臺灣博碩士論文加值系統

過去數十年來，新興病毒造成的感染症快速竄升。對於可能藏有未知病原的檢體，除了使用電子顯微鏡觀察之外，一般也利用微生物微陣列晶片輔助鑑定致病原。為了增進檢出率，許多分析前擴增方法被應用來放大致病原的核酸，例如representational difference analysis (RDA)，sequence-independent single primer amplification (SISPA)，或 random PCR，然而，敏感度仍十分有限。
　　因此本論文的目的就是發展一套有效的分析前擴增法，期望能靈敏地檢測未知致病原，第一部分會先收集懷疑帶有未知病原的臨床檢體，第二部分著重以全基因放大平台建立適當的前置擴增法。
　　從常規病毒診斷實驗室總共收集了676支懷疑帶有未知病原的臨床檢體，並進行病毒培養觀察是否有細胞病變，結果有兩個檢體經過四次繼代培養仍有細胞病變產生而且針對特定病原檢測結果為陰性。另外，亦有10支檢體採集自疑似感染症病患的急性期。
為建立有效的前置擴增法，分別採取兩個策略，一為利用micrococcal nuclease去除非病毒核酸，另一為引進全基因放大技術以增加靈敏度。micrococcal nuclease處理後的結果顯示大部分(至少97.7%)宿主基因體皆被清除但病毒顆粒內的病毒基因體並未受損。為了評估全基因放大技術擴增病毒基因體的效果，我們以冠狀病毒株JHM為受測對象，並分別測試Multiple strand displacement amplification and Genomeplex amplification兩種全基因放大技術。我們發現Multiple strand displacement amplification較適用於封閉型環狀DNA，線狀DNA在Genomeplex amplification效果較優。一般來說，增幅效果會因病毒基因體長短或濃度高低而有所影響。
　　我們隨後測試最佳化前置擴增法的敏感性，發現即便原始檢體所含的病毒量低到每400微升只含一千個病毒(冠狀病毒株JHM)顆粒仍然能夠有效放大(一萬倍以上)。我們將會以不同類型及大小的DNA及RNA病毒進一步評量此方法於臨床上的可適用性。希望在不久的將來此方法可以應用在疑似含有未知病原的檢體。

In the past decades, the infections caused by newly emerging viruses increased rapidly. For the specimens containing potential unknown pathogens, in addition to the electron microscopy examination, microbe-microarray hybridization is usually used to identify the causative pathogens. To further increase the detection rate, several pre-analysis amplification methods were included for amplification of the nucleic acids of causative pathogens, such as the representational difference analysis (RDA), sequence-independent single primer amplification (SISPA), or random PCR reaction. However, the detection sensitivity is still quite limited.
　　The specific aim of this study is thus set to the development of a method for efficient pre-amplification, which can help sensitive detection of causative unknown pathogens. Part I is to collect the clinical specimens suspected to contain unknown pathogens. Part II is to develop and optimize the pre-amplification method based on the whole genome amplification (WGA) platform. Part III is to evaluate the feasibility of the optimized pre-amplification method for the collected clinical specimens.
　　In total, 676 clinical samples suspected with unknown pathogens from routine diagnostic lab were processed for cytopathic effect (CPE) validation by virus culture. Only 2 samples showed clear CPE until 4th passage, but without specific virus antigen detected. In addition, 10 primary specimens were collected from suspected cases of unknown infectious disease, at the acute phase.
　　To set up the method for efficient pre-amplification, two approaches were used (1) using micrococcal nuclease to eliminate any non-virus nucleic acid, and (2) applying WGA platform to increase the sensitivity, respectively. The results from nuclease showed that most of the host genome (at least >99.7%) could be eliminated by the treatment, with the virus genome in the virions remained intact. To evaluate the efficiency of the WGA platform in amplification of viral genome, the coronavirus JHM strain was used for our testing. Two assays were included for the evaluation, the multiple strand displacement amplification and genomeplex amplification assay. We found that multiple strand displacement amplification was more suitable for closed circular form DNA, while linear form DNA was amplified more efficiently by PCR-based Genomeplex amplification. In general, the amplification efficiency depends on the length and the titer of viral genome analyzed.
　　By using coronavirus JHM strain as the target, the sensitivity of the optimized pre-amplification step included in the amplification procedure were as low as 103copies in 400ul specimen and it could be efficiently amplified (> 104 fold). The feasibility of its general clinical application will be further pursued in different types of viruses, including the DNA and RNA viruses with different genome sizes. We hope that the method could be applied for the specimens suspected to contain unknown infectious pathogens in the near future.

口試委員會審定書 i
致謝 ii
中文摘要 iii
英文摘要 v
目錄 I
圖目錄 IV
表目錄 V
第一章 緒論 6
1.1簡介病毒性新興感染症並舉例 6
1.2新興及再興感染症出現的主原因 7
1.3 針對未知病原常用之檢測方法介紹 7
1.3.1 pre-analytic amplification 方法應用於病毒核酸 8
1.3.2 概述 Next generation whole genome sequencing方法 9
1.4 鑑定未知病毒所面臨的問題 10
1.5 研究動機與目的 11
第二章 實驗材料與方法 14
2.1器材與試劑 14
2.1.1材料 14
2.1.2細胞 15
2.1.3病毒 16
2.1.4細胞培養耗材 16
2.1.5病毒培養耗材 16
2.2 方法 17
2.2.1細胞繼代培養 17
2.2.2病毒培養 17
2.2.4檢體種類和前處理 18
2.2.5去除細胞碎片及非病毒殘渣 19
2.2.6 Micrococcal nuclease treatment (NEB) 19
2.2.7 Viral Nucleic Acid Extraction 20
2.2.8病毒核酸反轉錄 21
2.2.9 Phenol-chloroform purification 21
2.2.10 Desalting 21
2.2.11 REPLI-g Ultra Fast mini kit whole genome amplify 22
2.2.12 GenomePlex Complete Whole Genome Amplification (WGA2) kit 23
2.2.13 Re-PCR 23
2.2.14 JHMV real-time PCR 24
2.2.15 High pure PCR clean up micro kit (Roche) 25
2.2.16酒精沉澱 25
2.2.17微陣列晶片(Microarray)檢測原理 25
第三章 結果 27
Part I: 檢體收集 27
3.1 病毒培養 27
3.2 未知病毒之臨床檢體採集 27
Part II: 建立核酸擴增方法之實驗步驟 28
3.3 以micrococcal nuclease清除檢體中的非病毒核酸 28
3.3.1 評估micrococcal nuclease清除宿主核酸的效果以及對病毒基因體的影響 28
3.4 應用whole genome amplification 以增加核酸檢測敏感度 30
3.4.1 REPLI-g Multiple strand displacement amplification 30
3.4.1-1 反應時間長短對ＭDA的影響 31
3.4.1-2 DNA模股類型對ＭDA的影響 31
3.4.2 GenomePlex Complete Whole Genome Amplification；WGA2 32
3.4.2-1 Fragmentation步驟對 WGA2擴增病毒核酸的必要性 32
3.4.2-2 Library preparation反應時間長短對WGA2的影響 32
3.4.3 結合兩種方法的優點:試驗1 33
3.4.4 結合兩種方法的優點:試驗2 35
3.5 測試方法的敏感度—以JHMV為例 35
3.5.1 研究方法之敏感度初步測試 35
3.5.2 修正 WGA2 Library preparation 步驟為兩次priming 36
3.5.3 最終測試 37
3.5.4 標準操作流程 38
Part III : Microarray analysis 39
第四章 討論 40
圖表附錄 44
參考資料 72

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