脑脊液病原体检测芯片技术平台的建立及其应用

发布时间：2018-08-07 10:58

【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。
[Abstract]:At present, the diagnosis of (CNS) infection in the central nervous system still depends on morphological examination, cerebrospinal fluid pathogen culture and immunological methods, which lack sensitivity, specificity, and time and effort. In particular, it is difficult to detect and diagnose pathogens that are difficult to culture. Because different pathogens have different treatment schemes, timely and accurate diagnosis can make successful treatment, and early treatment can reduce complications and mortality. Therefore, it is very important to establish an early parallel and rapid diagnosis method. Biochip technology, based on molecular biology and bioinformatics, is characterized by its high parallelism, diversity, miniaturization and automation in the typing and identification of pathogens, Many studies have been carried out on the detection of drug resistance and specific antibody, which shows a very broad prospect of application, which makes it possible to establish an early parallel and rapid diagnostic method. Objective: to establish two microarray detection systems based on positively charged nylon membrane and glass slide respectively, including oligonucleotide microarrays for detection of pathogen target genes and antigen microarrays for detection of pathogen antibodies. Methods: 1. Establishment and application of oligonucleotide microarray technology platform for cerebrospinal fluid pathogens (1) the design of oligonucleotide microarray firstly identified the common pathogenic bacteria in cerebrospinal fluid (CSF). The following bacteria were selected for the study: Acinetobacter calcium Acinetobacter Acinetobacter Acinetobacter, Bacteroides fragilis, Bacillus melanocytogenes, Escherichia coli, Enterococcus, Flavobacterium septicum Meningitis, Clostridium Necrosis, Haemophilus Listeria monocytogenes, Mycobacterium tuberculosis, Neisseria meningitidis, Streptococcus digestion, Pasteurella, Proteus, Pseudomonas, Staphylococcus aureus, Staphylococcus epidermidis, No Streptococcus lactobacillus, Salmonella, Streptococcus pneumoniae, etc. Searching for the gene sequence of these bacteria, selecting 16s rRNA gene as the target gene to be studied, downloading and retrieving all 16s rRNA gene sequences of these bacteria. After comparative analysis, the typical sequence of each bacteria is obtained. With the aid of computer software, a pair of universal primers were designed. Through the analysis of network Blast function and computer software, the evolutionary tree of 20 species of bacteria was constructed. Two specific probes were designed for each species of bacteria between upstream and downstream primers.
【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2006
【分类号】：R346

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