近年來由於抗生素的濫用，使得細菌產生多重抗藥性而形成超級細菌，抗藥性的形成會提升治癒的難度同時也增加了致死的機率，為公共衛生安全的一大危險，本實驗目標針對近年來常見的耐甲氧西林金黃色葡萄球菌 (methicillin-resistant S.aureus, MRSA)，由於產生抗藥性之特異蛋白 PBP 2a (penicillin-binding protein 2a)，其造成危害對於病人造成一定的威脅。在近年來常用的檢測方法聚合酶連鎖反應(PCR)會有費時且產生偽陽性的憂慮，若使用抗體免疫檢測法，隨然檢測簡單、靈敏，選擇性強，但依賴昂貴且重現性差的動物衍生抗體。本研究之材料具有易合成，和良好收集之特性，於磁性粒子表面修飾鏈親和素 (streptavidin)，與修飾有生物素 (biotin)的核酸適體進行結合，生物素與鏈親和素的結合是已知自然界中最強的非共價相互作用之一，修飾上對 PBP 2a 具有高特異性的核酸適體 (aptamer)，可有效捕捉細菌上特異性蛋白，且成本以及合成時間皆優於抗體檢測方法。結合一維聚丙醯胺膠體電泳分析，可於膠上快速識別107 CFU/mL細菌捕捉後PBP 2a蛋白，最後利用質譜法確定特異性蛋白並進一步利用質譜法可以實現105 CFU/mL鑑別生物標記胜肽，並經由蛋白質資料庫準確識別出是否具有青黴素結合蛋白 2a，並且在複雜細菌環境中仍然展現出有效的鑑別成果。

一、緒論 1
1、前言 1
2、細菌之抗藥性 1
3、抗生素之抗藥機制 3
4、細菌介紹 5
4.1、金黃色葡萄球菌 5
4.2、耐甲氧西林金黃色葡萄球菌 6
5、核酸適體 8
5.1、核酸適體篩選 8
5.2、核酸適體之應用 15
6、質譜檢測法 19
6.1、質譜檢測法於抗藥性細菌上之應用 22
7、研究動機 24
二、研究內容 27
1、藥品與儀器 27
1.1、藥品 27
1.2、儀器 28
1.3、儀器參數與條件 30
1.4、資料庫收尋參數設定 31
2、實驗方法 32
3、實驗步驟 32
3.1、細菌樣品製備 32
3.2、細菌蛋白質破菌 33
3.3、Rapid Gold BCA Assay 估計細菌蛋白質含量 35
3.4、細菌蛋白質水解 37
3.5、合成表面修飾核酸適體之磁性粒子 39
3.6、核酸適體修飾磁性粒子檢測細菌樣品 40
3.7、一維聚丙醯胺膠體電泳分析 40
三、結果與討論 45
1、利用不同方法破菌金黃色葡萄球菌 45
2、青黴素結合蛋白 2a標準品進行水解後質譜結果 46
3、核酸適體利用生物素與鏈親和素間交互作用鍵結於磁性粒子上結合時間優化 49
4、核酸適體修飾磁性粒子捕捉青黴素結合蛋白 2a 51
5、金黃色葡萄球菌與耐甲氧西林金黃色葡萄球菌分析 56
5.1、金黃色葡萄球菌與耐甲氧西林金黃色葡萄球菌進行水 解後質譜分析 56
5.2、抗藥性金黃色葡萄球菌利用核酸適體修飾磁性粒子捕捉後，結合一維聚丙醯胺膠體電泳分析鑑定識別 58
5.3、一維聚丙醯胺膠體電泳分析青黴素結合蛋白2a之偵測極限 60
6、利用質譜法進行蛋白分析 62
6.1、In-gel digestion 膠體消化分析 62
6.2、將蛋白從磁性粒子洗脫再進行水溶液消化 64
6.3、ON BEADS消化分析法 66
6.4、應用於不同MRSA臨床樣品之分析 69
6.5、MRSA、E.coli (大腸桿菌)、Acinetobacter baumannii (AB菌) 混合並利用核酸適體修飾磁性粒子捕捉 78
6.6、MRSA於人工尿液內進行處理及分析 79
6.7、核酸適體修飾磁性粒子材料之重複性 81
7、比較本篇研究之檢測方法與其他相關研究之偵測極限 82
四、結論 83
五、參考文獻 85
六、附錄 95

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