体和细小支原体。目前，我们正在制备DNA芯片，将淋球菌、沙眼衣原体、解脲杆菌、流感嗜血杆菌、副流感嗜血杆菌等细菌的DNA固定在芯片上。第三，我们建立了生殖支原体的定量检测方法。我们测定了男性NCNGU患者首过尿中支原体的细菌载量，发现支原体载量与尿道炎症状和炎症体征的存在密切相关。我们还在开发一种解脲支原体的定量检测方法。我们的目标是建立一套全面的尿道炎诊断系统，并对男性尿道炎患者的细菌载量进行定量评估。

In order to detect pathogens causing non-chlamydial non-gonococcal urethritis (NCNGU), we developed a molecular diagnosis system. First, we amplified the 16s rRNA genes from clinical specimens of men with NCNGU by the universal primers for mycoplasmas and ureaplasmas, and then sequenced the PCR products. We identified the pathogen based on the sequence of the amplified 16s rRNA gene. We detected only Mycoplasma genitalium, Mycoplasma hominis, Ureaplasma urealyticum and Ureaplasma parvum in the clinical specimens. Second, we fixed species-specific oligonucleotides of M. genitalium, M. hominis, U. urealyticum and U. parvum on the microtiter plate, and hybridized the PGR products with each of the oligonucleotides on the microtiter plate. Using this assay, we could identify only M. genitalium, M. hominis, U. urealyticum and U. parvum in the clinical specimens. Now we are preparing DNA microarrays, on which the DNAs of Neisseria gonorrohoeae, Chlamydia trachomatis, Bacteroides ureolyticum, Haemphilus influenzae, Haemphilus parainfluenzae, and other bacteria are fixed. Third, we developed a quantitative assay for M. genitalium. We determined the bacterial loads of the mycoplasma in the first-pass urine of men with NCNGU, and demonstrated that the bacterial loads were significantly related to the presence of urethritis symptoms and inflammatory signs. We are also developing a quantitative assay for U. urealyticum.We aim for the establishment of a comprehensive diagnosis system for urethritis and for the quantitative assessment of bacterial loads in men with urethritis.