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研究生:洪尚楷
研究生(外文):Shang-Kai Hung
論文名稱:生物晶片在病原微生物檢測之應用研究
論文名稱(外文):The Applications of Microarray-Diagnosis of Pathogenic Microorganisms
指導教授:陳瑞祥陳瑞祥引用關係黎慶
指導教授(外文):Ruey-Shyang ChenChing Li
學位類別:碩士
校院名稱:國立嘉義大學
系所名稱:生物科技研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:中文
論文頁數:89
中文關鍵詞:生物晶片病原微生物病原檢測EB病毒植物病原真菌
外文關鍵詞:microarrayEBV-chipMycochippathogenic microorganismsdiagnosis
相關次數:
  • 被引用被引用:3
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生物晶片是目前在基因相關研究上相當新穎且具有多功能的分析工具,本研究主要目的在利用其專一性高、偵測範圍廣等特性,研發可用於病毒及真菌等病原微生物之檢測用生物晶片,以供臨床診斷及植物檢疫等工作之需。本論文中,首先自EBV的基因體設計專一性PCR引子對,分段增幅成71個約1-3 kb的片段,並將這些經增幅後的片段,利用微矩陣儀 (microarrayer) 點製成EBV-chip。再以感染EBV的P3HR1細胞株為測試材料,萃取其mRNA,經反轉錄作用標定成cDNA探針後,與EBV-chip進行雜合反應及呈色作用,再經高解析度平台式掃描器和電腦軟體分析後的結果確定此EBV-chip的可行性;接著再以癌症病患身上取得的癌症組織為材料,利用EBV-chip來檢測EBV在不同癌症組織的分布情形。另一方面,本研究收集48株的植物病原真菌,利用通用性PCR引子,將其核糖體DNA (rDNA) 含內轉錄區間 (ITS1、ITS2及ITS-full) 片段增幅,再將此PCR增幅產物利用微矩陣儀點製成Mycochip。經以此Mycochip測試結果發現,生物晶片可用以檢測感病植物組織之病原真菌,並可同步加以鑑別不同種類病原真菌。綜合本研究結果顯示,生物晶片在病原微生物檢測應用上可克服目前利用PCR方式容易造成false-positive及in situ hybridization費時的缺點,更有可同步檢測多種病原菌及重複檢測不同病組織的優點;此外,更可藉由檢測過程同步進行基因表現的分析,顯示生物晶片在病原微生物檢測應用上極具發展的潛力。
Microarray is a novel and powerful tool for genomic studies. Based on its high capacity and specificity, the main objective of the present study is to develop a biochip for the diagnosis of pathogenic microorganisms. 71 EBV genomic DNA fragments of 1-3 kb in length were amplified by PCR using specific primers designed from EBV genome. These amplified products were spotted to make the EBV-chip by a microarrayer. To test the EBV-chip, mRNA extracted from EBV-infected P3HR1 cell line were transcribed into cDNA and labeled with biotin-16-dUTP. After hybridization and color development, the signals on EBV-chip were scanned and analyzed. The distribution of EBV in different tumor tissues were further assayed by the EBV-chip. On the other side, the Mycochip was made to detect and differentiate plant pathogenic fungi in the present study. Using the universal primers, ribosomal DNA (rDNA) including internal transcribed spacer (ITS) were amplified from 48 fungal isolates, and were spotted by a microarrayer. DNA extracted from tested fungal isolates and diseased plant tissues were PCR-amplified, labeled, and hybridized with the Mycochip. The results showed that different pathogenic fungi could be simultaneously detected and classified in diseased plant tissues. The Mycochip could also be used to differentiate fungal species form various sources. Based on the present study, the biochip has shown its great potential for the diagnosis of pathogenic microorganisms. Microarray technology also possessed several advantages over the current diagnostic methods, such as PCR and in situ hybridization, that were discussed in this study.
目 次
壹、緒論…………………………………………………………………..1
(一) 生物晶片簡介………………………………………………….2
(二) 研究動機及目的……………………………………………….4
貳、生物晶片檢測Esptein-Barr virus之應用………………………….7
一、前言……………………………………………………………..8
二、材料與方法……………………………………………………11
(一) P3HR1細胞株…………………………………………….11
(二) U937細胞株………………………………………………11
(三)癌症病理組織的取得及mRNA之萃取………………….11
(四) EBV-chip之製作…………………………………………13
(五) Microarray Hybridization…………………………………16
三、結果……………………………………………………………21
(一)未經TSA signal amplification的EBV-chip hybridizaton..21
(二) P3HR1和U937細胞株的EBV-chip hybridization……..22
(三)癌症組織的EBV-chip hybridization……………………...23
四、討論……………………………………………………………26
參、生物晶片檢測植物病原真菌之應用………………………………38
一、前言……………………………………………………………39
二、材料與方法……………………………………………………40
(一)供試植物病原真菌的收集………………………………..40
(二)植物病原真菌Genomic DNA的萃取……………………40
(三)植物病組織DNA之萃取…………………………………41
(四) Dot-blot Hybridization……………………………………41
(五) Mycochip之製作……………………………………….44
(六) Microarray Hybridization…………………………………46
三、結果……………………………………………………………50
(一)利用PCR檢測植物病原真菌…………………………….50
(二)利用Dot-blot hybridization檢測植物病原真菌………….51
(三)利用Dot-blot hybridization模擬生物晶片偵測植物
病原真菌…………………………………………………..52
(四)利用生物晶片 “Mycochip” 檢測植物病原真菌…..….53
四、討論……………………………………………………………56
肆、結論…………………………………………………………………78
伍、參考文獻……………………………………………………………81
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