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論文中文名稱:以雜交層析法及奈米金粒子偵測人類乳突腫瘤病毒16型之研究 [以論文名稱查詢館藏系統]
論文英文名稱:A Study of HPV16 DNA Detection Using Nanogold Particles and Hybrid Chromatography [以論文名稱查詢館藏系統]
院校名稱:臺北科技大學
學院名稱:工程學院
系所名稱:生物科技研究所
畢業學年度:97
出版年度:98
中文姓名:李蕙君
英文姓名:Hui-Chun Li
研究生學號:96688002
學位類別:碩士
語文別:中文
口試日期:2009-07-26
論文頁數:88
指導教授中文名:侯劭毅
指導教授英文名:Shao-Yi Hou
口試委員中文名:黃榮堂;王勝仕
口試委員英文名:Jung-Tang Huang;Sheng-Shih Wang
中文關鍵詞:人類乳突腫瘤病毒奈米金粒子雜交層析法
英文關鍵詞:gold nanoparticlesHPV 16Hybrid Chromatography
論文中文摘要:子宮頸癌是全球婦女排名第二的常見癌症,目前已有足夠證據顯示人類乳突腫瘤病毒感染是導致子宮頸上皮細胞內層腫瘤以及侵犯性子宮頸癌最重要的原因,幾乎所有的子宮頸癌都肇因於人類乳突腫瘤病毒的感染,其中以第16、18型最主要。本研究主要目的是以點漬分析法和雜交層析法偵測人類乳突腫瘤病毒第16型DNA。
本研究分為兩步驟,首先以HMY09和BMY11當引子進行聚合酶鏈鎖反應,將SiHa細胞染色體的晚期基因構築至大腸桿菌質體上。然後利用streptavidin和biotin與anti-streptavidin antibody的強結合力,設計一段互補部分晚期基因的biotin-DNA序列,能和固定在硝化纖維膜上的streptavidin或anti-streptavidin antibody結合;設計另一股部分互補晚期基因的硫醇-DNA,將末端修飾硫醇基的DNA夠固定在奈米金粒子的表面上。由於biotin-DNA和硫醇-DNA能和目標DNA雜交結合,利用點漬分析法和雜交層析法進而可以判讀DNA約略濃度。實驗結果證實,以點漬分析法在單股偵測可到100 amol,進一步銀還原能夠偵測到100 zmol;以雜交層析法在單股偵測至少能到160 zmol。而在雙股的構築質體偵測上,點漬分析法可偵測到100 amol,銀還原後可偵測到100zmol,雜交層析法可偵測到100 amol。
論文英文摘要:Human papillomavirus can lead to cancer of the cervix, vulva, vagina, and anus in women. High risk HPV type 16 and 18 infections have been commonly identified in cervical cancer. The purpose of this study is to detect DNA of HPV16 using gold nanoparticales dot blot test and hybrid chromatography test.
The assay could be implemented via three steps: First, we constructed an E.coli plasmid, pHC, containing SiHa cell chromosome HPV16 L1 region. Second, we designed biotin-DNA and SH-DNA probes as capture and detect probes, each complementary with L1 region. The former probe would associate with streptavidin and anti-streptavidin antibody on the nitrocellulose membrane, while the latter probe would associate with gold nanoparticles using covalent bond. Third, with hybridization, dot blot and chromatography methods were used to detect HPV. Base on the results, 100 amol of ssDNA and 100 amol of dsDNA were detected using the dot blot method. Furthermore, 160 zmol of ssDNA and 100 amol of dsDNA were detected using the chromatography method.
論文目次:目 錄

中文摘要 i
英文摘要 ii
誌謝 iii
目錄 iv
表目錄 vii
圖目錄 viii
第一章 緒論 1
1.1 前言 1
1.2 研究動機及目的 4
第二章 研究背景與原理介紹 5
2.1 人類乳突瘤病毒與子宮頸癌 5
2.2 現行HPV檢測方法及原理 7
2.2.1 細胞學檢查 7
2.2.2 薄層細胞學檢查 8
2.2.3 螢光檢測法 9
2.2.4 電腦輔助抹片判讀 9
2.2.5 HPV感染的分子診斷 9
2.3 奈米金粒子檢測技術介紹 15
2.3.1 膠體金結構 16
2.3.2 膠體金特性 16
2.3.3 膠體金粒子吸收光譜 16
2.3.4 膠體金製備方法 17
2.3.5 奈米金的應用 17
2.4 體外檢測診斷技術 21
2.4.1 體外檢測診斷技術分類 22
2.4.4 檢測診斷技術市場發展 25
第三章 實驗方法與步驟 27
3.1 實驗架構 27
3.2 以硫醇基將DNA 固定在金奈米粒子表面 29
3.3 HPV單股目標DNA點漬分析法 30
3.3.1 膜上固定avidin-biotin DNA probe偵測HPV單股目標DNA點漬分析法 30
3.3.2 膜上固定avidin偵測HPV單股目標DNA點漬分析法 32
3.3.3 膜上固定anti-avidin antibody偵測HPV單股目標DNA點漬分析法 33
3.4 雜交層析法偵測HPV單股目標DNA 35
3.5 重組質體構築 39
3.5.1 質體純化 (Plasmid purification) 41
3.5.2 SiHa cell 染色體純化 (Chromosome purification) 41
3.5.3 聚合酶鏈反應 (Polymerase chain reaction;PCR) 42
3.2.4 酶切 (Restriction enzyme cutting) 43
3.5.5 凝膠電泳 (Gel electrophoresis) 44
3.5.6 膠體純化 (Gel purification) 44
3.5.7 連接 (Ligation) 45
3.5.8 製備勝任細胞 (Preparing competent cell) 45
3.5.9 轉形(Transformation) 46
3.5.10 DNA定序 46
3.6 雜交層析法偵測構築之HPV雙股目標DNA 47
3.7 實驗藥品與設備 49
3.7.1 實驗藥品 49
3.7.2 實驗設備 50
第四章 結果與討論 52
4.1 以單股HPV 16目標DNA為模擬取得最佳化條件 52
4.1.1 不同pH值狀態下的影響 52
4.1.2 不同鹽濃度狀態下的影響 54
4.2 點漬分析法偵測HPV 目標DNA 55
4.2.1 膜上固定avidin偵測合成之單股HPV 16目標DNA 56
4.2.2 膜上固定anti-avidin antibody偵測合成之單股HPV 16目標DNA 57
4.3 雜交層析法偵測合成之單股HPV 16目標DNA 60
4.3.1 膜上固定avidin-biotin probe偵測合成之單股HPV 16目標DNA 60
4.3.2 膜上固定avidin偵測合成之單股HPV 16目標DNA 61
4.3.3 膜上固定anti-avidin antibody偵測合成之單股HPV 16目標DNA 62
4.4 點漬分析法偵測構築之雙股HPV 16目標DNA 64
4.5 雜交層析法偵測構築之雙股HPV 16目標DNA 66
第五章 結論 69
參考文獻 71
附錄 77














表 目 錄

表 3. 1:實驗中所有使用的探針引子序列 30
表 3. 2: 實驗藥品 49
表 3. 3: 實驗設備 50
表 4. 1: 以點漬分析法偵測 HPV 單股目標DNA,以不同物質固定在膜上做比較 60
表 4. 2:以雜交層析法偵測 HPV 單股目標DNA,以不同物質固定在膜上做比較 63
表 4. 3:HPV單股目標DNA偵測敏感度,不同偵測方法之比較。 64
表 4. 4:以雜交層析法在膜上固定偵測不同型態目標DNA之結果。 68
表 5. 1:實驗總結果 70


























圖 目 錄

圖 2. 1:人類乳突病毒致癌機制圖 6
圖 2. 2:子宮頸癌癌化過程在細胞型態學上的分類 8
圖 2. 3:hybrid capture II(HC2)概略步驟 10
圖 2. 4:NASBA偵測方法概要之示意圖 12
圖 2. 5: HPV DNA 檢測技術相對之敏感度 13
圖 2. 6:Corstjens 等人之方法示意圖 14
圖 2. 7:Mao等人之方法示意圖 15
圖 2. 8:概要描述各種代表性的金奈米探針的形成 18
圖 2. 9:260nm波長金奈米粒子探針與目標DNA結合後在不同溫度下之吸收情形 19
圖 2. 10:利用含銀離子的硝酸銀溶液可增強金奈米粒子偵測訊號 20
圖 2. 11:驗孕試紙原理示意圖 23
圖 2. 12:簡便使用型體外檢測診斷試劑側視圖 23
圖 3.1:實驗流程圖 28
圖 3. 2:HPV單股目標DNA以點漬分析法偵測,膜上固定avidin-biotin DNA probe示意圖 32
圖 3. 3:HPV單股目標DNA以點漬分析法偵測,膜上固定avidin示意圖 33
圖 3. 4:HPV單股目標DNA以點漬分析法偵測,膜上固定anti-avidin antibody示意圖 35
圖 3. 5:HPV單股目標DNA以雜交層析法偵測,膜上固定avidin-biotin DNA probe示意圖 37
圖 3. 6:雜交層析法,實驗裝置圖 37
圖 3. 7:HPV單股目標DNA以雜交層析法偵測,膜上固定avidin示意圖 38
圖 3. 8:HPV單股目標DNA以雜交層析法偵測,膜上固定anti-avidin antibody示意圖 39
圖 3. 9:質體構築之流程圖 40
圖 3. 10:引子與SiHa cell L1 region位置相對示意圖 43
圖 3. 11:雙股目標DNA位置與Au-SH DNA probe和biotin-DNA probe 偵測相對位置示意圖 48
圖 3. 12:HPV雙股目標DNA以雜交層析法偵測,膜上固定anti-avidin antibody示意圖 48
圖 4. 1:HPV單股目標DNA以點漬分析法偵測,膜上固定anti-avidin antibody,在不同pH值下實驗結果 53
圖 4. 2:HPV單股目標DNA以點漬分析法偵測,膜上固定anti-avidin antibody,在不同鹽濃度下之實驗結果 54
圖 4. 3:HPV單股目標DNA以點漬分析法偵測,膜上固定avidin,銀還原前實驗結果 56
圖 4. 4:HPV單股目標DNA以點漬分析法偵測,膜上固定avidin,銀還原後實驗結果 57
圖 4. 5:HPV單股目標DNA以點漬分析法偵測,膜上固定anti-avidin antibody,銀還原前實驗結果 58
圖 4. 6:HPV單股目標DNA以點漬分析法偵測,膜上固定anti-avidin antibody,銀還原後實驗結果 59
圖 4. 7:HPV單股目標DNA以雜交層析法偵測,膜上固定avidin-biotin probe之實驗結果 61
圖 4. 8:HPV單股目標DNA以雜交層析法偵測,膜上固定avidin之實驗結果 61
圖 4. 9:HPV單股目標DNA以雜交層析法偵測,膜上固定anti-avidin antibody之實驗結果 62
圖 4. 10:以點漬分析法偵測構築之HPV雙股目標DNA,膜上固定anti-avidin antibody銀還原前之實驗結果 65
圖 4. 11:以點漬分析法偵測構築之HPV雙股目標DNA,膜上固定anti-avidin antibody銀還原後之實驗結果 66
圖 4. 12:以雜交層析法偵測構築之HPV雙股目標DNA,膜上固定anti-avidin antibody之實驗結果 67
圖 4. 13:以雜交層析法偵測構築之HPV雙股目標DNA PCR產物,膜上固定anti-avidin antibody之實驗結果 68
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