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研究生:鄧明中
研究生(外文):Ming-Chung Deng
論文名稱:台灣豬瘟病毒之分子分析與診斷技術之開發
論文名稱(外文):Molecular Analysis of Classical Swine Fever Viruses in Taiwan and the Development of Rapid Diagnostic Method
指導教授:簡茂盛簡茂盛引用關係
學位類別:博士
校院名稱:國立中興大學
系所名稱:獸醫學系暨研究所
學門:獸醫學門
學類:獸醫學類
論文種類:學術論文
論文出版年:2008
畢業學年度:96
語文別:中文
論文頁數:84
中文關鍵詞:豬瘟病毒反轉錄恆溫環形核酸增幅法基因分型
外文關鍵詞:Classical swine fever viruslreverse transcriptase-oop-mediated isothermal amplificationgenotyping
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本研究主要針對台灣地區於1993至2001年間田間所分離共計36株之豬瘟病毒,分析比較其E2及NS5B基因序列,可將這些分離株區分為三個基因族群。其中一種古典型的病毒株分類屬於3.4基因亞型,與日本過去之分離株如神奈川株 (1974年) 及沖繩株 (1986年) 以及Paton等人所分析歸類之早期台灣分離株p97 以及 94.4同屬一個基因亞型。但此群病毒株於1996年以後則無法自田間分離到,而另由一種外來新的病毒株 (屬2.1基因亞型) 所取代。此新型病毒最早於1994年從花蓮地區之爆發場所分離到,之後便取代3.4基因亞型病毒而成為主宰台灣地區豬瘟之主要流行株。此外,在2001年時又從田間分離到一種與2.1基因亞型病毒之E2 、NS5B甚至全長基因序列分別只有94.1%至95.1%的相似度,因此我們將這群新分離之病毒分類為2.1b次亞型 (餘命名為2.1a subgroup)。為了探討這種病毒基因型間的消長與轉換,我們選定不同基因型之分離株進行動物接種實驗,並與傳統之標準強毒株ALD從臨床症狀、病程長短及血液白血球變化等方面進行比較,結果顯示不論是2.1或3.4基因亞型病毒株皆屬於中間毒力。而兔化豬瘟疫苗皆能有效保護豬隻不受不同基因型豬瘟病毒之攻擊。
由於豬瘟在我國是屬於法定重要傳染病,一旦疫情爆發時必須快速確診並採取防疫措施。過去傳統的診斷方法耗時費力,因此,本研究希望能藉由新型恆溫環形核酸增幅法 (RT-LAMP) 技術來開發新的臨床檢驗方法,並將此新方法與同樣診斷豬瘟病毒RNA之傳統RT-PCR、nested RT-PCR及qRT-PCR等方法比較敏感性與特異性,RT-LAMP比RT-PCR敏感1,000倍,而與nested RT-PCR及qRT-PCR相同都可偵測到稀釋105倍之病毒RNA。將豬瘟野外毒實驗接種及LPC免疫豬隻,於實驗早期 (第2及4天) 所採集之臟器樣品,經萃取總核酸後進行不同偵測病毒核酸方法之敏感性比較,結果同樣也顯示RT-LAMP的檢出率與敏感性,不論是野外毒或疫苗株,其偵測的結果與nested RT-PCR及qRT-PCR相似,均高於傳統RT-PCR。因此,應用RT-LAMP技術於豬瘟病例之診斷是可靠且敏感的。
台灣目前使用兔化豬瘟疫苗進行防疫,但由於豬瘟無法藉由血清學方法區別野外毒與疫苗株,因此本研究應用螢光標示引子,結合反轉錄恆溫環式核酸增幅法,開發出兼具方便快速、特異性及敏感性之田間豬瘟感染病例核酸區別檢測方法。與傳統應用鐵克曼探針區別不同基因型之方法比較,新技術在偵測野外毒及疫苗株之敏感性分別可達到稀釋105倍及104之病毒RNA,而鐵克曼探針及單純應用螢光標示引子不論是野外毒及疫苗株皆只能偵測到稀釋104倍,因此,結合螢光標示引子與反轉錄恆溫環式核酸增幅法可成功區別豬瘟野外毒及疫苗株之核酸,並兼具敏感性與快速性,而這項新技術未來將有助於田間豬瘟疫情之防治。
摘要……………………………………………………………………………………Ⅰ
英文摘要………………………………………………………………………………Ⅱ
目次……………………………………………………………………………………Ⅳ
表目次…………………………………………………………………………………Ⅵ
圖目次…………………………………………………………………………………Ⅶ
第一章 前言…………………………………………………………………………1
第二章 文獻探討……………………………………………………………………2
一 豬瘟之歷史…………………………………………………………………2
二 豬瘟病毒之特性……………………………………………………………2
三 病毒基因與蛋白之功能與角色……………………………………………5
四 豬瘟病毒之致病機制………………………………………………………8
五 豬瘟病毒之分子流行病學研究……………………………………………9
六 豬瘟病毒之診斷方法與技術………………………………………………10
第三章 台灣豬瘟病毒株分子分析與親緣樹建立…………………………………14
摘要…………………………………………………………………………14
英文摘要……………………………………………………………………15
一 前言…………………………………………………………………………16
二 材料與方法…………………………………………………………………18
三 結果…………………………………………………………………………20
四 討論…………………………………………………………………………26
第四章 台灣分離之不同基因型豬瘟病毒株之毒力測定…………………………28
摘要…………………………………………………………………………28
英文摘要……………………………………………………………………29
一 前言…………………………………………………………………………30
二 材料與方法…………………………………………………………………32
三 結果…………………………………………………………………………34
四 討論…………………………………………………………………………40
第五章 應用反轉錄恆溫環形核酸增幅法開發偵測豬瘟病毒核酸之檢驗法……42
摘要…………………………………………………………………………42
英文摘要……………………………………………………………………43
一 前言…………………………………………………………………………44
二 材料與方法…………………………………………………………………45
三 結果…………………………………………………………………………47
四 討論…………………………………………………………………………54
第六章 應用螢光標示引子及探針開發區別野外豬瘟病毒株與兔化豬瘟疫苗株
核酸之診斷方法……………………………………………………………56
摘要…………………………………………………………………………56
英文摘要……………………………………………………………………57
一 前言…………………………………………………………………………58
二 材料與方法…………………………………………………………………59
三 結果…………………………………………………………………………61
四 討論…………………………………………………………………………67
第七章 綜合討論……………………………………………………………………70
參考文獻……………………………………………………………………73
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