本文選題：16SrDNA　切入點：病原性細菌　出處：《第三軍醫(yī)大學》2005年碩士論文

【摘要】：背景致病菌的快速檢測是臨床微生物學診斷迫切需要解決的一個難題;目前,臨床細菌學檢測仍主要依賴基于觀察細菌形態(tài)的檢查、基于分析細菌代謝特性的生化試驗和基于檢測特定抗原或抗體的免疫血清學方法。生化鑒定需要對細菌進行分離培養(yǎng),耗時長(3～7d);血清學試驗在感染初期往往是陰性;16SrDNA 序列的變異能夠反映細菌的種屬關(guān)系,已經(jīng)用于細菌的分類研究中;基于PCR 的基因診斷技術(shù)已廣泛應用于臨床病原體的診斷,單純的PCR 診斷特異性差,假陽性多見,核酸雜交檢測能較好地克服這一問題;基因芯片是一種高通量、特異、快速的核酸雜交檢測技術(shù)。將PCR 和基因芯片兩種技術(shù)結(jié)合在一起,則可能實現(xiàn)對致病菌的快速檢測。本研究的目的是探討應用16SrDNA 檢測臨床常見感染性細菌的可行性。 目的 建立靈敏、特異的檢測16SrDNA 多態(tài)性的寡核苷酸芯片的技術(shù)路線。 方法 1、利用通用引物擴增部分常見病原性細菌的16SrDNA,并克隆、測序,以用做標準陽性對照。 2、收集RDP-II 數(shù)據(jù)庫中的臨床常見細菌的16SrDNA 的序列信息,結(jié)合我們克隆的標準對照的測序結(jié)果設(shè)計相應的探針。 3、片段化雜交用的PCR 擴增產(chǎn)物。 4、利用PCR 方法標記待檢測的靶序列,與玻片表面的探針進行固相雜交。 結(jié)果 1、利用篩選的通用引物,成功克隆了22株常見細菌的近全長(約1.5kb)的16SrDNA基因。 2、找到了一種在U(T)堿基處特異性片段化PCR 產(chǎn)物的方法。 3、通過優(yōu)化探針設(shè)計、雜交條件、產(chǎn)物高效標記與片段化等方法,初步制備出可以檢測細菌16SrDNA 寡核苷酸芯片并建立了相應的檢測方法。
[Abstract]:Background the rapid detection of pathogenic bacteria is an urgent problem to be solved in clinical microbiological diagnosis. At present, clinical bacteriological detection is still mainly based on the observation of bacterial morphology. Biochemical tests based on the analysis of metabolic characteristics of bacteria and immune serological methods based on the detection of specific antigens or antibodies. Biochemical identification requires isolation and culture of bacteria, The variation of 16s rDNA sequence in serological tests is often negative in the early stage of infection, which can reflect the species relationship of bacteria and has been used in the taxonomy of bacteria. The gene diagnosis technology based on PCR has been widely used in the diagnosis of clinical pathogens. Simple PCR has poor specificity and false positivity, which can be overcome by nucleic acid hybridization. Gene chip is a high throughput, specific and fast nucleic acid hybridization technique. PCR and gene chip are combined together. The aim of this study was to explore the feasibility of 16SrDNA for the detection of common clinical infectious bacteria. Purpose. To establish a sensitive and specific oligonucleotide chip for the detection of 16SrDNA polymorphism. Method. 1. The 16s rDNA of some common pathogenic bacteria was amplified by universal primers, cloned and sequenced to be used as standard positive control. 2. The 16SrDNA sequences of common clinical bacteria in RDP-II database were collected, and the corresponding probes were designed based on the results of our cloned standard control sequencing. 3, PCR amplification products for fragment hybridization. (4) PCR method was used to label the target sequence to be detected, and a solid phase hybridization was carried out with the probe on the glass surface. Results. 1. The nearly full-length (1.5 kb) 16SrDNA gene of 22 strains of common bacteria was successfully cloned by using universal primers. 2. A method for specific fragmentation of PCR products at UT base was found. 3. By optimizing the probe design, hybridization conditions, high efficiency labeling and fragmentation of the product, the microarray of bacterial 16SrDNA oligonucleotide was prepared and the corresponding detection method was established.
【學位授予單位】：第三軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2005
【分類號】：R378

【參考文獻】

相關(guān)期刊論文 前10條

1 吳蓉,府偉靈,陳鳴;2種不同類型的核酸探針對壓電基因傳感器陣列檢測特異性的影響[J];重慶醫(yī)學;2004年08期

2 焦振泉,劉秀梅;16s rRNA序列同源性分析與細菌系統(tǒng)分類鑒定[J];國外醫(yī)學(衛(wèi)生學分冊);1998年01期

3 李聰智,譚德明,魯猛厚,吳英,周文紅,劉國珍,劉安國;聚合酶鏈反應檢測細菌16S rRNA基因[J];湖南醫(yī)科大學學報;1999年02期

4 張萬江,鮑朗,王曉櫻,謝勇恩,陳瑋,張會東,于向華;結(jié)核桿菌檢測基因芯片的制備研究[J];華西醫(yī)科大學學報;2002年02期

5 翟俊輝,郭兆彪,宋亞軍,王津,張敏麗,楊瑞馥,韓俊;16SrDNA基因芯片檢測臨床常見感染性細菌[J];臨床檢驗雜志;2002年03期

6 靳曉紅,劉元東,彭佐林,苗仲水,吳雪瓊,張俊仙,楊坤,程緒浩;PCR-SSCP快速鑒別結(jié)核、非結(jié)核分枝桿菌臨床分離株的研究[J];臨床檢驗雜志;2002年04期

7 彭道泉,趙水平;循證醫(yī)學的辯證思維[J];醫(yī)學與哲學;1999年10期

8 底秀娟,馬桂芳,劉彩蓮;一種傷寒沙門氏菌分型的新方法[J];中國公共衛(wèi)生;2001年07期

9 劉吉榮,吳婉芳,秦雨春,張玉,馬官福,郭章溉,張梓荊;原核生物16S rDNA的PCR-ELISA檢測方法的研究[J];中華兒科雜志;1998年04期

10 滕懿群,孫眉月,尚世強,洪文瀾,金益人,陳黎勤;核糖體DNA聚合酶鏈反應診斷新生兒敗血癥[J];中華兒科雜志;1998年07期

，

本文編號：1655347