結核病是目前全球傳染病中死亡率最高的疾病，由於抗藥菌株的產生以及與愛滋病的共同感染而使得結核病的控制面臨更嚴厲的挑戰。目前，結核病的預防主要是施打卡介苗，卡介苗是世界上使用最廣泛的疫苗之一，其優點是沒有施打時間的限制、便宜、副作用小、穩定性高。卡介苗目前仍然是採用培養後統計可培養之菌落數來判定疫苗之效價。但由於卡介苗菌的生長十分緩慢，培養時間有時甚至長達二個月，製程監控不易達到效果。本研究的目的即是利用同步定量聚合酶連鎖反應技術檢測BCG 16S ribosomal RNA及IS6110基因，並分別比較檢測BCG菌之DNA及RNA檢體的敏感度及專一性，以建立一個快速、靈敏度高且專一性高的BCG效價檢測方法。在研究中已成功建立檢測之標準曲線，並能在8天培養後檢測出BCG菌的生長，可以有效率的減少BCG疫苗菌數的檢測時間。因此，期望未來能夠將此技術應用做為BCG疫苗生產製程中管控的工具，以取代曠日費時的傳統檢測方式，並期望將來可以將此檢測系統應用於結核病之臨床檢測。 / Tuberculosis is one of the important infectious diseases with the highest mortality rate worldwide. The control of tuberculosis infection is facing an even more strict challenge because of the evolving of drug-resistant clones and the co-infection of AIDS. Currently, the prevention of tuberculosis is mainly done by BCG vaccination, which is one of the most widely used vaccines. The advantages of BCG vaccine are its low cost, low side effects, and high stability. At present, the BCG potency is analyzed by calculating the growth of colonies after culture. However, the growth of BCG is pretty slow, sometimes it can take as long as two months and has made the in process control very difficult. The aim of this study was to establish a rapid, sensitive, and specific real-time quantitative PCR (Q-PCR) method for the detection of BCG potency and separately compares BCG DNA and RNA samples for BCG 16S ribosomal RNA and IS6110 genes for Q-PCR detection system. We have successfully set up the standard curves for the detection of BCG using Q-PCR, and the method could detect the growth of BCG after 8 day of cultivation. Using Q-PCR system, the detection of BCG vaccine is more efficient and the detection time can be largely reduced. We hope we will be able to apply this method as a tool for the in process control during the BCG vaccine production and to replace the time-consuming traditional culture method. / 目錄
誌謝 ------------------------------------------------------------------------ i
中文摘要 ------------------------------------------------------------------------ ii
英文摘要 ------------------------------------------------------------------------ iii
目錄 ------------------------------------------------------------------------ iv
表目錄 ------------------------------------------------------------------------ vi
圖目錄 ------------------------------------------------------------------------ viii
第一章 緒論------------------------------------------------------------------ 1
第二章 文獻回顧------------------------------------------------------------ 4
第一節 結核病 (Tuberculosis)-------------------------------------------- 4
第二節 分歧桿菌 (Mycobacteria)---------------------------------------- 6
第三節
卡介苗 (Bacille Calmette-Guérin vaccine ; BCG vaccine) 的發展---------------------------------------------------------------
8
第四節
聚合酶連鎖反應 (Polymerase chain reaction；PCR) 及其應用------------------------------------------------------------------
9
第五節
同步定量聚合酶連鎖反應 (Real-time quantitative polymerase chain reaction; 定量PCR)-------------------------
10
第三章 材料與方法--------------------------------------------------------- 13
一 BCG菌製備與培養----------------------------------------------- 13
二 定量PCR的primer與probe設計----------------------------- 13
三 定量PCR的primer與probe最適化-------------------------- 14
四 BCG DNA檢體萃取---------------------------------------------- 14
五 BCG RNA萃取與反轉錄---------------------------------------- 15
六 定量PCR反應條件----------------------------------------------- 16
七 傳統PCR反應條件----------------------------------------------- 16
八 洋菜膠體 (agarose gel) 電泳分析----------------------------- 16
九 BCG定量標準曲線製作與製圖-------------------------------- 17
第四章 結果與討論--------------------------------------------------------- 18
一 定量PCR primer 與 probe的設計----------------------------- 18
二
16S rRNA及IS6110 定量PCR中primers與 probes 之最適化---------------------------------------------------------------
19
三 建立BCG疫苗之定量PCR檢測標準曲線------------------- 20
四 以16S rRNA primer進行BCG疫苗培養分析--------------- 22
五 以定量PCR檢測BCG疫苗菌株之最佳檢測濃度--------- 23
六
依16S rRNA及IS6110定量PCR建立BCG疫苗效價之檢測------------------------------------------------------------------
25
第五章 結論與建議--------------------------------------------------------- 27
參考文獻 ------------------------------------------------------------------------ 30
附錄一 16S ribosomal RNA 基因序列---------------------------------- 81
附錄二 IS6110基因序列--------------------------------------------------- 83
表目錄
表一 臨床中重要的分歧桿菌------------------------------------------ 37
表二 分歧桿菌之致病力------------------------------------------------ 38
表三
Primer Express軟體設計之16S rRNA及IS6110 primers與probes------------------------------------------------------------
39
表四 定量PCR primer與probe最適化測試濃度組合------------ 40
表五 反轉錄反應藥品配方--------------------------------------------- 41
表六 定量PCR試劑配方------------------------------------------------ 42
表七 定量PCR反應條件------------------------------------------------ 43
表八 傳統PCR試劑配方------------------------------------------------ 44
表九 16S rRNA定量PCR之primer與probe最適化結果------- 45
表十 IS6110定量PCR之primer與probe最適化結果------------- 46
表十一 以16S rRNA及IS6110定量PCR檢測DNA檢體結果---- 47
表十二 以16S rRNA及IS6110定量PCR檢測RNA檢體結果---- 48
表十三
16S rRNA及IS6110定量PCR標準曲線之有效檢測範團及靈敏度比較------------------------------------------------------
49
表十四
16S rRNA定量PCR檢測BCG疫苗在不同培養條件下之生長情形------------------------------------------------------------
50
表十五 以16S rRNA定量PCR檢測BCG疫苗之最佳檢測濃度--- 51
表十六 以IS6110定量PCR檢測BCG疫苗之最佳檢測濃度-------- 52
表十七 16S rRNA定量PCR檢測BCG疫苗之效價------------------ 53
表十八 IS6110定量PCR檢測BCG疫苗之效價---------------------- 55
圖目錄
圖一 TaqMan Probe之定量PCR的作用原理反應流程--------- 57
圖二
ABI 7700 Sequence Detection System之同步偵測光學路徑原理---------------------------------------------------------------
58
圖三 定量PCR偵測螢光後之Threshold cycle圖---------------- 59
圖四 PCR過程及定量PCR偵測原理------------------------------- 60
圖五
Primer Express軟體中，設計primer與probe的 Preferences操作界面----------------------------------------------
61
圖六
以傳統PCR測試16S rRNA primer之PCR產物agarose gel電泳分析--------------------------------------------------------
62
圖七
以傳統PCR測試IS6110 primer之PCR產物agarose gel電泳分析------------------------------------------------------------
63
圖八 16S rRNA定量PCR primer與probe 最適化結果----------- 64
圖九 IS6110定量PCR primer與probe 最適化結果--------------- 65
圖十
16S rRNA定量PCR primer與probe最適化結果產物agarose gel 電泳分析---------------------------------------------
66
圖十一 16S rRNA定量PCR以DNA建立檢測標準曲線之結果--- 67
圖十二 16S rRNA定量PCR以DNA檢體所建立之標準曲線----- 68
圖十三 16S rRNA定量PCR以RNA檢體建立標準曲線之結果-- 69
圖十四 16S rRNA 定量PCR以RNA檢體所建立之標準曲線---- 70
圖十五 IS6110定量PCR以DNA檢體建立標準曲線之結果------ 71
圖十六 IS6110 定量PCR以DNA檢體所建立之標準--------------- 72
圖十七 IS6110定量PCR以RNA檢體建立標準曲線之結果------ 73
圖十八 IS6110定量PCR於RNA檢體所建立之標準曲線---------- 74
圖十九
以16S rRNA 定量PCR檢測BCG疫苗DNA檢體之生長------------------------------------------------------------------------
75
圖二十
以16S rRNA 定量PCR檢測BCG疫苗RNA檢體之生長------------------------------------------------------------------------
76
圖二十一 建立16S rRNA 定量PCR之BCG菌最佳檢測濃度-------- 77
圖二十二 建立IS6110定量PCR之BCG菌最佳檢測濃度------------ 78
圖二十三 以16S rRNA定量PCR檢測BCG疫苗效價----------------- 79
圖二十四 以IS6110定量PCR檢測BCG疫苗效價---------------------- 80
| Identifer | oai:union.ndltd.org:TW/094FY005108005 |
| Date | January 1994 |
| Creators | 李煒明, Wam-Ming Li |
| Contributors | 廖明一, Ming-Yi Liau |
| Publisher | 輔英科技大學, 生物技術系碩士班 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | 中文 |
| Detected Language | English |
| Type | 碩士 |
| Format | 84 |
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