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研究生：	黃莉媖 Huang, Liyin
論文名稱：	以核醣體核酸基因內轉錄區之序列鑑定臨床黴菌 Identification of Medically Relevant Molds by Sequence Analysis of the Internal Transcribed Spacer Regions 1 and 2
指導教授：	張憲彰 Chang, Hsien-Chang 張長泉 Chang, Tsung Chain
學位類別：	碩士 Master
系所名稱：	工學院 - 醫學工程研究所 Institute of Biomedical Engineering
論文出版年：	2003
畢業學年度：	91
語文別：	中文
論文頁數：	91
中文關鍵詞：	核醣體核酸內轉錄區、黴菌、真菌感染、資料庫
外文關鍵詞：	internal transcribed spacer region, fungal infection, database, mold
相關次數：	點閱：62 下載：1
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　　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?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



表目錄

表1.1  真菌的分類 ----------------------------------------------------------- 2
表1.2  真菌病感染的特徵 -------------------------------------------------- 3
表1.3  真菌染色染劑與特徵 ---------------------------------------------- 11
表1.4  真菌絲孢綱的分型 ------------------------------------------------- 12
表1.5  抗真菌藥物對不同伺機性真菌的最低抑菌濃度 (MIC) ---- 15
表3.1  本實驗中所採用的參考菌株 ------------------------------------- 35
表3.2  本實驗中所採用的臨床菌株 ------------------------------------- 38
表3.3  真菌核醣體核酸 (rDNA) 內轉錄區的通用引子 ------------ 41
表4.1  標準菌株 (type strain) 和參考菌株 ( reference strain) 內轉錄
　　　ITS序列長度和相似度值比較 ---------------------------------- 49
表4.2  架構ITS1資料庫的菌種 ----------------------------------------- 64
表4.3  架構ITS2資料庫的菌種 ----------------------------------------- 65
表4.4  Paecilomyces 不同菌種間 ITS1和ITS2 序列之相似度值- 66
表4.5  Aspergillus 不同菌種間 ITS1和ITS2 序列之相似度值  -- 67
表4.6  Fusarium 不同菌種間 ITS1和ITS2 序列之相似度值 ---- 68
表4.7  Microsporum 不同菌種間 ITS1和ITS2 序列之相似度值 - 69
表4.8  Curvularia 不同菌種間 ITS1和ITS2 序列之相似度值 -- 71
表4.9  Trichophyton 不同菌種間 ITS1和ITS2 序列之相似度值 - 72
表4.10  Scytalidium 不同菌種間 ITS1和ITS2 序列之相似度值 -- 73
表4.11 以架構之ITS 序列資料庫鑑定臨床分離株的誤判結果  --- 74
表5.1  GenBank資料庫中缺乏 ITS 序列的臨床絲狀黴菌 .-------- 77



圖目錄
圖1.1  傳統方法鑑定真菌菌種的流程圖 ------------------------------- 10
圖1.2  本實驗研究架構圖 ------------------------------------------------- 18
圖3.1  核醣體核酸 (rDNA) 結構及針對內轉錄區的通用引子 --- 41
圖3.2  瓊脂凝膠電泳的架設 .---------------------------------------------- 42
圖4.1  Aspergillus fumigatus BCRC 32120 rDNA-ITS1定序圖形檔 - 46
圖4.2  Aspergillus fumigatus BCRC 32120 rDNA-ITS1定序文字檔 - 46
圖4.3  Aspergillus fumigatus BCRC 30099, 30502, 32149, 和32120不
　　　同菌株之 ITS1及部分18S和5.8S rDNA 之序列 ---------- 47
圖4.4   Aspergillus fumigatus BCRC 30502經修正的 rDNA- ITS1 序
　　　列  ---------------------------------------------------------------------- 48
圖4.5  Aspergillus fumigatus BCRC 30099, 30502, 32149, 和32120不
　　　同菌株之完整內轉錄區 ITS1 序列比對  --------------------- 48
圖4.6   Acremonium strictum 不同菌株之 ITS 序列 PileUp 分析  -- 55
圖4.7   Acremonium strictum 不同菌株之 ITS1 區域進行 PrettyBox
　　　排列並顯示共同鹼基 (consensus base)  ------------------------ 56
圖4.8   Acremonium strictum 不同菌株之 ITS2 區域進行 PrettyBox
　　　排列並顯示共同鹼基 (consensus base)  ------------------------ 56
圖4.9   Curvularia geniculata 不同菌株之 ITS 序列 PileUp 分析  -- 57
圖4.10  Curvularia geniculata不同菌株之 ITS1 區域進行 PrettyBox
　　　排列並顯示共同鹼基 (consensus base)  ------------------------- 57
圖4.11  Curvularia geniculata不同菌株之 ITS2 區域進行 PrettyBox
　　　排列並顯示共同鹼基 (consensus base)  ------------------------- 58
圖4.12  Curvularia lunata不同菌株之 ITS 序列 PileUp 分析  ---- 59
圖4.13  Curvularia lunata不同菌株之 ITS1 區域進行 PrettyBox 排
　　　列並顯示共同鹼基 (consensus base)  ---------------------------- 59
圖4.14  Curvularia lunata不同菌株之 ITS2 區域進行 PrettyBox 排
　　　列並顯示共同鹼基 (consensus base)  ---------------------------- 60
圖4.15  Trichophyton mentagrophytes var. interdigitale不同菌株之 ITS
　　　序列 PileUp 分析 .------------------------------------------------- 61
圖4.16 Trichophyton mentagrophytes var. interdigitale不同菌株之ITS1
　　　區域進行 PrettyBox 排列並顯示共同鹼基 (consensus base)
　　　--------------------------------------------------------------------------- 62
圖4.17  Trichophyton mentagrophytes var. interdigitale不同菌株之ITS2
　　　區域進行 PrettyBox 排列並顯示共同鹼基 (consensus base)
　　　--------------------------------------------------------------------------- 63
                                    

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校內：2004-02-13公開
校外：2004-02-13公開

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