微生物非培养鉴定技术在临床标本检查中的应用研究

感染性疾病的病原学诊断仍以微生物的分离培养作为“金标准”，但能培养成功的仅为少数。绕过分离培养环节，以微生物rRNA／rDNA基因作为种属鉴别序列，设计通用引物（universal primer，up），用PCR扩增标本中微生物的16SrRNA或16S～23SrRNA序列，通过毛细管电泳（CE）进行单链构象多态性（SSCP）和限制性片段长度多态性（RFLP）分析，筛选基因的点突变以达到快速鉴定微生物（到属和种）。用PCR—CE—SSCP和PCR—CE—RFLP分析系统检测了呼吸道、消化道、女性生殖道标本中感染的病原菌；用PCR-CE-SSCP系统检测了男性泌尿道溶脲脲原体两个生物群。建立PCR—CE—RFLP分析系统，用非培养法鉴定技术检测脓汁、肠道和泌尿生殖道标本，检测并鉴定了临床标本中的多种病原菌；对人体肠道菌群进行定性和定量分析，用该法可协助腹泻的病原学诊断；检测生殖道常见的致病菌；用PCR—CE—SSCP系统建立了检测溶脲脲原体两个生物群的方法。微生物的非培养鉴定技术比传统方法缩短20h，为临床感染症的诊断提供快速、准确的依据。结果可见，利用16S～23SrRNA间区基因PCR—CE—RFLP和PCR—CE—SSCP系统可以达到对临床常见病原菌的快速种属鉴定，比传统的细菌培养法具有快速、准确、灵敏的优点，可用于临床感染症的病原学诊断。微生物的非培养鉴定技术将替代培养法而成为病原学诊断新的金标准。

Applied Study of Methodological for Un-Culture Microorganisms Identification on the Clinical Samples

Etiological diagnosis of infectious diseases is substantially depended on culture-based procedure, which is regarded as a golden standard. But culturable microorganisms occupy a small part of microorganism world. This project circumvents the culture protocol and utilizes the rRNA/rDNA sequences as labels to identify microbes. The basic principle is using designed universal primers (UP)to amplify 16S rRNA and 16S-23S rRNA spacer region genes. The PCR products are subjected to capillary electrophoresis (CE) analysis in view of single strand conformation polymorphism (SSCP) and restricted fragment length polymorphism (RFLP), and relevant information can be employed to identify the target microorganisms to their genera and species levels. PCR-CE-SSCP and PCR-CE-RFLP was used to analyze bacterial samples from respiratory tract, alimentary tract and female genital tract. Two subgroups of Ureaplasma urealyticum from male genital tract was analyzed by PCR-CE-SSCP. A set of PCR-CE based analysis platform was set up. Employing this culture-independent method, we successfully identified various bacteria from diverse clinical samples. Quantitative and qualitive analysis of intestinal flora, could facilitate the etiological diagnosis of diarrhea differentiation. Genital tract infection could be easily diagnosed and two subgroups of Ureaplasma urealyticum were clas-sified correctly. Our platform can save detection time nearly 20 h compared to traditional methods, and can provide timely and correct reports for diagnosis purpose. This PCR-CE-SSCP/RFLP based identification system can easily identify clinical microorganisms to species and genera levels, and is rapid, precise and sensitive. This system is amenable to clinical diagnosis utilization. Hopefully, the culture-independent method will be developed to a new microorganism identification golden standard.