Development of rapid and comprehensive molecular diagnosis system for genitourinary infections

我们设计了一对通用引物，可以扩增所有可能引起泌尿生殖道感染的病原体的16 SrRNA基因。我们用通用引物PCR扩增16 SrRNA基因的DNA片段，然后将PCR产物与固定在平板上的每个寡核苷酸杂交，以鉴定病原体。为了建立一套快速、全面的泌尿生殖道感染诊断体系，我们设计了人乳头瘤病毒、单纯疱疹病毒、阴道毛滴虫的特异性引物，用于扩增其他微生物（包括病毒和原虫）的DNA片段。我们开发了多重PCR系统，可以在一个试管中有效地扩增微生物的特异性DNA片段。淋病奈瑟菌、沙眼衣原体、生殖支原体和解脲支原体被认为是引起性传播感染的病原体。为了快速检测这些病原体，因此，我们开发了基于实时PCR的检测方法，该方法具有针对每种病原体的特异性引物。该方法可定量检测尿液中的每种病原体。淋病在日本是一种临床问题。在临床环境中，需要快速检测药物对抗生素的敏感性，以优化淋球菌感染的化疗。我们正在开发用于检测与氟喹诺酮类和β-内酰胺类耐药相关的突变的分子诊断系统。

We have designed universal primers which can amplified the 16S rRNA genes of all possible pathogens causing genitourinary tract infections. We have amplified the DNA fragments of the 16s rRNA genes by PCR with the universal primers, and then hybridized the PCR products with each of the oligonucleotides fixed on the plate to identify the pathogens. We have aimed for the establishment of a rapid and comprehensive diagnosis system for genitourinary tract infections.To amplify DNA fragments from other microorganisms, including virus and protozoan, we have also designed the primers specific for human papilloma virus, herpes simplex virus, and Trichomonas vaginalis. We have developed the multiplex PCR system to efficiently amplify respective DNA fragment specific for the microorganisms in one tube.Neisseria gonorrohoeae, Chlamydia trachomatis, Mycoplasma genitalium, and Ureaplasma urealyticum are recognized as pathogens causing sexually transmitted infections. To detect these pathogens rapidly, therefore, we have developed the real-time PCR-based assay with the primers specific for each pathogen. This assay allows quantitative detection of each pathogen in urine samples.Emergence and increase of antibiotics-resistant N. gonorrhoeae is a clinical concern in Japan. In clinical settings, rapid detection of drug susceptibilities to antibiotics is needed for optimal chemotherapy for gonococcal infections. We are developing the molecular diagnosis system for detecting some mutations associated with resistance to fluoroquinolones and beta-lactams.