【摘要】：目前，中樞神經(jīng)系統(tǒng)(CNS)感染的病原體診斷仍然靠形態(tài)學(xué)檢查、腦脊液病原體培養(yǎng)以及免疫學(xué)的方法，這些方法缺乏敏感性、特異性，而且費(fèi)時(shí)費(fèi)力，尤其是一些難以進(jìn)行培養(yǎng)的病原體感染，更難檢測(cè)和確診。由于不同的病原體有不同的治療方案，及時(shí)準(zhǔn)確的診斷才能進(jìn)行成功的治療，而早期治療能降低并發(fā)癥和死亡率，因此，建立一種早期平行快速診斷方法至關(guān)重要。建立在分子生物學(xué)和生物信息學(xué)基礎(chǔ)上的生物芯片技術(shù)，以其高度并行性、多樣性、微型化和自動(dòng)化的突出特點(diǎn)，在病原體的分型、鑒定、耐藥性檢測(cè)及特異性抗體檢測(cè)等方面進(jìn)行了多種研究，顯示出了非常廣闊的應(yīng)用前景，使建立一種早期平行快速的診斷方法成為可能。 研究目的：分別以帶正電荷的尼龍膜和玻片為基片，建立兩套微陣列芯片檢測(cè)系統(tǒng)，包括檢測(cè)病原菌靶基因的寡核苷酸微陣列和檢測(cè)病原體抗體的抗原微陣列。 研究方法： 1．腦脊液病原菌寡核苷酸微陣列技術(shù)平臺(tái)的建立及其應(yīng)用 (1)寡核苷酸微陣列的設(shè)計(jì) 首先確定腦脊液常見(jiàn)病原菌的種類，，選擇以下細(xì)菌作為研究的對(duì)象：醋酸鈣不動(dòng)桿菌、脆弱類桿菌、產(chǎn)黑色素類桿菌、大腸桿菌、腸球菌、腦膜炎敗血性黃桿菌、壞死梭桿菌、流感嗜血桿菌、單核細(xì)胞增生性李斯特菌、卡他莫拉氏菌、結(jié)核分枝桿菌、腦膜炎奈瑟氏球菌、消化鏈球菌、巴斯德菌、變形桿菌、假單胞菌屬、金黃色葡萄球菌、表皮葡萄球菌、無(wú)乳鏈球菌、沙門氏菌屬、肺炎鏈球菌等。通過(guò)網(wǎng)絡(luò)檢索查找這些細(xì)菌的基因序列，確定選擇16S rRNA基因?yàn)樗芯康陌谢颍⑾螺d檢索到這些細(xì)菌的所有16S rRNA基因序列，經(jīng)過(guò)比較分析，得到每種細(xì)菌的典型序列，借助于計(jì)算機(jī)軟件，設(shè)計(jì)出一對(duì)通用引物，通過(guò)網(wǎng)絡(luò)Blast功能和計(jì)算機(jī)軟件的分析，構(gòu)建出20種細(xì)菌的進(jìn)化樹(shù)，在上、下游引物之間，針對(duì)每種細(xì)菌設(shè)計(jì)出兩條特異性探針。
[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.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2006
【分類號(hào)】：R346

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 潘繼紅,韓金祥;cDNA微陣列與寡核苷酸芯片的制備方法[J];國(guó)外醫(yī)學(xué)(分子生物學(xué)分冊(cè));2003年01期

2 鄧范艷,陳宇,王宇;肺炎支原體IgM抗體檢測(cè)對(duì)肺炎支原體腦炎或腦膜炎診斷的臨床價(jià)值[J];黑龍江醫(yī)藥科學(xué);2002年02期

3 王曉紅 ,楊智宏 ,王岱明 ,朱啟昒 ,董佐權(quán);流行性腮腺炎腦膜腦炎血清及腦脊液特異性IgM抗體檢測(cè)[J];臨床兒科雜志;2002年05期

4 王易偉,鐘敏;腦脊液結(jié)核抗原與抗結(jié)核抗體的臨床診斷價(jià)值[J];臨床肺科雜志;2002年02期

5 劉毅,韓金祥;16SrRNA基因在腦脊液細(xì)菌鑒定中的應(yīng)用[J];臨床檢驗(yàn)雜志;2002年04期

6 劉毅,韓金祥,朱波,王世立,張更林;單純皰疹病毒I型Stocker株糖蛋白G基因在PBV221原核表達(dá)載體的表達(dá)與純化[J];山東大學(xué)學(xué)報(bào)(醫(yī)學(xué)版);2005年06期

7 丁亞平,陳立炎,張文,曹恒杰,倪世明,周枚芬,梁好,凌志光,耿永堯,王升啟;同時(shí)檢測(cè)血清中多種抗體的蛋白質(zhì)微陣列研究[J];生物化學(xué)與生物物理進(jìn)展;2002年04期

8 潘繼紅,韓金祥;基因芯片的制備方法[J];生命的化學(xué);2002年03期

9 高志賢,楊明星,王濤,王升啟;用于檢測(cè)葡萄球菌腸毒素的免疫芯片技術(shù)[J];中國(guó)生物工程雜志;2004年08期

10 張然,岳廣智,俞永新;流行性乙型腦炎特異性IgM、IgG診斷試劑盒的研制和應(yīng)用[J];中國(guó)生物制品學(xué)雜志;1995年01期

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