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系統識別號 U0026-0812200912055297
論文名稱(中文) 以核糖體RNA基因內轉錄區序列及寡核酸晶片鑑定皮癬菌
論文名稱(英文) Identification of Dermatophytes by Sequence Analysis of the Ribosomal DNA Spacer Regions and by an Oligonucleotide Array
校院名稱 成功大學
系所名稱(中) 醫學檢驗生物技術學系碩博士班
系所名稱(英) Department of Medical Laboratory Science and Biotechnology
學年度 94
學期 2
出版年 95
研究生(中文) 李欣潔
研究生(英文) Hsin-Chieh Li
學號 t3693103
學位類別 碩士
語文別 中文
論文頁數 88頁
口試委員 指導教授-張長泉
口試委員-許明隆
口試委員-吳俊忠
口試委員-李淑英
中文關鍵字 皮癬菌  寡核苷酸晶片  核糖體核酸基因內轉錄區  鑑定 
英文關鍵字 oligonucleotide array  identification  ribosomal DNA internal transcribed spacer  dermatophytes 
學科別分類
中文摘要 皮癬菌 (dermatophytes)包含3屬 (genera),分別為表皮癬菌屬 (Epidermophyton)、小芽胞菌屬 (Microsporum)和毛癬菌屬 (Trichophyton),其中有許多菌種會感染人類。本研究利用核糖體核酸基因內轉錄區 (internal transcribed spacer, ITS)序列及DNA晶片來鑑定這三菌屬的菌株。本研究定序標準菌株 (type strain)和參考菌株 (reference strain)的ITS序列,並結合由GenBank取得的參考菌株序列構成一個ITS序列資料庫,藉此資料庫以鑑定不同菌種。以ITS序列鑑定128株臨床分離之目標菌株 (target strain),靈敏度為100% (128/128)。在DNA晶片方面利用ITS序列設計專一性寡核酸探針 (oligonucleotide probe),點漬在尼龍膜上以鑑定皮癬菌。待鑑定菌種之ITS區域經PCR放大後,與晶片上的探針進行雜合反應,再使用酵素呈色法檢測雜合反應。本研究對17種皮癬菌設計了20個專一性探針,將其點製成0.5 cm × 0.5 cm的微陣列晶片,在測試了198菌株 (67參考菌株,131臨床分離株)後,此晶片之靈敏度為99.5% (197/198)。並測試90株非目標菌株 (non-target strain),特異性為97.8% (88/90)。若以Nested PCR增幅ITS區域,再進行雜合反應,此晶片之檢測極限 (detection limit)為1 pg (Epidermophyton floccosum ATCC 9646)至10 fg菌體DNA (Microsporum gypseum BCRC 30542)。以此晶片直接檢測臨床檢體 (表皮皮屑,指甲,及頭髮),其檢出率為84.2% (16/19)。本研究結果顯示,以DNA晶片及ITS序列鑑定臨床上常見之皮癬菌,兼具快速及正確之特性。

英文摘要 Many species of dermatophytes (Epidermophyton, Microsporum and Trichophyton) are human pathogens. The aims of this study was to construct a database of ribosomal DNA internal transcribed spacer (ITS) sequences and an oligonucleotide array to identify 17 species of dermatophytes. The ITS sequences of type strains and reference strains were sequenced and combined with ITS sequences of reference strains available in GenBank, ITS sequence database were constructed. The ITS sequences of dermatophytes had high intraspecies similarities and low interspecies similarities, and this characteristic was used to design species-specific oligonucleotide probes. A total of 128 target clinical isolates were identified by ITS sequencing and a sensitivity of 100% (128/128) was obtained. In addition, 20 species-specific oligonucleotide probes were used to fabricate a DNA chip (0.5 cm × 0.5 cm) to identify 17 species of dermatophytes. The ITS regions of dermatophytes were amplified by PCR and hybridized to probes immobilized on nylon membrane chip. A collection of 198 strains (67 reference strains and 131 clinical isolates) were identified by the chip, and a sensitivity of 99.5% (197/198) was obtained. In addition, 90 non-target strains were tested and a specificity of 97.8% (88/90) was obtained. By using nested PCR, the detection limit of the chip ranged from 1 pg (Epidermophyton floccosum ATCC 9646) to 10 fg (Microsporum gypseum BCRC 30542) of genomic DNA. The oligonucleotide chip was also used to directly detect dermatophytes in clinical specimens (skin scale, nail, and hair), and a detection rate of 84.2% (16/19) was obtained. In conclusion, ITS sequencing and the oligonucleotide array hybridization are rapid and accurate methods for identification and detection of clinically important dermatophytes.

論文目次 中文摘要...................................................................................................................Ⅰ
英文摘要...................................................................................................................Ⅱ
誌謝............................................................................................................................Ⅲ
目錄............................................................................................................................Ⅳ
表目錄........................................................................................................................Ⅶ
圖目錄........................................................................................................................Ⅸ

緒論
皮癬菌的分類..............................................................................................................1
皮癬菌的感染..............................................................................................................1
致病性皮癬菌的分佈..................................................................................................1
治療皮癬菌感染的藥物..............................................................................................3
皮癬菌的抗藥性..........................................................................................................3
皮癬菌的鑑定方法......................................................................................................4
核糖體核酸基因內轉錄區 (rRNA gene internal transcribed spacer, ITS)...................5
研究目的......................................................................................................................6
研究架構......................................................................................................................7

材料與方法
實驗菌種及臨床檢體..................................................................................................8
DNA萃取 (DNA extraction).........................................................................................9
ITS區域的增幅...........................................................................................................10
ITS區域的定序...........................................................................................................11
ITS區域之序列資料庫建構.......................................................................................12
增幅ITS區域以進行晶片雜合反應...........................................................................13
探針設計及製備.........................................................................................................13
晶片製備.....................................................................................................................14
晶片雜合反應.............................................................................................................15
晶片雜合反應結果判讀.............................................................................................17
靈敏度 (sensitivity)以及特異性 (specificity)定義......................................................17

結果
ITS區域的定序以及ITS序列資料庫的建立.............................................................18
小芽胞屬菌種 (Microsporum spp.)之鑑定................................................................18
毛癬菌屬菌種 (Trichophyton spp.)之鑑定...............................................................19
ITS序列鑑定判讀依據..............................................................................................20
以ITS序列資料庫鑑定臨床分離株..........................................................................21
探針設計及篩選........................................................................................................23
種特異性 (species-specific)探針................................................................................23
寡核酸晶片鑑定菌株之結果................................................................................24
寡核酸矩陣晶片之偵測極限 (detection limit)......................................................25
以寡核酸晶片直接檢測臨床檢體中之皮癬菌....................................................25

討論
以ITS序列資料庫鑑定皮癬菌...................................................................................27
具有二種基因型 (genotypes)之菌種.........................................................................28
探針設計.....................................................................................................................29
以寡核酸晶片鑑定皮癬菌.....................................................................................30
參考文獻.....................................................................................................................32

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