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研究生：	郭明澤 Kuo, Ming-Tse
論文名稱：	以淚液拉曼光譜及寡核苷酸DNA陣列晶片評估感染性角膜炎 Assessment of Microbial Keratitis using Raman Spectra of Tears and Oligonucleotide DNA Array
指導教授：	張長泉 Chang, Tsung-Chain 張憲彰 Chang, Hsien-Chang
學位類別：	博士 Doctor
系所名稱：	工學院 - 醫學工程研究所 Institute of Biomedical Engineering
論文出版年：	2011
畢業學年度：	99
語文別：	英文
論文頁數：	85
中文關鍵詞：	淚液、感染性角膜炎、拉曼光譜、寡核苷酸DNA陣列晶片
外文關鍵詞：	tears, microbial keratitis, Raman spectrum, oligonucleotide DNA array
相關次數：	點閱：81 下載：1
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早期正確診斷與治療是避免感染性角膜炎造成失明的關鍵。傳統診斷包括病史詢問推斷，細隙燈下病灶形態的研判，角膜病灶刮除清創採樣以進行抹片鏡檢及微生物的培養。拉曼平台能使微量物質甚至是液態下表現其特有的光譜鑑識指紋，而寡核苷酸陣列晶片具備高度物種辨識特性。故本研究應用拉曼光譜顯微鏡以及寡核苷酸陣列晶片對感染性角膜炎致病原因輔助鑑別診斷，發展感染性角膜炎病因診斷上，不同階段及不同層次之快速鑑別診斷方法。在拉曼平台上，吾人以合成淚液含參考菌株進行體外模擬試驗，測試並發展出最佳之光譜鑑別方式。在寡核苷酸陣列晶片研究上，吾人以感染性角膜炎常見感染菌為研究標的，對已設計及新設計之探針，以純培養之參考菌株，訓練及測試出高敏感度及高特異性之陣列晶片。臨床檢體之實測上，本試驗透過淚液檢體、角膜刮除檢體與培養後的微生物檢體為標的檢體，以拉曼光譜顯微鏡平台及寡核苷酸陣列晶片對感染性角膜炎之病患進行快速病因鑑別診斷。吾人以毛細管採集淚液樣本，對健康自願者、非感染性角膜炎之眼表疾患、及感染性角膜炎患者，將1.5微升淚滴在拉曼檢測基材上，以拉曼光譜顯微鏡獲取光譜訊號。以Microsoft Excel 2007軟體建構光譜資料庫，並運用Matlab R2008a軟體進行多變項分析。將感染性角膜炎患者清創後之剩餘刮除檢體，以單對分別(singleplex)或多對一起(multiplex)對rRNA基因或其內轉錄區放大，研究寡核苷酸陣列晶片之最佳診斷條件。根據目前的結果，淚液拉曼光譜平台具備30分鐘內判定感染與否的診間輔助診斷應用潛力，惟提升至分辨菌屬的解析度，仍有待累積更多相同感染菌個案方能有效驗證，如綠膿桿菌角膜炎。感染性角膜炎寡核苷酸陣列晶片之部份，尤其是真菌與棘狀阿米巴，顯示出高度臨床應用價值，可縮短診斷感染菌類或菌種時間至4-6小時。若加上影像判別處理，將使有效診斷涵蓋面擴及常見之細菌性角膜炎致病菌種。

Microbial keratitis is a vision-threatening disease. It needs prompt etiologic diagnosis and treatment. Conventional diagnostic approach for microbial keratitis needs direct microscope evaluation and laboratorial experts’ identification by culture using the corneal or conjunctival scraping samples. The invasiveness needs to be concerned, the sensitivity and specificity for this approach are low, and the time may be not rapid enough for some patients. Raman spectroscopy is a qualitatively and quantitatively optical technique by the scattered light to detect the optical fingerprints of molecules in little amount of the sample, even in the liquid status. DNA array, which probes designed in the rRNA gene or the inter-transcribed regions of rRNA gene, is a technology with high species-discrimination performance for cultured microbes. We applied both technologies to assess their roles in the assessment of microbial keratitis. Our motivation is to develop alternative and rapid methods for diagnosis of microbial keratitis. We consecutively collected the tear samples by microcapillary tubes for acquirement of tear Raman spectra from normal volunteers, patients of ocular surface diseases without microbial keratitis, and patients of microbial keratitis. The corneal scraping samples, one half for conventional microbiological survey and the other half for DNA array diagnosis, were collected from patients of ulcerative keratitis, who presumed microbial infection. For 1.5-μl teardrop on the Au/Ti substrate, tear Raman spectra were acquired by Raman microspectroscopy. The spectral database was constructed by Microsoft Excel 2007. The tear Raman spectra for microbial keratitis was analyzed by Matlab 2008a. The singleplex and multiplex PCR for these samples were tuned to find the optimal pathogenic discrimination for corneal scraping samples from presumed microbial keratitis. The reference strains of microbes and clinical isolates are used for the probe design, training, and testing in the DNA array of microbial keratitis. Integration of oligonucleotide probes for pathogens in microbial keratitis, including common bacteria, fungi, and acanthamoeba showed high sensitivity and specificity for discrimination of cultured microbes, especially at the addition of image diagnostic algorithm. The clinical feasibility was tested by comparison with clinical data. The sensitivity and specificity of the DNA array for microbial keratitis will be analyzed by Microsoft Excel 2007. From the results of clinical samples, the Raman platform implied the potential of 30-minute microbial keratitis recognition in the clinical office. However, more clinical cases with the same infectious entity should be cumulated continually for the future goal of pathogen identification, such as Pseudomonas keratitis. Oligonucleotide DNA array for microbial keratitis showed powerfully clinical application, especially in fungal keratitis. We believe the 2 novel diagnostic modalities will greatly influence on the future diagnostic quality of microbial keratitis, preventing more patients from visual loss.

Chapter 1 Introduction	......................................................................	1
1 Microbial keratitis (MK)	..............................................................	1
2 Conventional diagnostic modalities for MK .................................	2
3 A potential diagnostic target for MK - Tears ………………..…..	3
4 Two novel modalities with potential for diagnosis of MK ............	4
4.1 Raman spectrum and Raman microspectroscopy …..……..…..	4
4.2 Oligonucleotide DNA array .......................................................	6
5 Objectives .....................................................................................	7

Chapter 2 Materials and methods .......................................................	8
1 Preparation of simplified synthetic tears …………………..........	8
2 Reference strains and clinical isolates ..........................................	10
3 Participants for human subject ......................................................	10
4 Collection of human tears .............................................................	10
5 Collection of corneal scrapes for the survey of microbial Keratitis 11
6 Dried teardrop preparation for Raman spectral acquisition …......	12
7 DNA preparation ...........................................................................	14
8 Amplification for array hybridization ...........................................	15
9 Design of oligonucleotide probes ..................................................	16
10 Fabrication of DNA array ............................................................	17
11 Array hybridization .....................................................................	17
12 Analysis of discrepant results ......................................................	17
13 Statistical analysis .......................................................................	18

Chapter 3 Tear Raman Analytical Model for MK ...............................	19
1 Aim of this study  ........................................................................	19
2 Materials and methods ...................................................................	19
2.1 Experimental design ...................................................................	19
2.2 Definition of ulcerative keratitis with or without infection ........	20
3 Results ...........................................................................................	21
3.1 Raman spectral sampling of a dried teardrop …..………….…...	21
3.2 Spectral characteristics in different zones of a dried model teardrop 21
3.3 Normalized spectral characteristics in different zones……........	23
3.4 Determination of the optimal sampling zone ..............................	24
3.5 Test of the model by human teardrops in acute ulcerative keratitis 25
4 Discussion .....................................................................................	26

Chapter 4 tear Raman spectra for differentiation of ulcerative keratitis 31
1 Aim of this study ...........................................................................	31
2 Materials and methods ...................................................................	31
2.1 Collection of clinical data ..........................................................	31
2.2 Definition of ulcerative keratitis with or without infection ........	32
2.3 Data analysis of the Raman spectra of teardrop samples ............	33
3 Results ...........................................................................................	34
3.1 Clinical data analysis ..................................................................	34
3.2 Comparison of Raman spectra of different tear samples ............	37
3.3 Extraction of global features of Raman spectra…………….…. 	39
3.4 Comparison of infectious and non-infectious UK……….…..…	40
3.5 Estimation of mean PC scores for subgroups of IUK………......	41
4 Discussion .....................................................................................	43

Chapter 5 Complex DNA array for cultured microbes common in MK 46
1 Aim of this study ..........................................................................	46
2 Materials and methods ..................................................................	46
2.1 Microbial strains ........................................................................	46
2.2 Fabrication of oligonucleotide DNA array .................................	50
2.3 Objective identification for DNA array aided by image processing 53
3 Results ...........................................................................................	53
3.1 Identification of common microbes in MK by DNA array .........	53
3.2 Validity of the complex DNA array for microbial keratitis ........	54
4 Discussion .....................................................................................	55

Chapter 6 Oligonucleotide DNA array for fungal keratitis .................	58
1 Aim of this study ...........................................................................	58
2 Materials and methods ..................................................................	58
2.1 Framework of this study .............................................................	58
2.2 Performance validation of the array ...........................................	60
2.3 Amplification of the fungal ITS .................................................	61
2.4 Fabrication of array ....................................................................	62
3 Results ...........................................................................................	62
3.1 Performance validation of the array by cultured microbes .........	62
3.2 Clinical and FK-DNA array data for presumed microbial keratitis 64
3.3 Analysis of diagnosis by FK-DNA array …………………........	66
4 Discussion .....................................................................................	69

Chapter 7 Conclusions and prospect ...................................................	71

References ...........................................................................................	73
		
Appendix .............................................................................................	80
                                    

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校內：2015-09-06公開
校外：2016-09-06公開

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