臺灣博碩士論文加值系統

本實驗針對養殖龍膽石斑虹彩病毒之特性進行研究，進而利用分子生物學的技術建立病毒的偵測方法，實驗自2005年10月至2006年3月間，於台灣南部養殖海水魚養殖地區進行採樣，並以虹彩病毒專一性的引子進行PCR的偵測，結果以龍膽石斑、黃錫鯛、金目鱸、赤鰭笛鯛及高背鰏等魚種，均呈虹彩病毒陽性反應。在組織病理切片的觀察，罹患虹彩病毒龍膽石斑的脾臟、腎臟、鰓、心臟和肝臟中可見嗜鹼性腫大細胞。利用Giemsa染色，證明此病變細胞有腫大的情形。在致病性實驗方面，以腹腔注射的方式感染健康的龍膽石斑，在感染後之第9天累積死亡率可達到90％。利用虹彩病毒專一性引子對P3/P4 IRB5，以PCR反應放大病毒核酸ATPase部分基因片段（709bp）進行基因序列比對，顯示龍膽石斑虹彩病毒並不屬於目前已知虹彩病毒科的任何一屬，而與新命名的Tropivirus屬較類似。在龍膽石斑虹彩病毒檢測方式的建立方面，利用PCR的方法將探針進行DIG標定。首先以點墨雜交法（Dot blot hybridization）檢測探針之靈敏度，結果顯示高於1pg/μl的病毒核酸重組質體均可被測得；以此方法可以檢驗病魚是否有受虹彩病毒的感染，但仍有少數偽陽性的產生。在原位雜交反應（In situ hybridization）的檢驗，亦可成功的偵測到病魚的脾臟、腎臟、心臟及鰓被病毒感染的現象，靈敏度較傳統的組織切片高。在PCR檢驗方法之建立，利用已發表的虹彩病毒專一性引子對進行檢測，結果以ATPase 3和P3 IRB5/P4 IRB5兩種引子對較適合運用於偵測龍膽石斑虹彩病毒上。本實驗亦利用Real-time PCR的方法檢測病魚內臟中虹彩病毒的表現量，結果以脾臟及腎臟之病毒量最高，達1010 viral genome copies/g以上；而鰓、心臟及肝臟部位之病毒量雖然較脾臟及腎臟低，但仍達到109 viral genome copies/g以上。運用這些檢測方法，可實際偵測龍膽石斑是否罹患虹彩病毒。

The present study attempts to investigate the characteristics of the iridovirus that infect king grouper（Epinephelus lanceolatus）in pond culture and than to develope a molecular technique that could detect the virus sensitively. The diseased fish were collected during October, 2005 to March, 2006 from growth the ponds in southern coastal areas of the Taiwan. The virus infection was proved by PCR methods using a pair of primer that has been demonstrated RSIV specific. The results showed those of diseased fish including king grouper、goldlined seabream（Rhabdosargus sarba）、giant seaperch（Lates calcarifer）、crimson snapper（Lutjanus erythropterus ）and common slipmouth（Leiognathus equulus）were infected by RSIV. The virus infected cells showed distend and basophilic, and can be seen in the spleen, kidney, gill, heart and liver. Pathogenicity of the virus was proved by infect the juvenile king grouper, the juvenile of king grouper about 5-9g was using intra-peritoneal injection method with a solution 20×diluted virus extract. The mortality of infected fish was about 90% in 9 days, post infection. The PCR product by using a P3/P4 IRB5 primer pair was sequenced and analyzed to construct the phylogenetic relationships with other iridovirus. The result indicated the king grouper iridovirus（KGIV）may belong to the genus Tropivirus. DIG-labelled DNA probes prepared by PCR method. By dot blot hybridization, that were and used to detect the the viral DNA. The probe was proved can detect the viral DNA at a level of 1pg/μl. The probe is sensitive to detect the virus infected fish though some false positive may happen. Using the iridovirus specific primer to establish the PCR diagnosis showed that primers set ATPase 3 and P3 IRB5/P4 IRB5 are more suitable for detection of KGIV. Quantitative analysis the viral genome in various tissue of the infected fish by real-time PCR, the spleen and kidney that were found 1010 viral genome copies/g tissue where the major organs the virus proliferate. The minor organs such as gill, heart and gill also have 109 viral genome copies/g tissue. We suggest that PCR method is more suitable for detecting the king grouper whether suffer from iridovirus. By these methods that can examination of the king grouper whether suffers from iridovirus infection.

目錄
頁次
謝誌……………………………………………………………………………… I

目錄……………………………………………………………………………… II

表目錄…………………………………………………………………………… VI

圖目錄…………………………………………………………………………… VII

中文摘要………………………………………………………………………… 1
英文摘要………………………………………………………………………… 3
第一章 前言…………………………………………………………………… 5
1.1龍膽石斑的習性與養殖現況……………………………………………… 5
1.2虹彩病毒的特性與分類…………………………………………………… 6
1.3熱帶性虹彩病毒的特性…………………………………………………… 7
1.4熱帶性虹彩病毒之地理分佈與宿主範圍………………………………… 8
1.5熱帶性虹彩病毒的症狀…………………………………………………… 10
1.6熱帶性虹彩病毒的檢測方法……………………………………………… 10
1.6.1外表病徵觀察法……………………………………………………… 11
1.6.2組織病理切片………………………………………………………… 11
1.6.3電子顯微鏡技術……………………………………………………… 11
1.6.4酵素連結免疫分析法（ELISA）.…………………………………… 12
1.6.5 DNA探針……………………………………………………………… 12
1.6.6聚合鏈連鎖反應(Polymerase chain reaction，PCR)…………… 13
1.7熱帶性虹彩病毒的防治方法……………………………………………… 13
1.8實驗目的…………………………………………………………………… 14
第二章 材料與方法…………………………………………………………… 15
2.1台灣熱帶性虹彩病毒流行病學之研究…………………………………… 15
2.1.1採樣…………………………………………………………………… 15
2.1.2病理組織切片觀察（石蠟切片技術）……………………………… 15
2.1.2.1固定、脫水、包埋及切片………………………………………… 15
2.1.2.2染色………………………………………………………………… 15
2.1.2.2.1蘇木精和伊紅（Hematoxylin ＆ Eosin）染色法…………… 15
2.1.2.2.2Giemsa stain…………………………………………………… 16
2.2虹彩病毒片段基因的選殖………………………………………………… 16
2.2.1去氧核醣核酸（Total DNA）的萃取………………………………… 16
2.2.2聚合酶連鎖反應（PCR）….…………………………………………… 17
2.2.3膠體純析 ( Gel elution )…………………………………………… 17
2.2.4 DNA接合 ( DNA ligation )………………………………………… 18
2.2.5質體轉型 ( Transformation )……………………………………… 18
2.2.6質體抽取(Plasmid extraction).…………………………………… 18
2.2.7限制酵素切割 (Restriction enzyme digestion)………………… 19
2.2.8核酸序列之分析與比對……………………………………………… 19
2.3龍膽石斑虹彩病毒之致病性研究………………………………………… 19
2.3.1實驗動物……………………………………………………………… 19
2.3.2龍膽石斑病毒液之製備……………………………………………… 19
2.3.3龍膽石斑虹彩病毒之致病性試驗…………………………………… 20
2.4龍膽石斑虹彩病毒之檢測方式與分析…………………………………… 20
2.4.1點墨雜交（Dot blot Hybridization）…………………………… 20
2.4.1.1探針之製備與純化………………………………………………… 20
2.4.1.2點墨雜交反應（Dot blot Hybridization）…………………… 21
2.4.2原位雜交反應（In situ hybridization）………………………… 21
2.4.3龍膽石斑虹彩病毒PCR偵測方法……………………………………… 22
2.4.4絕對定量PCR（Absolute Real-time quantitation PCR）………… 23
2.4.4.1龍膽石斑病毒表現量之分析……………………………………… 23
第三章 結果…………………………………………………………………… 25
3.1龍膽石斑虹彩病毒症流行病學之研究…………………………………… 25
3.2組織病理觀察……………………………………………………………… 25
3.3虹彩病毒片段基因的選殖………………………………………………… 26
3.3.1ATPase之片段基因PCR轉殖和序列分析……………………………… 26
3.3.2虹彩病毒ATPase片段基因序列比對………………………………… 26
3.4龍膽石斑虹彩病毒致病性之研究………………………………………… 27
3.5龍膽石斑虹彩病毒檢測方式之建立與分析……………………………… 28
3.5.1點墨雜交試驗（Dot blot hybridization）………………………… 28
3.5.2原位雜交試驗（In situ hybridization）………………………… 29
3.5.3龍膽石斑虹彩病毒PCR偵測方法……………………………………… 29
3.5.4龍膽石斑病毒表現量之分析………………………………………… 30
圖表……………………………………………………………………………… 31
第四章 討論…………………………………………………………………… 48
第五章 參考文獻……………………………………………………………… 55
附錄……………………………………………………………………………… 63
A.儀器和材料…………………………………………………………………… 63
B.實驗藥品……………………………………………………………………… 63

表目錄
頁次
表1. 93年－95年台灣南部各海水養殖魚塭及澎湖箱網養殖地區採樣記
錄表………………………………………………………………………… 32

圖目錄
頁次
圖1.虹彩病毒感染之龍膽石斑外表及內部病徵……………………………… 33
圖2.虹彩病毒感染之龍膽石斑脾臟及腎臟組織病理切片圖
（H＆E stain）……………………………………………………………… 34
圖3.虹彩病毒感染之龍膽石斑心臟及肝臟組織病理切片圖
（H＆E stain）……………………………………………………………… 35
圖4.虹彩病毒感染之龍膽石斑脾臟及腎臟組織病理切片圖
（Giemsa stain）…………………………………………………………… 36
圖5.虹彩病毒感染之龍膽石斑鰓部組織病理切片圖
（Giemsa stain）…………………………………………………………… 37
圖6. 1.5％洋菜膠電泳分析圖，利用限制酶切割證實重組質體含虹彩病毒
ATPase片段基因…………………………………………………………… 38
圖7.利用Multalin軟體，比較不同魚類虹彩病毒ATPase片段基因之序列
比對………………………………………………………………………… 39
圖8. 13種虹彩病毒ATPase片段基因親緣關係之比較……………………… 40
圖9.龍膽石斑虹彩病毒致病性試驗之累積死亡率圖………………………… 41
圖10. 1.5％洋菜膠電泳分析圖，利用PCR進行確認病原性試驗之病魚內
臟含虹彩病毒…………………………………………………………… 41
圖11. 1.5％洋菜膠電泳分析圖，利用PCR的方法將探針進行DIG標定…… 42
圖12.點墨雜交法（Dot blot hybridization）之靈敏度試驗圖………… 43
圖13.利用點墨雜交法（Dot blot hybridization）對病魚組織DNA進行偵
測………………………………………………………………………… 43
圖14.利用原位雜交法（In situ hybridization），偵測虹彩病毒在病魚
脾臟中分佈的情形………………………………………………………… 44
圖15.利用原位雜交法（In situ hybridization），偵測虹彩病毒在病魚腎臟
中分佈的情形…………………………………………………………… 45
圖16. 1.5％洋菜膠電泳分析圖，利用不同的虹彩病毒專一性引子，以PCR
的方法偵測魚類受虹彩病毒感染的情形……………………………… 46
圖17.利用Real-time PCR分析龍膽石斑之病毒表現量……………………… 47

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