臺灣博碩士論文加值系統

香蕉條紋病毒 (banana streak virus, BSV) 是香蕉的重要病毒之一，但目前BSV的檢測方法仍有極大的進步空間，原因是BSV會以內源性序列 (integrated sequence) 存在於香蕉的基因體中。香蕉的原始種源包括Musa acuminata (A蕉) 與 Musa balbisiana (B蕉) 兩種，現今蕉種多為這兩大原種雜交的多倍體後代，而BSV的內源性序列，則存在於B蕉的基因體中，這些內嵌序列並不會造成病害，但在生產香蕉組培苗時，會有機會活化而成為游離型 (episomal) 病毒，進而引起香蕉條紋病。因此在BSV的篩檢上，是否能區分內源性 (endogenous) 及游離型的序列將是檢測方法成功與否的重要關鍵。另一方面，由於BSV屬於複合群 (complex)，其中包含9種病毒，無論是基因型 (genomic) 或是血清型 (serological) 都存在高度差異性，如此又提升了檢測難度。台灣國內現行標準檢測技術為利用Reverse transcription (RT)-PCR來檢測活化病毒之RNA，在大量篩檢時會消耗大量人力物力資源，且有一定機率在檢測時出現偽陽性的結果。本文目的在於研發出一套有效率且能夠將偽陽性機率降到最低的檢測方法。本研究主要著眼於三個可能的發展方向：單株抗體檢測、即時定量聚合酶連鎖反應檢測及反向聚合酶連鎖反應檢測。單株抗體檢測是最直接的檢測方法，因為只要能抓出病毒抗原，即代表該植株為染病植株；real-time PCR檢測法則是希望能找出專屬於游離型病毒顆粒的基因片段，而不會檢測到內嵌於香蕉genome中的序列片段；全基因檢測則是一個較新的概念，目前國際上並沒有相關文獻顯示有其他團隊利用全基因檢測來進行BSV感染植株的篩檢，此方法是利用BSV的genome為環狀DNA的特性，設計反向引子對 (abutting primer pair)，藉此達到僅增幅出游離病毒顆粒genome的效果。本研究最後進行了針對香蕉條紋病毒的田間調查，證明了三個方法中，單株抗體檢測法以及反向PCR檢測法，皆能有效的檢測出病毒的存在，且能夠大大降低偽陽性的發生，並且提高檢測效率。

Banana streak caused by Banana streak virus (BSV) is one of the important diseases of bananas. Current detection methods of BSV need more improvement and accuracy because of the integrated sequences of BSV (or endogenous BSV, eBSV) in the genome of banana. Edible bananas are mainly triploids derived from two wild progenitors, Musa acuminate and Musa balbisiana, with A genome and B genome, respectively, and those integrants have been found in the B genome. in general, they are unharmful but only under certain stress conditions, such as tissue culture and hybridization, they would be activated to become episomal virus. Therefore, how to correctly distinguish the integrated sequences and episomal virus in banana is the top priority of reliable diagnostic methods of BSV. On the other hand, BSV is a species complex, which includes nine different virus species with high variation in both genomic and serological level which present challenges for correct diagnosis. In Taiwan, the standard BSV detecting method is Reverse transcription (RT)-PCR. this is a laborious and time consuming method with a certain risk to be false positive. This thesis is purpose to develop an efficiency and high accuracy detection method. This study adopts three different ways, monoclonal antibody (McAb), quantitative polymerase chain reaction (qPCR, or real-time PCR) and abutting polymerase chain reaction, to develop the method. Monoclonal antibody is the most direct method because only the disease plant has episomal virus particle. Real-time PCR is mainly relied on the sequence only exist in virus genome and not in the eBSV sequences. Abutting PCR is an idea, there are no reference about this method so far. BSV has a circular, double strand genome, with this character, this study designed an abutting primer pair. This abutting primer pair can only amplify the genome of BSV, and separate episomal virus from endogenous counterpart. According to the results of field survey, both indirect ELISA with monoclonal antibody and abutting PCR can effectively detect the banana streak OL virus. These two detection methods can decrease the false positive reaction, and improve the detection efficiency.

論文口試委員審定書 i
致謝 ii
中文摘要 iii
Abstract v
目錄 vii
表目錄 x
圖目錄 xi
研究動機 1
前人研究背景 3
一. 香蕉條紋病的發現及分布 3
二. 香蕉條紋病之病徵 4
三. 香蕉條紋病毒之病原特性 5
四. 香蕉條紋病毒之寄主及傳播媒介 6
五. 內源性香蕉條紋病毒序列 7
六. 香蕉條紋病毒之偵測方法 9
實驗設計、材料及方法 11
壹、病毒接種及檢測 11
一、病毒及媒介昆蟲來源 11
二、BSOLV接種 11
三、BSOLV檢測 11
(1) 總核酸萃取法 11
(2) 聚合酶連鎖反應 (Polymerase chain reaction, PCR) 12
四、香蕉條紋病毒全解序 12
貳、單株抗體生產 13
一、表現質體之建構 13
(1) 專一性引子對之設計 13
(2) 細菌培養基製備 13
(3) BSOLV鞘蛋白DNA序列增幅及定序 13
(4) 表現質體之建構 14
二、表現及純化重組鞘蛋白 15
(1) 表現重組蛋白 15
(2) 蛋白質電泳分析 (SDS-PAGE) 15
(3) 西方墨點 (Western blot) 分析 16
(4) 純化表現蛋白 17
三、合成多肽序列選擇 18
四、單株抗體製作及篩選 19
(1) 小鼠免疫注射 19
(2) 細胞培養基製備 19
(3) 融合瘤製作 20
(4) 分泌抗體之融合瘤細胞篩選 21
(5) 單株融合瘤細胞株系之篩選 22
(6) 目標抗體篩選 23
(7) 單株抗體亞型 (Isotype) 測定 23
(8) 單株抗體大量生產 24
(9) 硫酸銨沉澱法 25
(10) 單株抗體之純化 25
(11) 單株抗體力價測試 26
(12) 抗體專一性測試 26
參、real-time PCR 及PCR檢測 27
一、Primer及probe選擇 27
二、Primer測試 27
三、real-time PCR循環條件 28
四、標準曲線之建立 28
五、Primer敏感性測試 29
肆、Abutting PCR 29
一、相鄰引子對設計 30
二、引子對最佳條件測試 30
三、引子對專一性測試 31
伍、香蕉條紋病毒田間染病情況調查 31
結果 33
壹、台蕉5號蕉苗接種BSOLV結果 33
貳、抗體製作結果 33
一、表現質體之建構 33
(1) BSOLV鞘蛋白DNA序列增幅及定序 33
(2) 表現質體之建構 34
二、表現及純化重組鞘蛋白 34
(1) 誘導表現重組蛋白 34
(2) 重組鞘蛋白大量誘導及純化 34
三、合成多肽序列選擇 35
四、單株抗體製作及篩選 35
(1) 小鼠免疫注射 35
(2) 單株抗體細胞株 35
(3) 純化自腹水及細胞培養上清液之單株抗體力價測試 36
(4) 抗體專一性測試 36
參、Real-time PCR及PCR檢測 37
一、Primer溫度測試 37
二、real-time PCR結果 37
(1) TaqMan system 38
(2) SYBR green system 38
三、Primer標準曲線之建立 38
四、primer pair F6R1偵測極限測試 38
肆、Abutting PCR 39
一、相鄰引子對設計 39
二、引子對最佳條件測試 39
三、引子對專一性測試 40
伍、香蕉條紋病毒田間染病情況調查 41
一、DNase / two step rt-PCR檢測BSV之結果 41
二、Abutting PCR檢測 41
三、抗體檢測 42
四、Primer F6R1適用性測試 42
討論 43
參考文獻 51
表 61
圖 71
附錄 86

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