【作者】 刘毅；

【导师】 韩金祥；

【作者基本信息】 山东大学 ， 生物化学与分子生物学， 2006， 博士

【摘要】 目前，中枢神经系统(CNS)感染的病原体诊断仍然靠形态学检查、脑脊液病原体培养以及免疫学的方法，这些方法缺乏敏感性、特异性，而且费时费力，尤其是一些难以进行培养的病原体感染，更难检测和确诊。由于不同的病原体有不同的治疗方案，及时准确的诊断才能进行成功的治疗，而早期治疗能降低并发症和死亡率，因此，建立一种早期平行快速诊断方法至关重要。建立在分子生物学和生物信息学基础上的生物芯片技术，以其高度并行性、多样性、微型化和自动化的突出特点，在病原体的分型、鉴定、耐药性检测及特异性抗体检测等方面进行了多种研究，显示出了非常广阔的应用前景，使建立一种早期平行快速的诊断方法成为可能。 研究目的：分别以带正电荷的尼龙膜和玻片为基片，建立两套微阵列芯片检测系统，包括检测病原菌靶基因的寡核苷酸微阵列和检测病原体抗体的抗原微阵列。 研究方法： 1．脑脊液病原菌寡核苷酸微阵列技术平台的建立及其应用 (1)寡核苷酸微阵列的设计 首先确定脑脊液常见病原菌的种类，选择以下细菌作为研究的对象：醋酸钙不动杆菌、脆弱类杆菌、产黑色素类杆菌、大肠杆菌、肠球菌、脑膜炎败血性黄杆菌、坏死梭杆菌、流感嗜血杆菌、单核细胞增生性李斯特菌、卡他莫拉氏菌、结核分枝杆菌、脑膜炎奈瑟氏球菌、消化链球菌、巴斯德菌、变形杆菌、假单胞菌属、金黄色葡萄球菌、表皮葡萄球菌、无乳链球菌、沙门氏菌属、肺炎链球菌等。通过网络检索查找这些细菌的基因序列，确定选择16S rRNA基因为所要研究的靶基因，并下载检索到这些细菌的所有16S rRNA基因序列，经过比较分析，得到每种细菌的典型序列，借助于计算机软件，设计出一对通用引物，通过网络Blast功能和计算机软件的分析，构建出20种细菌的进化树，在上、下游引物之间，针对每种细菌设计出两条特异性探针。更多还原

【Abstract】 Currently, the detection of pathogens from central nervous system(CNS) is mainly depended on morphology, culture and immunology techniques, which are not only lack of sensitivity and specificity, but also laborious Shd time-consuming. Furthermore, it is more difficult for detecting fastidious and slowly growing microorganisms. Treatment plans for different pathogens are different and specific so that the accurate and rapid diagnosis become very important for successful treatment and care. Biochip technique based on molecular biology and bioinformatics has been used in pathogen typing, identification, detection of resistance and specific antibodies, which has demonstrated extensive perspective for clinical application, making it possible to establish the parallel and rapid diagnostic methods.Objective: To establish two kinds of microarray systems inclusing oligonucleotidemicroarray for detecting target genes of bacteria and antigen microarray for detectingantibodies of other pathegens.Methods:1 Establishment and application of the technical platform of oligonucleotidemicroarray for bacteria from CSFs(1) Design of oligonucleotide microarrayFirst of all, common bacteria from CSFs were selected and 20 species of bacteria were as targets for research, which were as follows: Acinetobacter calcoaceticus, Bacteroides fragilis, Escherichia coli, Enterococcus, Flavobacterium meningosepticum,更多还原