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研究生:鄭偉志
研究生(外文):Wei-Chih Cheng
論文名稱:透過表面增強拉曼光譜技術建立結核菌與非結分枝桿菌之分辨方法以及其抗藥性之快速檢驗
論文名稱(外文):Development of a rapid detection method by surface enhanced Raman scattering technique to discriminate mycobacteria and their antibiotic susceptibility
指導教授:林奇宏林奇宏引用關係
指導教授(外文):Chih-Hung Lin
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:生醫光電研究所
學門:工程學門
學類:生醫工程學類
論文種類:學術論文
論文出版年:2014
畢業學年度:103
語文別:中文
論文頁數:66
中文關鍵詞:結核菌拉曼光譜表面增強拉曼光譜抗藥性敏感性檢驗
外文關鍵詞:Mycobacteriium tuberculosisRaman scatteringSurface enhanced Raman scatteringantibiotic susceptibility
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臨床上常用的傳統結核菌群與非結核分枝桿菌檢驗方法及藥物敏感性測試,需要耗費較多的天數來鑑定。本研究利用表面增強拉曼散射技術,發展具有快速偵測、分子振動層級解析度等特性的方法,希望能提供臨床診斷結核菌額外輔助的資訊,並期許能加快診斷結核病的時效性。由實驗研究結果顯示:480 cm^-1波峰可用來鑑定分枝桿菌,而進一步可以730 cm^-1 及1330 cm^-1 拉曼位移來區分結核菌群和非結核分枝桿菌。此外,初步證實全藥物敏感的結核菌添加第一線抗結核藥物isoniaizd處理後,所取到的表面增強拉曼散射光譜在730 cm^-1發生明顯的下降。因此,本研究證實可以藉由表面增強拉曼散射技術區別結核菌群與非結核分枝桿菌;且初步證實可提供一個新穎、快速、簡易的平台運用於結核菌的藥物敏感性試驗。
Clinical diagnosis and drug susceptibility testing of Mycobacterium tuberculosis complex (MTBC) usually takes a long time using conventional methods. The goals of this study are to develop a simple and rapid method for differentiating MTBC and nontuberculous mycobacterium (NTM) and to explore the feasibility of detecting drug susceptibility. The surface enhanced Raman scattering (SERS) technique is a simple and rapid method that can provide molecular vibrational information for clinical diagnosis of MTBC and NTM. Our study results showed that wave number 480 cm-1 is a signature peak for mycobacteria, and 730 cm-1 and 1330 cm-1 can be used to differentiate MTBC and NTM. In addition, pansusceptible M. tuberculosis isolate treated with isoniazid, a first-line anti-tuberculosis drug, we observed intensity change of the SERS spectrum at 730 cm-1. We concluded that SERS technique is a rapid method and can be applied for MTBC diagnosis and drug susceptibility testing in clinical settings.
目錄
致謝 i
Abstract ii
中文摘要 iii
目錄 iv
圖表目錄 vi

第一章 研究動機 1
第二章 研究背景 2
2.1 結核病的流行病學 2
2.2 分枝桿菌的生理特性與結構[5] 4
2.3 分枝桿菌的分類 8
2.4 分枝桿菌臨床檢驗方法[4] 13
2.5 結核菌藥物感受性試驗 14
2.6 拉曼散射 18
2.6.1 拉曼散射歷史 18
2.6.2 拉曼散射原理[21,22] 19
2.7 表面增強拉曼散射 25
2.7.1 表面增強拉曼散射歷史 25
2.7.2 表面增強拉曼散射原理[23,25,28] 27
第三章 材料與方法 30
3.1 拉曼光譜儀及系統架構 30
3.2 奈米銀粒子陣列基板 31
3.3 分枝桿菌樣本前處理 32
3.3.1 透過SERS光譜區分MTBC和NTM 32
3.3.2 第二次上清液730cm-1訊號的回復 34
3.3.3 結核菌偵測的極限(極小)菌量 35
3.3.4 分枝桿菌培養基的SERS訊號 37
3.4 利用SERS作結核菌的抗藥性快速檢測 38
3.4.1 製備抗結核菌的藥品 38
3.5.2 SERS抗藥性快速檢測流程與方法 38
第四章、實驗結果 41
4.1 透過SERS光譜區別MTBC和NTM 41
4.2分枝桿菌培養基的SERS訊號 49
4.3 臨床分枝桿菌的盲樣測試 51
4.4結核菌藥物敏感性試驗 52
4.4.1 結核菌在二次水中加藥作用 53
4.4.2 結核菌在7H9 培養基中加藥作用6天 54
4.4.3 結核菌在7H9 培養基中加藥作用5天和3天 56
4.4.3 結核菌偵測極小菌量 57
第五章、討論 60
5.1 透過SERS光譜來區分MTBC和NTM 60
5.2 730 cm-1訊號的回復 61
5.3 結核菌加入INH後,SERS光譜的變化 61
參考文獻 63

圖表目錄
圖1 分枝桿菌細胞壁基本組成[5]:MAPc、MA-AG-PG 6
圖2 分子散射示意圖 20
圖3 分子散射電子能階示意圖 23
圖4 史托克和反史托克拉曼散射相關圖 24
圖5 螢光和拉曼訊號的分子能階圖與光譜[23] 25
圖6 SERS相關文獻成長圖[27] 26
圖7 表面增強拉曼散射的電磁場增強效應[23] 28
圖8 拉曼光譜儀系統簡圖 30
圖9 奈米銀粒子陽極氧化鋁基板[29] (Ag/AAO substrate) 31
圖10 分枝桿菌前處理流程圖 41
圖11 Sup 1、Sup 2及Sup 3及Sus 1、Sus 2及Sus 3的SERS訊號 42
圖12(A) 結核菌ATCC 25177的Sup1 SERS訊號 43
圖12(B) 結核菌ATCC 27294的Sup1 SERS訊號 44
圖13 結核菌群(A)與非結核菌(B)的Sup1 SERS光譜比較 45
圖14 CAP臨床結核菌的Sup1 SERS訊號 46
圖15 CAP臨床結核菌與ATCC標準結核菌的Sup1 SERS光譜比較 47
圖16 CAP臨床非結核分枝桿菌(NTM) Sup1 SERS訊號 48
圖17 M. tuberculosis的Sup1訊號與MTB media訊號比較 49
圖18 生長在不同培養基下,所取得的M. tuberculosis的sup1光譜 50
圖19 盲樣測試CAP臨床分枝桿菌結果 51
圖20 結核菌Sup 2 的730 cm-1訊號回復 52
圖21 結核菌加藥實驗前處理流程圖 53
圖22 結核菌加藥作用6天之光譜變化 54
圖23 結核菌在7H9 培養基加藥作用6天結果 55
圖24 結核菌在7H9 培養基加藥作用5天結果 56
圖25 結核菌在7H9 培養基加藥作用3天結果 57
圖26 結核菌最小偵測量 58
圖27 結核菌在7H9 培養基加藥作用3天結果,將起始菌數由6 x 109 cfu /ml減少為6 x 108 cfu /ml 59
圖28 加藥實驗:起始菌數6 x 109 cfu /ml與6 x 108 cfu /ml,比較730cm-1變化 60

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