臺灣博碩士論文加值系統

乳房炎的發生，最常見的是被細菌感染所造成，當病原菌侵入乳腺組織造成炎症的發生。本研究之目的在發展一生物晶片(oligonucleotide array)，期能正確鑑定乳房炎病原菌。這些病原菌包括: Corynebacterium bovis, Enterococcus faecalis, Escherichia coli, Listeria monocytogenes, Mycoplasma bovis, Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus chromogenes, Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus hyicus, Staphylococcus intermedius, Staphylococcus simulans, Staphylococcus xylosus, Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus parauberis, and Streptococcus uberis等。本研究以16S-23S 核糖體核酸基因間隔區(intergenic spacer region)為標的，設計寡核苷酸探針(probe)，點在尼龍膜(nylon membrane)上製成晶片。以PCR放大病原菌菌株之ITS區域後，以晶片雜合反應(hybridization) 鑑定這些菌種。本研究對18種乳房炎病原菌設計了35個特異性探針，將其點製成0.6 cm × 0.5 cm的陣列晶片，經鑑定322個目標菌株(target strains)及154非目標菌株(nontarget strains)後, 晶片之靈敏性(sensitivity)為100%，特異性為96.2%。顯示本研究的晶片能有效鑑定引起乳房炎的病原菌。

Cow mastitis is caused by a wide variety of microbial pathogens. In this study, an oligonucleotide array was developed to identify the pathogens causing mastitis. These pathogens include Corynebacterium bovis, Enterococcus faecalis, Escherichia coli, Listeria monocytogenes, Mycoplasma bovis, Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus chromogenes, Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus hyicus, Staphylococcus intermedius, Staphylococcus simulans, Staphylococcus xylosus, Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus parauberis, and Streptococcus uberis. Specific oligonucleotide probes were designed from the 16S-23S rRNA gene intergenic spacer (ITS) region and immobilized on Nylon membrane. The identification method consisted of PCR amplification of the ITS region using bacterial universal primers, followed by hybridization of the digoxigenin-labeled PCR product with oligonucleotide probes on the array. A total of 35 species-specific oligonucleotide probes were immobilized on the array (0.6 cm × 0.5 cm) to identify 18 species of bacteria. After testing a collection of 322 target strains and 154 nontarget strains, the sensitivity and specificity of the array was 100 % and 96.2 %, respectively. The current array has a great potential to rapidly and correctly identify bacteria causing bovine mastitis.

中文摘要...................................................I
英文摘要..................................................II
誌謝......................................................IV
目錄......................................................V
第一章 前言......................................1
1.2 實驗架構 2
第二章、文獻回顧............................5
2.1 乳牛乳房炎病原菌鑑定之重要性 5
2.2 乳牛乳房炎病原菌背景介紹 5
2.2.1 傳染性病原菌 (contagious pathogen) 5
2.2.2 環境性病原菌 (environmental pathogen) 5
2.2.3 機會性病原菌 (opportunistic pathogen) 6
2.2.4 其他 6
2.3 乳牛乳房炎病原菌特性探討 6
2.3.1 Corynebacterium bovis 6
2.3.2 Eneterococcus faecalis 6
2.3.3 Escherichia coli 6
2.3.4 Listeria monocytogenes 7
2.3.5 Mycoplasma bovis 7
2.3.6 Pseudomonas aeruginosa 8
2.3.7 Staphylococcus aureus 8
2.3.8 Staphylococcus chromogenes 8
2.3.9 Staphylococcus epidermidis 8
2.3.10 Staphylococcus haemolyticus 9
2.3.11 Staphylococcus hyicus 9
2.3.12 Streptococcus agalactiae 9
2.3.13 Streptococcus dysgalactiae 9
2.3.14 Streptococcus uberis 9
2.4 牛乳微生物之檢測方法 10
2.4.1 傳統方法 10
2.4.1.1 總菌數 10
2.4.1.2 直接鏡檢法 10
2.4.1.3 培養法 10
2.4.1.4牛乳微生物檢測生化反應 11
2.4.2 鑑定病原菌或產物的非培養技術 12
2.4.2.1顆粒凝集方法 (particle agglutination) 12
2.4.2.2免疫電泳法 (counter immunoelectrophoresis, CIE) 12
2.4.2.3酵素聯結免疫分析法 12
2.4.2.4單株抗體技術 (moncolonal antibodies) 12
2.4.2.5分子生物鑑定方法 13
2.5 Microarray 13
2.5.1 DNA array 菌種鑑定技術開發 13
2.5.2 DNA 微點陣的技術運用在微生物的檢測/鑑定 14
2.6 實驗原理 15
2.6.1 PCR 15
2.6.2 16S-23S 核糖體核酸基因序列 16
2.6.3雜交反應原理 16
2.6.4呈色反應原理 19
2.6.5定序分析 (DNA sequencing) 19
第三章 材料及方法.................................21
3.1 實驗材料 21
3.1.1實驗用菌株、培養及保存 21
3.1.2牛乳分離菌來源及保存 21
3.2 實驗方法 32
3.2.1 DNA 之萃取 32
3.2.2 ITS區域之擴增及定序 32
3.2.2.1 PCR試劑 34
3.2.2.2 PCR條件 34
3.2.3 參考菌株DNA分子之電泳分析 34
3.2.3.1瓊脂凝膠電泳 (agarose gel electrophoresis) 34
3.2.3.2聚合酶連鎖反應產物純化 34
3.2.3.3 ITS 選殖(cloning)及定序 35
3.2.4 參考菌株定序分析 35
3.2.5標準菌株資料庫建構 36
3.2.6 16S rRNA基因的定序 36
3.2.7目標菌種分子演化分析 (phylogenetic analysis) 36
3.2.8 增幅 ITS 區域以進行晶片雜合反應 36
3.2.9參考菌株探針設計及測試 36
3.2.10 晶片製備 39
3.2.11 晶片雜合反應 41
3.2.12 晶片雜合反應判定 43
3.2.13探針sensitivity及specificity定義 43
3.2.14生乳中病原菌ITS之擴增 45
3.2.14.1第一次PCR條件 45
3.2.14.2第二次PCR 條件 45
3.2.14.3 電泳 46
第四章 結果與討論...........................................47
4.1實驗參考菌株分析 47
4.2 目標菌DNA分子之電泳分析 47
4.3 標準菌株資料庫建構 50
4.4 目標菌種演化樹分析 (phylogenetic analysis) 探討 55
4.4.1 目標菌 ITS 序列之演化樹分析 55
4.4.2 目標菌 16S rDNA 序列演化樹分析 55
4.5參考菌株ITS序列比對結果 58
4.6 目標菌相似度值計算 63
4.6.1目標菌的 ITS 相似度 63
4.6.2 目標菌的 16S rDNA 相似度 63
4.6.3 目標菌的 ITS 與 16S 序列相似度比較 63
4.7 探針設計及製備 66
4.8菌種特異性探針 (species-specific probe)及雜合反應結果 68
4.9寡核苷酸晶片鑑定菌種之靈敏度(sensitivity) 73
4.10 鑑定非目標菌種之特異性探討 75
4.11 牛乳分離株雜合反應結果 80
4.12 晶片檢測牛乳中病原菌之結果 80
第五章 結論...........................................81
第六章、參考文獻...........................................82
作者簡介...............................................90





表目錄

表1A. 實驗使用之目標菌株.............................23
表1B. 實驗使用之非目標菌株..............................26
表2. 放大intergenic spacer (ITS)所使用之引子...............33
表3. 靈敏度以及特異性定義.............................44
表4. 標準菌株與參考菌株在GenBank上的編號...................51
表5. 目標菌株的ITS相似度比較................................64
表6. 目標菌株的16S相似度比較...............................65
表7. 晶片探針分配表...................................67
表8. 菌種特異性探針雜合反應結果...........................69
表9. 用來鑑定M. bovis的反向探針..........................72
表10. 寡核苷酸晶片鑑定目標菌種之靈敏度………..................74
表 11. Escherichia ITS相似度比較..........................77
表 12. Listeria ITS相似度比較...........................78
表13. 用來針測L. monocytogenes的探針........................79

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