近年來黴菌感染有增加的趨勢，快速的菌種鑑定有利於抗真菌藥物的適當使用，黴菌傳統的鑑定方法主要是依據菌株型態和生化試驗，通常需要數天或更久的時間。本研究評估核醣體核酸基因 (rDNA) 的內轉錄區 (internal transcribed spacer, ITS) ITS1 和 ITS2的序列，作為鑑定臨床黴菌之可行性。目前GenBank資料庫中，所提供的 ITS 序列仍不足，因此需架構更完整的 ITS 資料庫。本研究以PCR擴增198株 (69種) 參考菌株 (reference strain)的 ITS1和 ITS2 序列，定序之後經由排序整理，於其中選取110條 ITS1 和108條 ITS2序列，並和 GenBank中搜尋到的序列做比對。結果顯示大多數的黴菌其菌種內 (intraspecies) ITS1 和 ITS2 序列之相似度值 (similarity score) 大於 0.95，而不同種間之相似度值小於 0.95。 以此資料庫測試70株臨床分離株 (30種)，得到89% 之靈敏度 (sensitivity) 及90% 之特異性 (specificity)。本研究的結論是，利用ITS 序列分析來鑑定臨床黴菌是快速、簡單、且可靠的方法，能提供不同於傳統鑑定方法的另一種選擇。

The frequency of fungal infections has increased in recent years due to the increasing number of immunocompromised patients. Rapid identification of clinically relevant molds is useful for appropriate treatment with antifungal agents. The conventional methods for fungal identification mostly rely on morphological and biochemical tests; these methods are time-consuming and may be inaccurate. In this study, the feasibility of identification of medically important fungi by using the sequences of the internal transcribed spacer regions 1 and 2 (ITS1 and ITS2) was evaluated. The ITS data in GenBank were insufficient to identify clinically relevant fungi. The ITS1 and ITS2 regions from 198 reference strains (69 species) were amplified by PCR and sequenced; these sequences in combination with data in the GenBank were used to construct an ITS sequence database for the identification of molds. Totally 110 ITS1 and 108 ITS2 sequences were determined in this study. It was found that the ITS similarity scores among strains of the same species usually exceeded 0.95, whereas the scores were less than 0.95 among different species. The database was used to test 70 strains (30 species) of clinical isolates; a sensitivity of 89% and a specificity of 90% were obtained. It was concluded that the present method provides a rapid, simple, and reliable alternative to conventional methods for identification of medically important molds.

中文摘要 ------------------------------------------------------------------------- I
英文摘要 ------------------------------------------------------------------------ II
誌謝 ----------------------------------------------------------------------------- III
目錄 ----------------------------------------------------------------------------- IV
表目錄 ------------------------------------------------------------------------- VII
圖目錄 ------------------------------------------------------------------------ VIII
第一章 緒論 -------------------------------------------------------------------- 1
1.1 真菌的特性與分類 .-------------------------------------------------- 1
1.2 人類真菌病 ----------------------------------------------------------- 3
1.2.1 皮表真菌病 ----------------------------------------------------- 4
1.2.2 下表皮真菌病 -------------------------------------------------- 6
1.2.3..全身性真菌病 -------------------------------------------------- 7
1.2.4..伺機性真菌病 ------------------------------------------------- 8
1.3 真菌的鑑定方法 --------------------------------------------------- 10
1.3.1 傳統鑑定方法 ------------------------------------------------ 10
1.3.2 .DNA 探針測試 ---------------------------------------------- 13
1.3.3 血清試驗 ------------------------------------------------------ 13
1.4 抗真菌藥物治療 --------------------------------------------------- 14
1.5 真菌的院內感染 --------------------------------------------------- 16
1.6 研究目的及動機 --------------------------------------------------- 17
1.7 研究架構 ------------------------------------------------------------ 18
第二章 文獻回顧與實驗原理 ---------------------------------------------- 19
2.1 文獻回顧 ------------------------------------------------------------ 19
2.1.1 酵素檢測法 --------------------------------------------------- 19
2.1.2 抗體抗原反應 ------------------------------------------------ 19
2.1.3 Confocal Roman microspectroscopy ..---------------------- 20
2.1.4 分子檢測法 --------------------------------------------------- 20
2.2 聚合酶鏈反應原理 ------------------------------------------------ 25
2.3 電泳分析原理 ------------------------------------------------------ 26
2.3.1 凝膠電泳 ------------------------------------------------------ 26
2.3.2 凝膠電泳分離原理 ----------------------------------------- .27
2.3.3 等電聚焦 ------------------------------------------------------ 27
2.4 定序分析 ------------------------------------------------------------ 28
2.4.1 Maxam-Gilbert sequencing --------------------------------- 28
2.4.2 dideoxy method ----------------------------------------------- 29
第三章 實驗 ------------------------------------------------------------------- 30
3.1 實驗藥品及配製方法 --------------------------------------------- 30
3.1.1 實驗藥品 ------------------------------------------------------ 30
3.1.2 實驗藥品配製 ------------------------------------------------ 31
3.1.3 實驗設備 ------------------------------------------------------ 33
3.2 菌種來源 ------------------------------------------------------------ 35
3.3實驗 ------------------------------------------------------------------- 39
3.3.1 菌種培養及保存 --------------------------------------------- 39
3.3.2 DNA 萃取 --------------------------------------------------- 39
3.3.3 PCR 放大 ---------------------------------------------------- 40
3.3.4 瓊脂凝膠電泳 ------------------------------------------------ 42
3.3.5 PCR 產物純化 ---------------------------------------------- 43
3.3.6 定序分析 ------------------------------------------------------ 44
3.3.7 序列比對 ------------------------------------------------------ 44
第四章 結果與討論 ---------------------------------------------------------- 46
4.1 序列比對 ------------------------------------------------------------ 46
4.1.1 定序結果 ------------------------------------------------------ 46
4.1.2 同種序列比對 ------------------------------------------------ 47
4.2 建立資料庫 --------------------------------------------------------- 55
4.2.1 區分 genotypes ---------------------------------------------- 55
4.2.2 建構ITS序列資料庫 --------------------------------------- 63
4.2.3 不同菌種間ITS序列的比較 .----------------------------- 66
4.3 測試臨床菌株 ------------------------------------------------------ 74
4.3.1. 誤判之臨床分離株 ------------------------------------------- 74
4.3.2..無法區分之臨床分離株 ------------------------------------ 75
第五章 結論 ------------------------------------------------------------------- 76
5.1 以ITS1和ITS2序列鑑定臨床絲狀黴菌 ---------------------- 76
5.2 GenBank 資料庫中所提供的序列 ----------------------------- 77
5.3 與傳統生長型態鑑定方法結合 --------------------------------- 77
參考文獻 ----------------------------------------------------------------------- 78
附錄 ----------------------------------------------------------------------------- 82

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