利用分子信標結合整合式微流體系統於魚類病原體之快速檢測

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研究生：	蘇怡之
論文名稱：	利用分子信標結合整合式微流體系統於魚類病原體之快速檢測 Integrated Molecular Beacon-based Microfluidic System for Rapid Detection of Fish Pathogens
指導教授：	李國賓
口試委員:	吳旻憲林哲信
學位類別：	碩士 Master
系所名稱：	工學院 - 動力機械工程學系 Department of Power Mechanical Engineering
論文出版年：	2013
畢業學年度：	101
語文別：	英文
論文頁數：	79
中文關鍵詞：	神經壞死病毒、虹彩病毒、專一性磁珠、分子信標、微流體系統
相關次數：	點閱：2 下載：0
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神經壞死病毒與虹彩病毒皆為高傳染性及高致病性病毒，可感染多種魚類。以石斑魚魚苗為例，其感染神經壞死病毒或虹彩病毒，死亡率均高於百分之八十。高死亡率造成養殖漁業重大經濟損失。現有檢測技術如病毒培養、酵素免疫分析法、聚合酶鏈鎖反應/反轉錄聚合酶鏈鎖反應有些缺點，包含費時、操作過程複雜、需要大型專業儀器及訓練有素之專業人員操作實驗。此外，目前對已感染病毒的魚並沒有適當的治療方式。因此，快速及準確的診斷神經壞死病毒與虹彩病毒對於養殖漁業是非常重要的議題。若愈早診斷出受病毒感染的魚，可越早將其撲殺，以防止疾病的擴散，進而降低經濟損失。在本研究中，以整合式微流體系統，包括整合式機台、微流體晶片、專一性磁珠及專一性分子信標，能夠在三十分鐘內自動化診斷神經壞死病毒或虹彩病毒，大幅縮短檢測時間。在本研究中，偵測神經壞死病毒或虹彩病毒的最低偵測極限均為10 ng/μL。最重要的是，此整合式微流體系統可成功自魚苗魚體中偵測出受神經壞死病毒或虹彩病毒感染之魚苗。因此，本研究之微流體系統未來可運用於田間檢測魚類病原體的有效診斷工具。

Abstract    I
中文摘要    III
誌謝    IV
Table of Content    V
List of Figure    X
List of Table    XIII
Abbreviations    XIV
Nomenclature    XVI
Chapter 1: Introduction    1
1    MEMS and Bio-MEMS Technology    1
2    Molecular Beacon    3
3    Literature Review    4
3.1    Nervous Necrosis Virus    4
3.2    Iridovirus    5
3.3    Conventional Diagnosis Methods    6
4    Motivation and Objectives    7
Chapter 2: Theory    10
1 Microfluidic Control System    10
1.1 Suction-type Pneumatic-driven Micropump    10
1.2 Micro-mixer    12
2 Molecular Diagnosis    13
2.1 DNA and RNA    13
2.2 PCR and RT-PCR    14
2.3 Gel Electrophoresis    15
2.4 Fluorescence Resonance Energy Transfer    16
Chapter 3: Materials and Methods    23
1 Viral Strain    23
2 Probe Conjugated Magnetic Beads    25
2.1 Preparation of Probe Conjugated Magnetic Beads    25
2.2 Hybridization Condition of Probe Conjugated Magnetic Beads    26
3 Molecular Beacon    27
4 Chip Design    27
5 Fabrication of Microfluidic Chip    28
6 Experimental Procedure    29
7 Custom-made Control System    30
Chapter 4: Results and Discussion    36
1 NNV RNA2 Molecular Beacon Assays    36
1.1 Optimal Hybridization Temperature of Specific NNV RNA2 Probe Conjugated Magnetic Beads    37
1.2 Optimal Hybridization Temperature of Molecular Beacon    38
1.3 Optimal Hybridization Time of Molecular Beacon    39
1.4 Detection Limit of Molecular Beacon    40
1.5 Specificity Tests of Molecular Beacon    41
1.6 Diagnosed of Infected and Non-infected NNV Fish Sample by Specific Probe and Molecular Beacon    42
2 Iridovirus MCP Molecular Beacon Assays    44
2.1 Optimal Hybridization Temperature of Specific Iridovirus MCP Probe Conjugated Magnetic Beads    44
2.2 Optimal Hybridization Temperature of Iridovirus MCP Molecular Beacon    45
2.3 Optimal Hybridization Time of Molecular Beacon    46
2.4 Detection Limit of Molecular Beacon    47
2.5 Specificity Test of Molecular Beacon    48
2.6 Diagnosis of Iridovirus Infected and Non-infected Fish Sample    50
Chapter 5: Conclusions and Future Work    68
1 Conclusions    68
2 Future Work    69
References    71

                                

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