臺灣博碩士論文加值系統

狂犬病病毒 ( Rabies virus, RV )、犬瘟熱病毒 ( Canine distemper virus, CDV ) 、犬腺病毒 ( Canine adenovirus, CAV ) 與犬小病毒 ( Canine parvovirus, CPV ) 乃感染犬隻之重要病毒。其中RV乃人畜共通傳染病，台灣目前雖為狂犬病非疫區，但緊鄰之中國大陸仍為此病盛行區，事先防範加已迫再眉睫。此外，此四種病毒彼此間需要互相區別診斷，故發展出一套能快速偵測並區別此四種病毒之診斷方法，將有助於防疫與診療工作之進行。本實驗利用PCR或RT-PCR的方式偵測RV、CDV、CAV與CPV四種病毒，再進一步進行此四病毒之Multiplex RT-PCR，取其產物與所設計之專一性探針於晶片進行雜交，以確認感染的病毒為疫苗株或野外株。結果顯示，針對RV、CDV、CAV與CPV所設計出的四對引子，分別可專一性地增幅出符合預期大小的PCR產物，而以Multiplex PCR的方式亦能成功的區別出此四種病毒。另外針對RV、CDV、CAV、CPV分別設計出的共通性探針，發現皆具有與各自PCR產物進行雜交能力，利用此技術亦已完成CDV與CPV疫苗株與野外株之區別及CAV-2之鑑定；在敏感度方面，相較於僅以電泳分析PCR產物，結合基因晶片分析之敏感度提高至少100倍。綜合上述結果發現此套偵測系統確實符合快速、敏感、精確等優點，另外也適合於臨床檢體的偵測。

Rabies virus (RV), canine distemper virus (CDV), canine adenovirus (CAV) and canine parvovirus (CPV) are very important viral pathogens of dogs. Of all the four diseases, rabies belongs to zoonosis. Taiwan is at present free of this disease. However, rabies has been identified in many of the Asian countries including our neighbor mainland China. We are right now under the great risk of the outbreak of this disease. To be aware of the situation and be able to identify the disease once it is introduced, a well established diagnosis method is necessary. Moreover, the differential diagnosis of this four diseases is necessary . In this study, we perform the multiplex RT-PCR to detect and distinguish the four viruses. The PCR product was further used to hybridize specific probes by gene chip to confirm the origin of viruse e.g. field or vaccine . The results showed that the four pairs of primers designed in this study could detect RV, CDV, CAV, and CPV respectively. Moreover, all the viruses also could be detected and distinguished from each other successfully by multiplex RT- PCR. In combination with gene chip, RV, CDV, CAV, and CPV can be detected with their specific probes. Differentiation between vaccine and field strain viruses in the identification of CDV and CPV was successful. In the sensitivity assay, combination with gene chip was 100-fold more sensitive than gel electrophoresis. In the conclusion, the detection system developed in this study is rapid , sensitive and accurate method suitable for the diagnosis of clinical specimens.

目錄--------------------------------------------------------I
圖次--------------------------------------------------------IV
表次--------------------------------------------------------V
中文摘要----------------------------------------------------VI
英文摘要----------------------------------------------------VII
第一章、文獻探討--------------------------------------------1
第一節、狂犬病--------------------------------------------1
1. 1. 1狂犬病之歷史背景、臨床病徵與流行病學-------------1
1. 1. 2狂犬病病毒之性狀與特徵---------------------------3
第二節、犬瘟熱--------------------------------------------4
1. 2. 1犬瘟熱之歷史背景、臨床病徵與流行病學-------------4
1. 2. 2犬瘟熱病毒之性狀與特徵---------------------------6
第三節、犬小病毒感染症------------------------------------7
1. 3. 1犬小病毒感染症之歷史背景、臨床病徵與流行病學-----7
1. 3. 2犬小病毒之性狀與特徵-----------------------------8
第四節、犬腺病毒感染症------------------------------------9
1. 4. 1犬腺病毒感染症之歷史背景、臨床病徵與流行病學-----9
1. 4. 2犬腺病毒之性狀與特徵-----------------------------11
第五節、聚合酶鏈鎖反應的發展與應用------------------------12
第六節、多引子聚合酶鏈鎖反應的發展與應用------------------15
第七節、基因晶片之發展、應用與現況------------------------17
第二章、緒言------------------------------------------------26
第三章、材料與方法------------------------------------------30
第一節、實驗設計及流程圖----------------------------------30
3. 1. 1病毒偵測與區別診斷-------------------------------30
3. 1. 2 PCR產物確認與基因型區分-------------------------31
3. 1. 3敏感度分析---------------------------------------32
第二節、實驗材料與方法------------------------------------33
3. 2. 1病毒來源-----------------------------------------33
3. 2. 2寡核苷酸引子與探針之設計-------------------------33
3. 2. 2. 1寡核苷酸引子之設計----------------------------33
3. 2. 2. 2寡核苷酸探針之設計----------------------------34
3. 2. 3利用單一(反轉錄)聚合鏈鎖反應檢測病毒-------------35
3. 2. 3. 1 RNA病毒核酸之萃取----------------------------35
3. 2. 3. 2 DNA病毒核酸之萃取----------------------------35
3. 2. 3. 3反轉錄反應------------------------------------36
3. 2. 3. 4聚合酶鏈鎖反應--------------------------------36
3. 2. 3. 5聚合酶鏈鎖反應之電泳分析----------------------38
3. 2. 4 利用多引子 (反轉錄) 聚合酶鏈鎖反應檢測並區別病毒-38
3. 2. 4. 1 同時萃取RNA與DNA之病毒核酸-------------------38
3. 2. 4. 2 多引子反轉錄反應-----------------------------38
3. 2. 4. 3多引子聚合酶鏈鎖反應--------------------------39
3. 2. 5 以基因晶片偵測與區別病毒株或型別----------------39
3. 2. 5. 1 探針特異性試驗-------------------------------39
3. 2. 5. 2 PCR產物於電泳之敏感性分析--------------------39
3. 2. 5. 3 探針濃度之測試-------------------------------40
3. 2. 5. 4 雜交溫度之測試-------------------------------40
3 . 2. 5. 5 PCR產物與雜交溶液總體積之比例測試-----------40
3. 2. 5. 6 晶片雜交之敏感性分析-------------------------41
第四章、結果------------------------------------------------43
第一節、以單一PCR或RT-PCR技術偵測病毒---------------------43
4. 1. 1 以單一RT-PCR技術偵測狂犬病病毒------------------43
4. 1. 2 以單一RT-PCR技術偵測犬瘟熱病毒------------------43
4. 1. 3 以單一PCR技術偵測犬小病毒-----------------------43
4. 1. 4 以單一PCR技術偵測犬腺病毒-----------------------43
第二節、多引子RT-PCR技術之建立----------------------------44
第三節、以基因晶片確認PCR產物並分型-----------------------44
4. 3. 1各病毒探針之專一性測試---------------------------44
4. 3. 1. 1 狂犬病病毒-----------------------------------44
4. 3. 1. 2 犬瘟熱病毒-----------------------------------44
4. 3. 1. 3 犬小病毒-------------------------------------45
4. 3. 1. 4 犬腺病毒-------------------------------------45
4. 3. 2 以晶片偵測病毒核酸之敏感性分析------------------46
4. 3. 2. 1 雜交溫度之測試與PCR產物比例之測試------------46
4. 3. 2 2 探針濃度之測試-------------------------------46
4. 3. 2. 3 以電泳解析PCR產物之最小偵測極限-------------47
4. 3. 3. 4 以基因晶片解析PCR產物之最小偵測極限---------47
第四節、以基因晶片確認多引子RT-PCR產物並分型--------------47
第五章、討論------------------------------------------------63
附錄一、主要溶劑之配方--------------------------------------70
參考文獻----------------------------------------------------71

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