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研究生:周應欽
研究生(外文):Ying-Chin Chou
論文名稱:犬瘟熱病毒之分子檢測及其親緣關係之探討
論文名稱(外文):Detection of Canine Distemper Virus and Phylogenetic Analysis by Molecular Techniques
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:48
中文關鍵詞:犬瘟熱病毒隔絕式恒溫聚合酶連鎖反應反轉錄聚合酶連鎖反應
外文關鍵詞:canine distemper virusiiPCRRT-PCR
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犬瘟熱(canine distemper, CD)是犬隻中重要且盛行已久之疾病,所有年齡的犬隻皆具有感受性,尤其是3~6月齡的幼犬,感染後會引起全身性及中樞神經系統性之症狀,屬於高發生、高傳染及高死亡率之疾病。因其臨床屬多系統性(multi-systemic)之症狀,因此在檢測上通常需花費許多時間來等待結果,故在臨床診斷上通常較不易確診,須藉由分子生物學診斷技術來輔助並加以確診。本實驗主要是應用反轉錄聚合酶連鎖反應(Reverse Transcriptase Polymerase Chain Reaction, RT-PCR)及隔絕式恒溫聚合酶連鎖反應(Insulated Isothermal Polymerase Chain Reaction, iiPCR)來檢測臨床上疑似感染犬瘟熱病毒(canine distemper virus, CDV)的檢體,並比較其陽性率及其親緣關係。在RT-PCR檢測引子對的敏感性為100 pg/μL,特異性檢測中僅對CDV才可檢測出。在疑似病例檢測陽性率方面,結果發現RT-PCR的陽性率為42%(42/100),而iiPCR的陽性率為41%(41/100)。雖然應用iiPCR檢測確診時間較快,但反應後產物無法直接進行核酸序列分析來比較親緣關係,而RT-PCR能直接進行核酸序列分析來比較親緣關係。從陽性檢體中隨機抽取10株病毒株,進行核酸序列分析,其中7株病毒序列與各國分離株相似度為99.3 -94.9%,但發現有3株病毒株序列與其他7株病毒株序列在親緣關係上差異性較大,值得再進一步的探討分析。
Canine distemper virus (CDV) is an important disease and has a high prevalence for a long time. All ages are susceptible to infections, particularly in 3-6 months of age puppy. CDV is a high incidence, high prevalence and lethal disease, it causes systemic infection and central nervous system symptoms in affected dogs. CDV caused multisystem disorders and difficult to diagnose in clinical. This problem can be solved by molecular technique for disease diagnosis. The aim of this study was survey prevalence of suspected CDV infection by use reverse transcriptase polymerase chain reaction (RT-PCR), and insulated isothermal polymerase chain reaction (iiPCR), and established the phylogenetic relationship of wild-type CDV between different countries. The sensitivity of RT-PCR is 100 pg/L, the specificity was also established. The positive rate of RT-PCR and iiPCR were 42%, 41%, respectively. Although iiPCR is a time-saving technique, the reaction product can not subject to sequencing. Comparing the nucleic acid sequences obtained by RT-PCR with published strains in the GenBank. Seven of ten viral strain from positive specimen showed 99.3-94.9% identity with other country, but the other three wild-type strains had low identity. Further investigation is required for understanding the phylogenetic difference.
中文摘要……………………………………………………… Ⅰ
Abstract…………………………………………………………………Ⅲ
謝誌………………………………………………………………………Ⅴ
目錄…………………………………………………………………… VI

圖表目錄…………………………………………………………………Ⅷ
第1章 前言………………………………………………………… 1
第2章 文獻回顧……………………………………………… 3
2.1 犬瘟熱病毒之歷史背景與流行病學………………………… 3
2.2 犬瘟熱病毒之病毒特性……………………………………… 4
2.3 犬瘟熱病毒之傳播途徑與致病機轉………………………… 5
2.4 犬瘟熱病毒之臨床症狀……………………………………… 6
2.5 犬瘟熱病毒之臨床診斷……………………………………… 8
2.6 犬瘟熱病毒之實驗室診斷…………………………………… 8
2.7 犬瘟熱病毒之病理學診斷…………………………………… 9
2.8 犬瘟熱病毒之分子生物學診斷……………………………… 9
2.9 反轉錄聚合酶連鎖反應(RT-PCR)之簡介及應用………… 10
2.10 隔絕式恒溫聚合酶連鎖反應(iiPCR)簡介及應用……… 11
第3章 材料與方法…………………………………………………… 15
3.1 病材來源……………………………………………………… 15
3.2 犬瘟熱病毒核酸抽取與純化…………………………………… 15
3.3 應用RT-PCR檢測犬瘟熱病毒……………………………………15
3.3.1 犬瘟熱病毒檢測用引子對之設計………………………… 15
3.3.2 RT-PCR反應條件………………………………………………16
3.3.3 檢測用引子對敏感性試驗………………………………… 16
3.3.4 檢測用引子對特異性試驗………………………………… 17
3.4 應用iiPCR檢測犬瘟熱病毒………………………………………17
3.4.1 iiPCR檢測犬瘟熱病毒核酸之步驟………………………… 17
3.4.2 iiPCR檢測用引子對特異性試驗…………………………… 18
3.5 核酸定序及親緣分析…………………………………………… 19
3.5.1 核鞘蛋白基因(N gene)序列之增幅……………………… 19
3.5.2 RT-PCR產物之純化………………………………………… 19
3.5.3 接合作用(Ligation)…………………………………… 20
3.5.4 勝任細胞(competent cell)之製備……………………20
3.5.5 轉形作用(Transformation)………………………… 21
3.5.6 菌落之篩選……………………………………………………21
3.5.7 小量質體DNA之萃取與純化………………………………… 21
3.5.8 限制酶切割確認重組之質體DNA………………………… 22
3.5.9 核鞘蛋白基因(N gene)序列之親緣分析…………… 22
第4章 結果…………………………………………………………… 23
4. 1 應用RT-PCR檢測犬瘟熱病毒………………………………… 23
4.1.1 檢測犬瘟熱病毒之引子對敏感性試驗………………………23
4.1.2 檢測犬瘟熱病毒之引子對特異性試驗………………………24
4.1.3 疑似感染病例之RT-PCR檢測結果…………………………25
4.2 應用iiPCR檢測犬瘟熱病毒……………………………………26
4.2.1 iiPCR犬瘟熱病毒引子對之特異性…………………………26
4.2.2 疑似感染病例之iiPCR檢測結果…………………………27
4.3 多重檢體之陽性樣本檢測之結果……………………………28
4.3.1 RT-PCR多重檢體之陽性樣本檢測之結果………………28
4.3.2 iiPCR多重檢體之陽性樣本檢測之結果……………………29
4.4 犬瘟熱病毒親緣關係樹狀圖……………………………… 30
4.4.1 犬瘟熱病毒核苷酸親緣關係樹狀圖…………………… 30
4.4.2 犬瘟熱病毒胺基酸親緣關係樹狀圖…………………………32
4.4.3 犬瘟熱病毒核苷酸親緣關係相似度及歧異度分析……………34
第5章 討論……………………………………………………………36
參考文獻…………………………………………………………………40
作者簡介…………………………………………………………………49

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