臺灣博碩士論文加值系統

在我們的生活環境中，伴隨空氣品質的惡化，空氣中過量的真菌(特別是致敏性真菌)孢子濃度可能對人體健康產生不良影響，極需要建立相關致敏菌的檢測平台與評估其對國民健康的風險。傳統上，真菌的檢測和鑑定主要依賴形態特徵或生化特性等。本研究開發一種基礎於熔解曲線圖的分子指紋檢測與鑑定真菌，以助於過敏性真菌的疾病管理。High-resolution melting (HRM)技術，比傳統的凝膠電泳分析，快速、低成本且能更有效解析目標DNA序列的差異性。本研究使用BCRC 31123、BCRC 30812、BCRC 32888、BCRC 32054、BCRC 30099、BCRC 30473、BCRC 34548、BCRC 30010、BCRC 32490及BCRC 32628等7屬10種國內常見致敏真菌與HR-1儀器。分別使用通用引子對ITS1/ITS4、28SU1/28U2、IGSV3/IGSV4、Ascoll1/Ascoll2、1a-F2/Ascoll2及LR3R/LR7放大5.8S、LSU、ITS及IGS片段，在產物中鑲嵌LCGreen+ 螢光染劑，再以熔解曲線分析，結果顯示ITS1/ITS 4及28SU1/28U2兩組引子的熔解曲線微分縱剖圖(-dF/dT profile)較有辨識度、且能由其-dF/dT圖區分菌株成10類。接著選用三組引子ITS1/ITS4、28SU1/28U2及LR3R/ LR7，互相搭配進行Duplex PCR+HRM分析，結果顯示ITS1/ITS4、LR3R/LR7組的Duplex PCR產物之-dF/dT圖辨識度較Singlex PCR組高、且更能辨識10株菌區分成10類。而同時使用三組引子對ITS1/ITS4、28SU1/28U2及LR3R/LR7進行Multiplex PCR後再以熔解曲線分析，結果顯示對10株菌都具有最高的區別性及辨識度，其專一性高於Singlex PCR或Duplex PCR組。

In our living environment, excess amounts of airborne fungal spores, especially for the existences of allergic fungi, might have adverse effects on human health as the quality of air deteriorates. It is urgent to build a detection and identification platform and to assess the health risks and hazards for these allergic fungi. Traditionally, fungi are detedted and classfied mainly by their morphological characteristics and biochemical features, among others. This research is intended to employ the melting profiles as molecular fingerprints for fungal species detection and identification to assist the disease management of allergic fungi. High-resolution melting (HRM) analysis, compared with the traditional agarose gel electrophoresis, is rapid, cost- effective and more powerful in resolving the difference in the DNA sequences of interest. Totally, 10 allergic fungal strains, belonging to 7 genera, prevalent in Taiwan including BCRC 31123, BCRC 30812, BCRC 32888, BCRC 32054, BCRC 30099, BCRC 30473, BCRC 34548, BCRC 30010, BCRC 32490 and BCRC 32628 as well as HR-1 instrument were used. Six universal primer sets ITS1/ITS4, 28SU1/28U2, IGSV3/ IGSV4, Ascoll1/Ascoll2, 1a-F2/Ascoll2 and LR3R/LR7 were chosen to amplify the 5.8S, LSU, ITS, and IGS rDNA fragments, respectively. The results showed that the HRM profiles(-dF/dT profiles) of the LCGreen+ -labelled ITS or LSU amplicons, when primer ITS1/ITS4 or LR3R/LR7 was used, the resulting distinguishable profiles could be classified into 10 types. When primer sets ITS1/ITS4, 28SU1/28U2 and LR3R/LR7 were subsequently used in pairs for the Duplex PCR studies, the results indicated that the ITS-LSU amplicons, if primers ITS1/ITS4 and LR3R/LR7 were used, resulted in distinctive melting profiles and could group the examined strains into 10 types. In addition, the specificity was evidently higher than those of the Singlex PCR. Lastly, the result proved that the Multiplex PCR, with the primers ITS1/ITS4, 28SU1/28SU2 and LR3R/LR7, coupled with the HRM analysis was the most useful mean to differentiate the 10 examined strains.

壹、緒論...1
一、 前言...1
二、 研究背景與文獻回顧...2
1. 致敏性真菌...2
2. 真菌檢測...5
3. 研究動機與目的 ...9
貳、實驗材料與方法 ...11
一、 菌種與保存...11
二、 菌株培養...12
三、 菌株基因組DNA萃取...12
四、 引子對選擇...13
五、 聚合酶連鎖反應...14
六、 瓊脂膠體電泳...14
七、 高解析熔解曲線分析...15
參、實驗結果...16
一、利用單一組序列引子對致敏性真菌檢測...16
1. 10種真菌的單引子放大ITS片段之HRM分析...16
2. 10種真菌的單引子放大LSU片段之HRM分析...17
3. 8種真菌的單引子放大IGS片段之HRM分析...19
4. 10種真菌的單引子放大5.8S片段之HRM分析...20
二、利用兩組序列引子產物對致敏性真菌檢測...22
1. 10種真菌的雙引子放大ITS與LSU片段之HRM分析...22
2. 10種真菌的雙引子放大LSU片段之HRM分析...24
三、利用三組序列引子產物對致敏性真菌檢測...25
10種真菌的三引子放大ITS與LSU片段之HRM分析...25
肆、討論...28
一. DNA條碼選擇...28
二. 結合Duplex PCR及Multiplex PCR的HRM檢測...29
三. 電泳分析與HRM分析靈敏度的比較...31
四. 致敏真菌的分子檢測平台...31
圖次...34
表次...54
參考文獻...85
附錄...90

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