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研究生： 謝馨誼
Hsieh, Hsin-yi
論文名稱： 以寡核苷酸晶片檢測纖維性囊腫病患痰液檢體中之真菌
Development of an Oligonucleotide Array for Detection of Fungi in the Sputa of Patients with Cystic Fibrosis
指導教授： 張長泉
Chang, Tsung-chain
學位類別： 碩士
Master
系所名稱： 醫學院 - 醫學檢驗生物技術學系
Department of Medical Laboratory Science and Biotechnology
論文出版年： 2007
畢業學年度： 95
語文別： 中文
論文頁數： 72
中文關鍵詞： 纖維性囊腫 、寡核苷酸晶片
外文關鍵詞： cystic fibrosis, oligonucleotide array
相關次數： 點閱：104  下載：1 
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    • 纖維性囊腫(cystic fibrosis, CF)是由於纖維性囊腫跨膜傳導調節蛋白(cystic fibrosis transmembrane conductance regulator gene, CFTR)基因發生突變所造成，為白人族群最常見的遺傳疾病之一。CFTR能在上皮細胞扮演氯離子通道(chloride channel)之角色，當此蛋白質功能失常會造成病患呼吸道分泌異常濃稠的黏液，提供給微生物有利的生長環境，往往造成慢性感染，也是導致CF病患死亡的主因。最常從CF病人身上分離到之真菌為Aspergillus spp. 及 Candida spp.，臨床上主要是以痰液培養方式來鑑定，本研究則是發展寡核苷酸晶片來鑑定病患痰液中之真菌。本研究之晶片可鑑定20種真菌，經測試181株菌株(包含132株參考菌株及49株臨床分離株)，晶片之靈敏度為99.4% (180/181)。另外亦測試了142株非目標菌株(共136菌種)，晶片之專一性為98.6% (140/142)。晶片之偵測極限(detection limit)可達1 pg (Paecilomyces variotii CBS 370.70)至10 fg菌體DNA (Aspergillus fumigatus BCRC 30502)。進一步利用此晶片檢測58個CF病患痰液檢體，整體而言能同時檢測到痰液中多種真菌之存在，且往往比傳統培養方法檢測到更多菌種。本研究之寡核苷酸晶片為一種能快速檢測CF病患痰液中真菌之良好工具。

    Cystic fibrosis (CF) is one of the most common genetic disorders in white populations caused by mutations in the cystic fibrosis transmembrane regulator (CFTR) gene. CFTR functions as a chloride channel on the apical membranes of epithelial cells. Deficiency of CFTR impairs functions of the chloride channel and results in production of abnormally thick and sticky mucus. Airways with secretion of sticky mucus provide a favorable environment for infectious microorganisms that may cause chronic infections and these infections are the major cause of morbidity and mortality in CF patients. The most common fungi recovered from CF patients are Aspergillus spp. and Candida spp.. Sputum culture is the standard method used to detect fungal pathogens in clinical specimens. In this study, an oligonucleotide array was developed to detect fungi in the sputa of CF patients. A collection of 181 fungal strains (132 reference strains and 49 clinical isolates) representing 20 target species were analyzed by the array and a sensitivity of 99.4% (180/181) was obtained. A total of 142 nontarget strains (136 species) were also tested and a specificity of 98.6% (140/142) was obtained. The detection limit of the array ranged from 1 pg (Paecilomyces variotii CBS 370.70) to 10 fg (Aspergillus fumigatus BCRC 30502) of genomic DNA. Furthermore, 58 DNA samples extracted from the sputa of CF patients were analyzed by the array for direct detection of fungi in these samples. In general, the array method was capable of simultaneous detection of multiple fungal species in the sputum samples of CF patients, and usually detected more species than the culture methods. The present method is a good tool for rapid detection of fungi in the sputa of CF patients.

    目錄 中文摘要.................Ⅰ 英文摘要.................Ⅱ 誌謝.................Ⅲ 目錄.................Ⅳ 表目錄.................Ⅵ 圖目錄.................Ⅶ 第一章 緒論 1.1 纖維性囊腫.................1 1.2 感染/定著在纖維性囊腫病患呼吸道之真菌.................3 1.3 治療真菌感染之藥物.................5 1.4 真菌的鑑定方法.................6 1.5 核糖體核酸基因內轉錄區.................8 1.6 研究目的.................9 1.7 研究架構.................9 第二章 實驗材料與研究方法 2.1 實驗菌種及臨床檢體.................10 2.2 實驗方法 2.2.1 真菌培養及保存.................11 2.2.2 DNA萃取.................11 2.2.3 內轉錄區之擴增.................13 2.2.4 ITS區域定序.................16 2.2.5 探針設計.................17 2.2.6 寡核苷酸晶片製備.................18 2.2.7 晶片雜合反應 .................19 2.2.8 晶片雜合反應結果判讀.................21 2.2.9 靈敏度與專一性定義.................21 2.2.10 分子選殖.................22 第三章 實驗結果 3.1 探針篩選結果.................24 3.2 種特異性之探針.................24 3.3 寡核苷酸晶片之靈敏度與專一性.................26 3.4 寡核苷酸晶片之偵測極限.................27 3.5 以寡核苷酸晶片直接檢測痰液檢體中之真菌.................27 第四章 討論 4.1 以寡核苷酸晶片檢測纖維性囊腫病患痰液中之真菌.................32 4.2 探針設計.................33 4.3 以寡核苷酸晶片直接檢測痰液檢體中之真菌.................33 4.4 寡核苷酸晶片之應用與展望.................34 參考文獻.................36 表目錄 Table 1. Reference strains of target species used in this study.................44 Table 2. Clinical isolates of target species used in this study.................46 Table 3. Nontarget strains used for specificity test.................47 Table 4. Oligonucleotide probes used in the study.................51 Table 5. Identification of target species by the oligonucleotide array.................52 Table 6. Detection of fungi in the sputa of CF patients.................53 Table 7. Cloning and sequencing of the ITS regions of several selected sputum specimens that produced discrepant fungal identifications between the culture method and chip hybridization.................58 圖目錄 FIG. 1. The experimental flowchart of the study.................60 FIG. 2. The organization of the rRNA genes and the locations of the fungus-specific, universal primers.................61 FIG. 3. Layout of oligonucleotide probes on the array.................61 FIG. 4. Alignment of partial ITS2 sequences of six strains of Acrophialophora fusispora.................62 FIG. 5. Alignment of partial ITS1 sequences of eight strains of Talaromyces emersonii.................62 FIG. 6. Alignment of partial ITS1 sequences of eight strains of Talaromyces emersonii.................63 FIG. 7. Hybridization results of target species.................64 FIG. 8. The hybridization result of Exophiala dermatitidis LMA 40502454.................66 FIG. 9. The hybridization result of Acrophialophora levis CBS 484.70.................66 FIG. 10. The detection limit of the oligonucleotide array by using PCR for target DNA amplification.................67 FIG. 11. The detection limit of the oligonucleotide array by using nested PCR for target DNA amplification.................68 FIG. 12. Hybridization results of clinical specimens.................69

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