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研究生： 蕭淳仁
Hsiao, Chen-Ren
論文名稱： 以寡核苷酸微陣列晶片快速鑑定臨床上重要的黴菌
Rapid Identification of Medically Important Molds by an Oligonucleotide Array
指導教授： 張長泉
Chang, Tsung-Chain
學位類別： 碩士
Master
系所名稱： 醫學院 - 醫事技術學系
Department of Medical Technology
論文出版年： 2004
畢業學年度： 92
語文別： 中文
論文頁數： 141
中文關鍵詞： 黴菌 、鑑定 、寡核苷酸微陣列晶片
外文關鍵詞： oligonucleotide array, internal transcribed spacer, ITS, molds, identification
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    • 　　近幾年來，由於醫藥環境改變，黴菌感染有逐漸增加的趨勢，特別在免疫不全、癌症治療、骨髓以及器官移植等病人中，是重要的感染菌。快速的菌種鑑定有利於抗真菌藥物的適當使用。傳統的黴菌鑑定以菌種型態以及生化試驗為主，然而部分菌種生長緩慢或是型態類似，因此鑑定困難。本研究利用核糖體核酸內轉錄區(internal transcribed spacer, ITS)序列，其在同種間有高相似度而在異種間低相似度的特性，設計專一性探針，並將其點製在尼龍膜上成為晶片。不同菌種之ITS的PCR產物經加熱變性，與探針進行雜合反應後，再使用呈色法檢測雜合反應，以肉眼直接判讀。本研究一共對65菌種(34菌屬)篩選57個專一性探針，將其點製成約1.1 cm × 0.9 cm的微陣列晶片。以此晶片測試258株參考菌株，靈敏度為98.5% (196/199)，專一性為93.2% (55/59)；測試71株臨床分離株，靈敏度為94.4% (51/54)，專一性為88.2% (15/17)。臨床感染上重要的Aspergillus，本研究針對其中7菌種(參考菌株53株，以及臨床分離株22株)加以鑑定，靈敏度達到100%。依照本研究的結果，以寡核苷酸微陣列晶片鑑定臨床黴菌，可以快速以及正確的鑑定不同的菌種，是一個具有發展潛力的檢驗工具。

    　　Mold infection is increasing in recent years. Because of the advance of medical science and the growing population of immumocompromised, bone marrow transplantation and solid organ transplantation patients, fungi become important pathogens. Accurate and rapid identification of molds causing infections is important for appreciate patient treatment with antifungal agents. The conventional identification methods of molds heavily rely on the morphological characteristics and biochemical tests. Some fungal species may grow slowly and are morphologically indistinguishable, so the conventional methods are sometimes not suitable. We utilized ribosomal RNA internal transcribed spacer (ITS) sequence and an oligonucleotide array to identify clinically relevant molds. Specific probes were designed from the ITS sequences and were spotted onto a nylon membrane. The ITS regions of molds were amplified by PCR and hybridized to probes on the membrane chip. The hybridization results were visualized by color development on the chip. In this study, 57 specific probes were spotted on an 1.1 cm × 0.9 cm chip, which was used to identify 65 species, belonging to 34 genera. A total of 258 reference strains were tested and a sensitivity of 98.5% (196/199) was obtained, whereas the specificity was 93.2% (55/59). Seventy one clinical isolates were tested and the sensitivity was 94.4% (51/54), however the specificity was 88.2% (15/17). The sensitivities for the identification of 7 Aspergillus species including 53 reference strains and 22 clinical isolates were both 100%. In conclusion, oligonucleotide array is a powerful tool to rapidly and accurately identify most important mold pathogens.

    目錄 中文摘要 ………………………………………………………………Ⅰ 英文摘要 ………………………………………………………………Ⅱ 誌謝 ………………………………………………………………Ⅲ 目錄 ………………………………………………………………Ⅳ 表目錄…………………………………………………………………Ⅷ 圖目錄…………………………………………………………………Ⅸ 第一章緒論 …………………………………………… 1 1.1 前言 …………………………………………………1 1.2 真菌的分類…………………………………………… 1 1.3 治療真菌感染的藥物……………………………………3 1.4 真菌感染的用藥…………………………………………5 1.5 黴菌的鑑定方法…………………………………………5 1.6 核糖體核酸基因內轉錄區 (rDNA internal transcribed spacers, ITS).10 1.7 微陣列晶片(Microarray)………………………………13 1.8 研究動機…………………………………………………15 1.9 研究架構…………………………………………………15 第二章實驗原理 ………………………………………………17 2.1 聚合酶連鎖反應 (polymerase chain reactrion, PCR) …………17 2.2 瓊膠電泳 (agarose gel electrophoresis)…………17 2.3 寡核苷酸微陣列晶片反應原理 ………………………18 2.3.1 雜合反應原理……………………………………………18 2.3.2 呈色反應原理…………………………………………18 2.4 定序分析 (DNA sequencing) ………………………20 2.4.1 Maxam-Gilbert sequencing method…………………20 2.4.2 Sanger method…………………………………………21 第三章 實驗材料及研究方法 ……………………………………22 3.1 實驗菌種……………………………………………… 22 3.2 實驗藥品……………………………………………… 30 3.3 實驗藥品配製 …………………………………………………… 32 3.4 實驗設備……………………………………………… 36 3.5 實驗方法……………………………………………… 38 3.5.1 黴菌培養………………………………………………38 3.5.2 黴菌保存……………………………………………38 3.5.3 抽取菌體DNA…………………………………………38 3.5.4 DNA內轉錄區之擴增(PCR反應)…………………… 39 3.5.5 PCR產物確認…………………………………………42 3.5.6  PCR產物純化…………………………………………43 3.5.7  PCR產物定序…………………………………………44 3.5.8  序列分析……………………………………………44 3.5.9 探針設計 ………………………………………………45 3.5.10  雜合反應…………………………………………… 46 第四章 實驗結果 ……………………………………………… 54 4.1 探針篩選結果……………………………………………54 4.2 具種特異性(species-specific)探針…………………54 4.3 可鑑定二種(或以上)黴菌之探針…………………… 62 4.4 參考菌株之雜合反應結果………………………………67 4.5 臨床分離株之雜合反應結果 ………………………70 4.6 植物黴菌分離株雜合反應結果 …………………………73 第五章 討論………………………………………………………75 5.1 參考菌株測試結果 ………………………………… 75 5.2 臨床分離株鑑定結果 ……………………………………77 5.3 植物黴菌分離株鑑定結果………………………………80 5.4 Aspergillus 之鑑定結果…………………………82 第六章 結論…………………………………………………………85 6.1 寡核苷酸晶片鑑定臨床致病性黴菌……………………85 6.2 鑑定靈敏度及專一性……………………………………85 6.3 探針及鑑定菌種…………………………………………86 6.4 序列相近的菌種之探針設計……………………………86 參考文獻………………………………………………………………87

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