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研究生:徐翠玲
研究生(外文):Tsuei-Ling Shiu
論文名稱:台灣貓隻貓冠狀病毒之檢測及其親緣性關係之探討
論文名稱(外文):Detection of Feline Coronavirus and Phylogenetic Analysis at Taiwan Cats
指導教授:廖明輝廖明輝引用關係
指導教授(外文):Ming-Hui Liao
學位類別:碩士
校院名稱:國立屏東科技大學
系所名稱:獸醫學系所
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:2013
畢業學年度:101
語文別:中文
論文頁數:65
中文關鍵詞:貓冠狀病毒貓腸道型冠狀病毒貓傳染性腹膜炎病毒S基因
外文關鍵詞:Feline Coronavirus (FCoV)Feline enteric coronavirus (FECV)Feline infectious peritonitis virus(FIPV)S gene
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貓冠狀病毒(Feline Coronavirus;FCoV)為具有封套的正向單股RNA病毒。根據血清學上可分為兩種血清型別,即血清型第一型(FCoV type I)與血清型第二型(FCoV type II)貓冠狀病毒,此兩型皆會感染野生貓科動物及家貓。已有文獻指出兩種血清型別確定能造成貓腸道型冠狀病毒(Feline enteric coronavirus;FECV),及貓傳染性腹膜炎病毒(Feline infectious peritonitis virus;FIPV)。傳染性腹膜炎屬於高致死性疾病,伴隨著免疫病理性疾病,而發展為血管炎、貧血、嗜中性球增多症、淋巴球減少症及多重器官之肉芽腫性炎症病變。在本實驗中,藉由巢式反轉錄聚合酶鏈鎖反應(RT-nPCR),針對臨床上,貓隻疑似感染貓冠狀病毒之檢體進行檢測,並利用S基因進行血清型別分析,以了解FCoV血清型於台灣貓隻感染之情形。目前已採集4年臨床檢體,共計391隻樣本,檢測結果有105隻(26.9%)為貓冠狀病毒陽性。再從105隻陽性檢體進行血清型別分析,其結果發現結果發現 33(31.5%)隻為血清型第一型, 而血清型第二型則為9(8.6%)隻,同時感染血清型第一型與第二型者為5隻(4.8%)。在親源關係方面,FCoV陽性株與各國株比對結果,與美國株(FJ938055)及馬來西亞株(FJ938055)較為相近,相似度為97-100%。FCoV type I與各國株比對結果,與韓國株(JN654410)較為相近,相似度為93.7%。FCoV type II與各國株比對結果,與中國株(AY452031)與疫苗株(DQ286389)較為相似,相似度達100%。本研究結果可提供疾病防治相關單位的參考依據。
Feline Coronavirus (FCoV) is an enveloped virus with a single-stranded, positive-sense RNA genome. There are two serotypes of FCoVs, namely type I and type II FCoVs, and both can infect wild and domestic Felidae. Two serotypes of feline enteric coronavirus (FECV) have been identified, both being able to cause feline infectious peritonitis virus (FIPV) . FIP is a highly lethal, immunopathological disease accompanied by the development of vasculitis, anemia, neutrophilia, lymphopenia, and multifocal granulomatous inflammatory lesions in various organs. In the study, Reverse transcriptase nested-polymerase chain reaction (RT-nPCR) was applied to the detection of FCoV from the clinical suspected cases. In order to understand the prevalence of FCoV infection in the domestic cats in Taiwan serotyping of the positive specimens with another RT-nPCR targeted to the S gene was performed. Reverse transcriptase nested-polymerase chain reaction RT-nPCR was applied to the detection of FCoV from the clinical suspected cases. For a period of 4 years, 391 samples were collected and 105 were found positive for FCoV RNA.The serotyping revealed that among the 105 positive cases 33 (31.5%) were type I, 9 (8.6%) were type II, and 5 (4.8%) were infected with both types. All nucleic acid sequences of this study comparing with published strains on the GenBank, the wild-type strains comparing with American (FJ938055), Malaysia (FJ938055) strain have 97-100% identity in FCoV. The type I strains comparing with Korea (JN654410), have 93.7% identity in FCoV. The type II strains comparing with china (AY452031), DF2 strain (DQ286389) have 100% identity in FCoV. These results could provide the information for disease diagnosis and controlling authorities and public health protection to base their decisions on for these diseases future prevention.
中文摘要................................................Ⅰ
Abstract................................................Ⅲ
謝誌....................................................Ⅴ
目錄....................................................VI
圖次....................................................Ⅸ
表次....................................................Ⅸ
第1章 前言..............................................1
第2章 文獻回顧..........................................4
2.1 貓冠狀病毒命名及分類................................4
2.1.1 貓冠狀病毒........................................4
2.1.2 貓腸道型冠狀病毒..................................5
2.1.3 貓傳染性腹膜炎病毒................................6
2.1.4 貓冠狀病毒之血清型別..............................7
2.2 基因體特性與蛋白質結構..............................8
2.3 流行病學............................................10
2.4 診斷................................................11
2.4.1 分子生物學診斷....................................11
2.4.2 滲出液分析........................................11
2.4.3 血液學及血清生化學................................12
2.4.4 診斷試劑..........................................13
2.4.5 組織病理學檢查....................................13
2.5 治療................................................14
2.6 防治................................................14
2.7 天擇理論............................................15
第3章 材料與方法........................................17
3.1 病材來源............................................17
3.2 貓冠狀病毒核酸萃取..................................17
3.3 貓冠狀病毒檢測......................................18
3.3.1 引子設計..........................................18
3.3.2 反轉錄聚合酶鏈鎖反應..............................18
3.3.3 巢式聚合酶連鎖反應................................19
3.3.4 貓冠狀病毒核酸檢測之敏感性試驗....................19
3.3.5 貓冠狀病毒核酸檢測之特異性試驗....................20
3.4 貓冠狀病毒血清型別..................................21
3.4.1 引子設計..........................................21
3.4.2 反轉錄聚合酶鏈鎖反應..............................22
3.4.3 巢式聚合酶連鎖反應................................23
3.4.4 PCR產物之純化.....................................23
3.5 基因選殖............................................24
3.5.1 接合作用..........................................24
3.5.2 勝任細胞製備......................................24
3.5.3 轉形作用..........................................24
3.5.4 菌落篩選..........................................25
3.5.5 小量質體DNA之萃取與化.............................25
3.6 核酸定序及分析......................................26
3.6.1 DNA定序...........................................26
3.6.2 序列比對..........................................26
3.6.3 族群遺傳之分析....................................26
第4章 結果..............................................28
4.1 貓冠狀病毒之PCR鑑定.................................28
4.1.1 貓冠狀病毒引子對敏感度試驗結果....................29
4.1.2 貓冠狀病毒引子對特異性試驗結果....................30
4.1.3 貓冠狀病毒之陽性率................................31
4.1.4 貓冠狀病毒之季節陽性率分析........................32
4.1.5 貓冠狀病毒相似度及歧異度分析......................33
4.1.6 貓冠狀病毒之親緣樹狀圖分析........................34
4.1.7 貓冠狀病毒之序列分析..............................36
4.2 貓冠狀病毒血清型別之PCR鑑定.........................37
4.2.1 多貓家庭之血清型別結果............................38
4.2.2 貓冠狀病毒血清型別之相似度及歧異度分析............39
4.2.3 貓冠狀病毒血清型別之親源樹狀圖分析................41
4.3 族群遺傳分析之結果..................................42
第5章 討論..............................................43
參考文獻................................................48
附錄....................................................60
作者簡介................................................64

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