臺灣博碩士論文加值系統

纖鍊病毒發現於1997年，是非病原性的單鍊DNA病毒，在人群中極為盛行。早期的檢測研究，多針對轉譯區中的N22片段，然而，DNA雖是纖鍊病毒的遺傳物質，其轉譯區卻有相當高的變異性，也因此嚴重低估了其盛行率，對東台灣原住民族群僅有的一篇研究也是如此。本論文的主要目的，即釐清纖鍊病毒在東台灣原住民族群中真正的盛行率，並且更進一步探討其基因型、分群以及地域特異性。
我們針對高度保守的非轉譯區，設計引子進行檢測，發現在東台灣原住民族群的盛行率高達95 %，但與各國相較則未見顯著差異。我們也發現在東台灣原住民身上，明顯有多株變體混合感染，因此擴增並選殖了包含完整轉譯區的片段加以定序，以確認各變體的基因型。
由我們的一份血清檢體中，至少可辨別出13株病毒變體，依據親緣關係分析，其中11株可細分為兩型，各自有8株與3株，且此二者獨自成為一個單系群，不屬於任何已知的分類，是纖鍊病毒的新成員。其餘兩株，則分屬纖鍊病毒既有五大群中的第一與第三群。
我們比較同一型的8株病毒變體，發現75 %以上的胺基酸突變，侷限在不到一號展讀框兩成長度的超歧異區，這暗示了纖鍊病毒可能具有準物種的特性。

Torque Teno virus (TTV), the first circular negative-stranded human DNA virus, was isolated in 1997. It has a very high prevalence among general population but without evidence of leading to any disease process. It is extremely variable and shows a wide range of tissue tropisms and persistent infection in healthy individuals.
Early studies of prevalence were mostly performed with a polymerase chain reaction (PCR) amplification of the N22 region, a fragment in open reading frame 1 (ORF 1), after the first isolated clone. However, some studies have showed that the viral detection based on this fragment strongly underestimate the prevalence of TTV. In the present study, we found 95% (133/140) prevalence in eastern Taiwanese indigenous population by using newly designed PCR primers deduced from the highly conserved untranslated region (UTR), and that is no significant difference with other countries.
Entire ORFs from one serum sample was amplified with UTR primers by inverted long PCR, and followed by cloning and sequencing. Thirteen TTV isolates were obtained. One of them belongs to group 1, one to group 3, and eleven to group 6, a new member of TTVs. Comparison of the 8 isolates belonging to group 6, 75-95% mutations on amino acid level were restricted to the hypervariable region of ORF 1, hence suggesting that TTV circulation may have a quasispecies nature.

總目次
中文摘要………………………………………………………………I
英文摘要………………………………………………………………II
致謝……………………………………………………………………III
總目次…………………………………………………………………IV
圖目次…………………………………………………………………VIII
表目次…………………………………………………………………IX
術語對照與名詞縮寫…………………………………………………X
第一章 緒論……………………………………………………………1
1.1 前言………………………………………………………………1
1.2 文獻探討…………………………………………………………2
1.2.1 TTV的發現與命名……………………………………………2
1.2.2 TTV的病毒形態與基因組結構………………………………3
1.2.3 TTV的分類……………………………………………………6
1.2.4 TTV的傳播途徑與生命週期…………………………………9
1.2.5 TTV的臨床症狀………………………………………………10
1.2.6 TTV的流行病學研究…………………………………………11
1.2.7 台灣地區對於TTV的研究現況………………………………13

第二章 材料與方法……………………………………………………14
2.1 檢體採集………………………………………………………14
2.2 純化病毒核酸…………………………………………………14
2.3 多重序列排比…………………………………………………15
2.3.1 完整基因組序列排比………………………………………15
2.3.2 ORF1序列排比………………………………………………15
2.4 引子設計………………………………………………………16
2.4.1 盛行率引子…………………………………………………16
2.4.2 長距PCR引子………………………………………………16
2.5 盛行率檢測……………………………………………………16
2.6 長距PCR………………………………………………………18
2.7 PCR產物純化…………………………………………………18
2.8 TA選殖…………………………………………………………19
2.8.1 額外Taq反應…………………………………………………19
2.8.2 拓樸選殖……………………………………………………20
2.8.3 載體轉殖……………………………………………………20
2.8.4 菌落培養……………………………………………………20
2.8.5 質體純化……………………………………………………21
2.9 定序反應………………………………………………………21
2.10 評估資訊豐度………………………………………………22
2.11 親緣關係分析………………………………………………22
2.11.1 序列編輯…………………………………………………22
2.11.2 建構親緣關係樹…………………………………………23
2.11.3 拔靴法……………………………………………………24
第三章 結果…………………………………………………………25
3.1 盛行率測定……………………………………………………25
3.2 完整展讀框定序………………………………………………25
3.3 親緣關係分析…………………………………………………27
3.3.1 多重序列排比………………………………………………27
3.3.2 資訊豐度評估………………………………………………27
3.3.3 親緣關係樹…………………………………………………28
第四章 討論…………………………………………………………34
4.1 TTV混合感染…………………………………………………34
4.2 TTV的分群……………………………………………………34
4.3 引子專一性……………………………………………………35
4.4 盛行率的地理差異……………………………………………38
4.5 準物種特性？…………………………………………………39
第五章 參考文獻……………………………………………………40
中文文獻……………………………………………………………40
A-C…………………………………………………………………40
D……………………………………………………………………41
E-F…………………………………………………………………42
G-H…………………………………………………………………43
I-K…………………………………………………………………45
L……………………………………………………………………46
M……………………………………………………………………47
N-O…………………………………………………………………48
P……………………………………………………………………50
Q-T…………………………………………………………………51
U-Y…………………………………………………………………53
Z……………………………………………………………………54

圖目次
圖一、電子顯微鏡下之TTV顆粒……………………………………4
圖二、TTV的表現產物………………………………………………5
圖三、根據ORF1的TTV分群…………………………………………8
圖四、根據3.2 kb基因組的TTV分群………………………………8
圖五、PCR產物電泳圖………………………………………………17
圖六、盛行率片段定序原始資料……………………………………26
圖七、概似映射分佈…………………………………………………29
圖八、鄰聚法親緣關係樹……………………………………………31
圖九、最大概似法親緣關係樹………………………………………32
圖十、最大儉約法親緣關係樹………………………………………33
圖十一、鄰聚法親緣關係樹中的第一子群…………………………37

表目次
表一、IUB 鹼基簡併碼一覽表……………………………………16
表二、TTV盛行率與統計差異……………………………………26
表三、東台灣原住民TTV ORF1之核酸相同度……………………28
表四、東台灣原住民TTV ORF1之胺基酸相同度…………………28
表五、53A25群之ORF1胺基酸置換表……………………………39

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