【作者】 蒋志云；

【导师】 王继春；

【作者基本信息】 中国医科大学 ， 病原生物学， 2008， 硕士

【摘要】 目的80年代以来,性传播疾病（sexually transmitted diseases,STD）发病率日趋上升,其中淋菌性尿道炎（gonococcal urethritis）和非淋菌性尿道炎（nongonococcalurethritis,NGU）发病率较高,前者由淋病奈瑟菌（Neisseria gonorrhoeae,Ng）感染引起,而后者主要由解脲脲原体（Ureaplasma urealyticum,Uu）、人型支原体（Mycoplasma hominis,Mh）等感染引起。由于引起泌尿生殖道感染的病原体复杂、多样,为临床治疗带来困难,因此迫切需要寻求一种特异性强、敏感性高同时又快速、经济的病原学检测方法,为临床病原学治疗提供实验依据。目前临床用于泌尿生殖道感染病原体检测的方法有培养技术、免疫技术和PCR技术等。这些技术在临床诊断中已发挥了巨大的作用,但仍存在一些缺点。培养技术繁琐而费时;免疫技术要有特异的抗血清。PCR技术本身的优越性是无可厚非的,但是使用不当很容易引起交叉污染,出现假阳性;如果反应条件控制不好也可能出现假阴性。这些缺点需要应用新的技术去弥补。近几年发展起来的基因芯片技术为泌尿生殖道感染的病原学诊断提供了一种强有力的手段。其快速、高效、高通量、并行化的检测特点在病原学诊断方面具有独特的优势。基因芯片技术在病原学诊断上的应用研究虽已取得很大进展,但目前尚无成熟的泌尿生殖道感染病原体检测基因芯片应用于临床,且我国学者在这方面的研究开展较少。本实验制备了一种可同时检测Ng、Uu和Mh三种病原体的复合基因芯片,以期为临床提供一种高通量、并行化的泌尿生殖道感染病原体的检测方法,并为我国日趋严峻的STD防治工作的改善奠定理论和实验基础。实验材料和方法一、实验材料标准菌株:淋病奈瑟菌ATCC 29106,购于中国药品生物制品检定所;解脲脲原体ATCC 33697,人型支原体ATCC 23114均购于首都儿科研究所。临床标本来自中国医科大学第一附属医院皮肤性病科门诊就诊患者。支原体基础培养基PPLO购于北京泽平生物技术研究所,淋球菌琼脂基础培养基购于中国药品生物制品检定所,Uu和Mh的选择性鉴定固体培养基（二合一培养基）由上海恩康生物科技有限公司友情提供。PCR试剂盒（TaKaRa）引物与探针（上海生工合成）PCR扩增仪（Biometra Personal PCR system,Germany）,生物芯片点样仪（MicroGrindⅡ600,Biorobtics Ltd,England）,激光共聚焦扫描仪(Gene TAC^TMLSⅣ,Gnomic Solutions Inc.USA)二、实验方法1、用液体和固体两种培养基培养Uu和Mh两种支原体标准菌株,用固体平板培养基在烛缸内培养Ng标准菌株。2、临床标本采集:用无菌棉拭子取男性尿道女性宫颈分泌物。3、应用生物信息学软件设计通用引物和特异探针,分析其合理性。4、采用酚-氯仿法提取病原体DNA模版。5、不对称PCR制各样品靶序列。6、制备病原体检测芯片:片基处理,按预先设计好的微阵列矩阵使用生物芯片点样议将探针点在芯片上,水化,烘烤固定。7、通过重复杂交、分析测序结果和对比临床回复的结果来检测芯片的重复性和可靠性。结果1、标准菌株在两种培养基中均生长良好,提取模版DNA,PCR扩增目的条带,产物经聚丙烯酰胺凝胶电泳后银染色,产物条带清晰,长度与理论设计一致。2、探针设计合理,与其它病毒、细菌等无同源性,待检测病原体各探针之间有极高的特异性。3、芯片杂交温度为60℃,反应1h所获得的杂交信号较强。4、芯片杂交结果重复性较好,探针检测重现率97.5%,与门诊回复结果符合率为96%,反馈的测序结果经BLAST软件对比分析,与其芯片检测结果完全一致,验证了芯片的可靠性。结论1、泌尿生殖道病原体检测基因芯片设计合理,检测结果理想。2、该芯片具有较好的重复性和可靠性,为临床泌尿生殖道感染病原体检测新技术的开发提供了实验依据。更多还原

【Abstract】 ObjectiveSince 1980s, the incidence of sexually transmitted diseases (STD) has gradually increased, and gonococcal urethritis and non-gonococcal urethritis (NGU) are of higher morbidity. The former is caused by Neisseria gonorrhoeae (Ng), and the latter mainly by Ureaplasma urealyticum (Uu), Mycoplasma hominis (Mh) or other Pathogens infections. The cause of the genitourinary tract infection pathogens is complex and diverse, which make clinical treatment difficult. Thus, it is urgent to find a pathogenic detection method with strong specificity, high sensitivity, time saving, and low expenditure, it will lay an experimental basis for the clinical treatment of etiology.At present some pathogen detection methods, such as culture, immunohistochemical techniques, PCR technology, are clinically used for genitourinary tract infections. The above technologies have played a tremendous role in the clinical diagnosis. However, there are still some shortcomings in different methods. For example, culture is cumbersome and time consuming; and specific antisera are needed in immune tests. The advantages of PCR technology itself are understandable, but cross-contamination and false positive results appear if to be used improperly. Moreover, some false negative results may occur if reaction conditions are not strictly controlled. These shortcomings need to overcome by new technology.In recent years, newly developed gene chip technology provides a powerful diagnostic tool for genitourinary tract infection etiology. Characteristics of fast, efficient, high-throughput, parallel detection of gene chip in etiological diagnosis is of the unique advantage. It has been made that considerable progress in applied research of gene chip on etiological diagnosis. Unfortunately, no practical gene chip is used in clinical detection for genitourinary tract infection pathogens to this day, and little is known about this research field in our country. To provide a high-throughput and parallel detection method for clinical diagnosis of genitourinary tract infections, we prepared a multiple-purposed gene chip in this project. This newly developed chip could simultaneously detect three kinds of pathogens, i.e. Ng, Uu and Mh that cause gonococcal urethritis and non-gonococcal urethritis, respectively. Our works will lay a foundation in both theories and technologies for the improvement of increasingly serious situation about STD prevention and control work in our country.Materials and Methods MaterialsStandard strains: Neisseria gonorrhoeae ATCC 29106 was purchased in National Institute for the Control of Pharmaceutical and Biological Produce; Ureaplasma urealyticum ATCC 33697, Mycoplasma hominis ATCC 23114 were purchased in the Capital Institute of Pediatric.Clinical samples were collected from Department of dermatonosis and cypridopathy, The First Hospital affiliated China Medical University.Mycoplasma basal medium PPLO was purchased in Beijing Zeping-Biotechnology Institute, Neisseria gonorrhoeae agar medium drugs was purchased in National Institute for the Control of Pharmaceutical and Biological Produce, and Mh-Uu selective identification solid medium (two in one-medium) was kindly provided by Shanghai Enkang Biological Technology.PCR Kit (TaKaRa).Primers and probes (Shanghai Public Health Synthesis). PCR Amplification (Biometra Personal PCR system, Germany), biochips plotter (Micro Grind II 600, Biorobtics Ltd, England), confocal laser scanner (Gene TACTMLS IV, Gnomic Solutions Inc. USA.Methods1. Two standard strains of Mh and Uu were cultured in solid and liquid culture medium, respectively. And standard strain of Ng was cultured with solid medium in the candle-cylinder.2. Clinical samples (secretions) were collected from male urethra and female cervix, using sterile cotton swab.3. A pair of universal primers and 3 specific probes were designed with bioinformatics software, and their rationality was analyzed.4. Pathogen DNA templates were extracted using the phenol-chloroform.5. Preparation of sample target sequences using asymmetric PCR.6. Preparation of pathogen detection gene chips: after film-processing, probes were added on the chip using biochips plotter according to pre-designed micro-array matrix, then the chips were hydrated and fixed.7. Repeatability and validity were detected through repeated hybridizations, sequencing analyses, and comparisons to clinical report results.Results1. Two standard strains grew well in both solid and liquid mediums. Following template DNA extraction, PCR amplification polyacrylamide gel electrophoresis and silver-staining, expected product bands with designed length were clearly observed.2. Probe design was reasonable, and had no homology compared with other viruses or bacteria. There was high specificity among different probes of detected pathogens.3. When chip hybridization temperature was 60℃for 1h, the hybridization signal was significant. 4. The results of chip hybridizations repeated well. The reproducibility rate of probe detection was 97.5%, and coincidence with out-patient reports was 96%. Analyzed by software BLAST, the results of sequencing were identical with that of chip detection. The above findings proved that chip is validity and practical.Conclusion1. The design of gene chip for urogenital tract pathogen detection is reasonable, and results are ideal.2. The chip is of good repeatability and reliability, which will lay an experimental foundation for the development of new techniques to detect clinical urogenital tract infection pathogens.更多还原