指導教授：張本恆臺灣大學：獸醫學研究所陳敏修Chen, Min-HsiuMin-HsiuChen2014-12-012018-07-092014-12-012018-07-092014http://ntur.lib.ntu.edu.tw//handle/246246/264502台灣分離之九孔鮑 (Haliotis diversicolor supertexta)疱疹病毒 (AbHV) DNA經定序後，其中一段5781 bp基因序列，與澳洲的鮑魚疱疹病毒 (strain Victoria/AUS/2007, AbHV-1)的DNA聚合酶基因DNA序列有99% (5767/5779)的相同度 (identity)，胺基酸序列有99% (1923/1926)的高度相同度，與感染牡蠣等雙殼貝類的牡蠣疱疹病毒 (ostreid herpesvirus 1, OsHV-1) DNA聚合酶胺基酸序列則30% (563/1856) 的相同度。在本研究中，依據AbHV DNA聚合酶基因序列，開發了一個以PCR反應檢測九孔鮑疱疹病毒感染的方法；以引子對40f與146r進行PCR反應，預期產物大小為606bp。PCR檢測九孔鮑疱疹病毒具特異性，與其他疱疹病毒包括牡蠣疱疹病毒 (OsHV-1)、錦鯉疱疹病毒 (koi herpesvirus, KHV)、鰻魚疱疹病毒 (eel herpesvirus)、雞傳染性喉氣管炎病毒 (avian infectious laryngotracheitis virus, ILTV)進行PCR反應，結果都呈陰性反應。結合PCR與組織病理學檢查，能提供可靠的九孔鮑疱疹病毒感染的診斷。在本研究中，依據病毒DNA聚合酶基因序列，亦開發了一個以恆溫環形核酸增幅法 (loop-mediated isothermal amplification, LAMP)檢測九孔鮑疱疹病毒；最佳反應條件為溫度63℃下，反應60分鐘。LAMP反應結果可用電泳分析或添加螢光劑觀察。LAMP法檢測九孔鮑疱疹病毒具特異性，與其他疱疹病毒包括牡蠣疱疹病毒 (OsHV-1)、錦鯉疱疹病毒 (KHV)、鰻魚疱疹病毒 (eel herpesvirus)、雞傳染性喉氣管炎病毒 (ILTV)進行LAMP反應，結果都呈陰性反應。本試驗所開發的LAMP檢驗法敏感性為PCR的100倍，敏感性較SYBR Green PCR少10倍。顯示LAMP檢驗法是一個簡單、迅速、有高度特異性與敏感性及可靠的偵測九孔鮑疱疹病毒技術。A 5781-base pair (bp) fragment of genomic DNA was obtained from Taiwanese abalone herpesvirus and showed 99% (5767/5779) identity in the nucleotide sequence and 99% (1923/1926) in the amino acid sequence with the DNA polymerase gene of the abalone herpesvirus strain Victoria/AUS/2007. Identity of the amino acid sequence was 30% (563/1856) with the DNA polymerase of ostreid herpesvirus 1. In this study, a PCR-based procedure for detecting herpesvirus infection of abalone, Haliotis diversicolor supertexta, in Taiwan was developed. The method employed primer sets targeting the viral DNA polymerase gene, and was able to amplify DNA fragments of the expected size from infected samples. Primer sets of 40f and 146r were designed for amplification of an expected PCR product of 606 bp. Combining the new PCR protocol with histopathology, this assay can serve as a reliable diagnostic method for herpesvirus infections in abalone. A loop-mediated isothermal amplification (LAMP) assay was developed for the detection of abalone herpesvirus DNA. Two pairs of primers were designed, based on the sequence of the DNA polymerase gene of abalone herpesvirus. The reaction temperature and time were optimized at 63℃ and 60 min, respectively. LAMP amplicons were analyzed by 2% agarose gel electrophoresis or by visual inspection of a colour change emitted by fluorescent dye. The method developed was specific for the detection of abalone herpesvirus, without cross-reactions with other tested herpesviruses including ostreid herpesvirus 1 (OsHV-1), European eel herpesvirus, koi herpesvirus (KHV) and an avian herpesvirus. The LAMP assay was 100 folds more sensitive than a conventional PCR and 10 folds less sensitive than a SYBR Green PCR. These results indicate that the developed LAMP assay is a simple, rapid, sensitive, specific and reliable technique for the detection of abalone herpesvirus.摘要…………………………………………………………….……….…… i Abstract………………………………………………………….………………. ii 目錄………………………………………………………….……….………… iii 表目錄……………………………………………………….…………………. vii 圖目錄………………………………………………………….………………. viii 第一章 前言…………………………………………………….…………….. 1 第二章 文獻回顧…………………………………………………..…………. 3 2.1九孔鮑 (Haliotis diveircolor supertexta)簡介…… 3 2.1.1 分類……….……………………………...………………………….. 3 2.1.2 分佈……….……………………………...………………………….. 3 2.1.3 環境與習性..…………………………...………………………….. 4 2.1.4 養殖概況…….………………………...…………………………….. 5 2.2 鮑魚之病毒性疾病…..………..……...…………………………….. 6 2.2.1 肌肉萎縮病 (Amyotrophia)……….…...…………………………….. 6 2.2.2球狀病毒與弧菌混合感染 (Spherical virus co-infection with vibrio bacteria)..….………...…………….………………….………….. 7 2.2.3 鮑肌肉萎縮症病毒 (Abalone shriveling syndrome-associated virus, AbSV)..….………...…………….………………….……………….. 7 2.2.4鮑魚疱疹病毒 (Abalone herpesvirus, AbHV)........... 8 2.3軟體動物雙貝類 (bivalves)及鮑魚之Herpesvirus….……….……….. 9 2.3.1 Herpesvirus分類………....……………………………..……….. 9 2.3.2 牡蠣疱疹病毒 (Ostreid herpesvirus 1, OsHV-1)..…….. 10 2.3.3九孔鮑疱疹病毒(Abalone herpesvirus, AbHV)..………...….. 11 2.4 聚合酶鏈反應 (Polymerase chain reaction, PCR) 技術.….. 12 2.4.1 聚合酶鏈反應 (Polymerase chain reaction, PCR) 發展背景 12 2.4.2 聚合酶鏈反應原理…………………………………………......….. 13 2.4.3 聚合酶鏈反應應用……………………………………………..….. 13 2.5 SYBR Green即時聚合酶鏈反應 (SYBR Green Real-time PCR) 技術 14 2.5.1 即時聚合酶鏈反應 (Real-time PCR) 原理…. 14 2.5.2 SYBR Green即時聚合酶鏈反應……………………..…. 14 2.5.3 SYBR Green即時聚合酶鏈反應的應用………………. 15 2.6恆溫環形核酸增幅法 (loop-mediated isothermal amplification, LAMP)..….………...…………….………………….……………….. 15 2.6.1 恆溫環形核酸增幅法原理…………………………………….. 15 2.6.2 恆溫環形核酸增幅法產物之檢測………………..….. 17 2.6.3恆溫環形核酸增幅法之優點………………………….…….. 17 2.6.4恆溫環形核酸增幅法之應用………………………………….. 18 2.7電子顯微鏡應用於動物疾病之診斷………………………... 19 2.7.1電子顯微鏡簡介………………………….…………..……………. 19 2.7.2電子顯微鏡應用於微生物傳染性疾病診斷……….. 19 2.7.3穿透式電子顯微鏡之病毒樣品的準備…………….…. 21 2.7.4病毒顆粒大小 (size)之測量……………………..…. 22 2.8離心分離方法…………………………..…..………….…. 22 2.8.1離心分離方法簡介……………………..…………………….…. 22 2.8.2蔗糖梯度離心法……………………..…………………….…. 23 第三章 九孔鮑疱疹病毒 (AbHV) DNA polymerase基因PCR檢測方法之開發…...... 25 3.1摘要………………………………………………………………….. 25 3.2前言................................... 25 3.3材料與方法………........................... 27 3.3.1材料來源…….............………………………...... 27 3.3.2 病毒分離、負染色與DNA萃取.............. 27 3.3.3 病毒DNA定序……...….................... 28 3.3.4 以聚合酶鏈反應 (PCR) 偵測AbHV.......... 28 3.3.5 組織病理學和PCR一致性分析............... 29 3.4 結果……....………………………………………….…………….. 29 3.4.1 核酸定序及序列比對分析 (sequence alignment)..... 29 3.4.2 AbHV的PCR檢測…………………………….......………. 30 3.4.3 組織病理學檢查……....................... 30 3.4.4 電子顯微鏡檢查....................…..…. 30 3.4.5 組織病理學與PCR一致性分析................. 31 3.5 討論..............................……..…. 31 第四章 九孔鮑疱疹病毒恆溫環形核酸增幅法 (LAMP) 檢測方法之開發 34 4.1摘要………………………………………………………………….. 34 4.2前言……............................……..….. 34 4.3材料與方法……….....................…..……….. 36 4.3.1 樣品及DNA萃取…...………………………..……..……..….. 36 4.3.2 LAMP primers……..........……………………..….. 36 4.3.3 LAMP的反應條件……......…………………….………..….. 36 4.3.4 AbHV LAMP檢驗法的特異性分析…......……………….….. 37 4.3.5 LAMP、常規PCR與SYBR Green PCR的敏感性與偵測極限 37 4.3.6以養殖現場九孔鮑進行LAMP檢驗法的評估…………..….. 38 4.4 結果……....……………………………………………………….. 38 4.4.1 AbHV 的LAMP反應條件最佳化…………….…………….. 38 4.4.2 LAMP與常規PCR的敏感性…………………….......………. 39 4.4.3 LAMP法檢測AbHV的特異性分析…………….....………. 39 4.4.4 SYBR Green PCR………………………………........………. 39 4.4.5以現場九孔鮑評估AbHV LAMP檢測法的敏感性………. 39 4.5 討論.............................……..…. 40 第五章 結論…………..……………………………………………….…….. 42 參考文獻……………………………………………………………….……… 433189126 bytesapplication/pdf論文公開時間：2019/07/22論文使用權限：同意有償授權(權利金給回饋學校)疱疹病毒九孔鮑DNA聚合酶聚合酶鏈反應 (PCR)恆溫環形核酸增幅法 (LAMP)SYBR green即時聚合酶鏈反應thesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/264502/1/ntu-103-D94629005-1.pdf