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論文中文名稱:運用16S核醣DNA/RNA探針結合金奈米粒子之側流免疫分析檢測沙門氏菌 [以論文名稱查詢館藏系統]
論文英文名稱:Salmonella detection using 16S ribosomal DNA/RNA probe-gold nanoparticles and lateral flow immunoassay [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:工程科技研究所
畢業學年度:101
出版年度:102
中文姓名:劉正哲
英文姓名:Cheng-Che Liu
研究生學號:99679018
學位類別:博士
語文別:中文
口試日期:2013-07-26
論文頁數:92
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:黃志宏;葉明功;高治華;黃光策;吳宛儒
口試委員英文名:Chih-Hung Huang;Ming-Kung Yeh;Jyh-Hwa Kau;Kuang-Tse Huang;Wan-Ru Wu
中文關鍵詞:沙門氏菌檢測16S核醣核酸金奈米粒子側流免疫分析
英文關鍵詞:Salmonella detection16S rRNAgold nanoparticleslateral flow immunoassay
論文中文摘要:本實驗開發一種靈敏、簡單且快速,可用於檢測沙門氏菌的側流免疫分析。其原理是將奈米金粒子接合上一股可互補沙門氏菌16S 核醣DNA與核醣RNA的核酸探針。首先在模擬目標序列的實驗中,運用合成之單股DNA為目標模板進行前測試驗,檢出極限值為5 fmol。本分析法實際應用於培養之沙門氏菌樣本測試,可快速於30分鐘內測得107菌數所萃取之核酸。若輔以銀還原放大訊號,則檢測極限可達104菌數,低於文獻所報導人類攝入沙門氏菌產生臨床癥候之感染菌量—105菌數。此外,相較於其他9株腸內菌(測試結果),本實驗所使用之探針對沙門氏菌確具專一性。故此分析法不僅可應用於微生物檢測,對於食品安全或臨床診斷亦為有效工具,且其兼具靈敏度、專一性及方便操作等優點,適合提供發展中國家之大規模篩選使用。
論文英文摘要:We develop an ultrasensitive, simple, and fast lateral flow immunoassay for Salmonella detection using gold nanoparticles conjugated with a DNA probe, which is complementary to the 16S ribosomal RNA and DNA of Salmonella. The detection limit is 5 fmol for the synthetic single-stranded DNA. For Salmonella cultured samples, the nucleic acids from 107 bacteria were rapidly detected in 30 minutes. After silver enhancement, the detection limit was as low as 104 cells which is lower than 105 bacteria cells, the human infective dose of food-borne Salmonella. Furthermore, the probes used in this study are specific to Salmonella compared to 9 other species of Enterobacteriaceae. This approach would be useful for microbial detection in regards to food safety or clinical diagnosis. It is also suitable for large-scale screening in developing countries because it is sensitive, specific and convenient.
論文目次:摘 要 i
ABSTRACT ii
誌 謝 iii
目 錄 v
圖目錄 vii
表目錄 ix
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 7
2.1核醣RNA簡介 7
2.2沙門氏菌(Salmonella)簡介 8
2.3現行之沙門氏菌標準檢測法 12
2.3.1生化反應檢測法(Biochemical test)之應用 12
2.3.2聚合酶鏈鎖反應應用於沙門氏菌檢測 13
2.4實驗室已發展於檢測沙門氏菌之免疫分析應用 16
2.4.1菌落拓印法(Colony-print immunoassay) 17
2.5本實驗發展之16S核醣核酸探針-金奈米粒子側流免疫分析 (16S ribosomal DNA/RNA probe-gold nanoparticles and lateral flow immunoassay) 18
2.5.1金奈米粒子(Gold nanoparticles)之特性 19
2.5.2側流免疫分析(Lateral flow immunoassay)簡介 21
2.5.3金奈米探針—核醣核酸探針與金奈米粒子的修飾結合 25
2.5.4銀還原訊號放大(Silver enhancement) 27
第三章 實驗方法與步驟 29
3.1實驗研究架構 29
3.2實驗材料與試劑 31
3.3實驗儀器與設備 33
3.4探針設計應用軟體 34
3.5菌種序列與資料庫來源 35
3.6紙膜製作 36
3.7細菌培養 37
3.8核酸萃取純化 38
3.9 16S核酸探針修飾至金奈米粒子表面 39
3.10銀還原染色強化訊號 40
3.11檢測流程 41
3.12訊號定量分析(Quantitative analysis) 42
第四章 實驗結果與討論 43
4.1 16S核酸探針設計 43
4.1.1序列收集與比對(Alignment) 44
4.1.2二聚體自由能(Dimer dG)預測 49
4.1.3本實驗之探針於rRNA二級結構上之結合位預測 50
4.2合成之單股目標DNA測試與定量分析 52
4.3應用於培養之沙門氏菌檢測 54
4.4本分析法之專一性測試 56
第五章 結論 61
參考文獻 63
附 錄 75
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