臺灣博碩士論文加值系統

魚類病毒性神經壞死症(Viral Nervous Necrosis disease,VNN disease)一直以來都是影響亞洲地區石斑魚苗大量死亡的主要原因之一。魚類病毒性神經壞死症目前沒有良好的治療方式，所以只有先從預防做起，故必須先對魚類病毒性神經壞死症的傳染途徑進行探討，然而在育苗階段餌料生物非常重要，所以希望通過本實驗來解決石斑魚在育苗階段所遇到的問題。本實驗主要是研究餌料生物汙染魚類神經壞死病毒的時間長短，並以橈足類浸泡病毒液後採樣檢測，接著再以RT-PCR (reverse transcriptase polymerase chain reaction, RT-PCR)、Nested-RT-PCR (nested reverse transcriptase polymerase chain reaction, Nested-RT-PCR)進行檢測，以便了解魚類病毒性神經壞死症可以在橈足類身上停留多長的時間。並且以原位雜交來研究橈足類攜帶魚類神經壞死病毒所在的位置，進而期望解決魚類病毒性神經壞死症在石斑魚育苗階段的高感染率。結果顯示浸泡過病毒液的橈足類用RT-PCR、Nested-RT-PCR檢測後，到第八天的檢測結果，則沒有在餌料生物上檢測到魚類神經壞死病毒，接著再進一步用原位雜交探討病毒停留在餌料生物的組織位置。以上結果都顯示汙染魚類神經壞死病毒的餌料生物，經汙染後都可以檢測到魚類神經壞死病毒，但是到了第192小時則檢測不到魚類神經壞死病毒RNA。所以養殖業者若能以乾淨的餌料生物或者用流水畜養後，來飼育石斑魚幼苗，便可有效預防因餌料生物所引起的魚類病毒性神經壞死症的傳染。

VNN disease (Viral nervous necrosis disease) has always been one of the main reasons which cause the numerous deaths for grouper fry in Asian area. There is no good remedy for the disease in the present, so the only way is the prevention. It’s necessary that we have to understand the route of infection about VNN disease during the fish fry rearing stage. We hope to resolve the problem that happens in the grouper fry cultivation through this experiment. The experiment is mainly to understand how long the NNV (Nervous necrosis virus) will stay after the feed organism took a NNV bath and we use copepods for our experiment sample testing. The following step is using RT-PCR, Nested-RT-PCR to examine the sample in order to know how long will the NNV stay on the copepods and take in situ hybridization to find out the location in which copepods carry the NNV. By this step, we hope to solve the high infection rate of VNN disease in the grouper fry stage. The result shows that 192hrs, when we use RT-PCR, Nested-RT-PCR to examine the copepods which took a NNV bath, we don’t get any reaction of NNV on the feed organism. The last step is using in situ hybridization to comprehend the locations of the tissues where the virus stays in the feed organism. To sum up, all of the results reveal that we can examine the NNV on all feed organism which took a virus bath, but after 192hrs, there is no any reaction of the virus RNA anymore. So if fish farmers can use the running water or free-virus feed organism to cultivate grouper fry, they can prevent the infection of VNN disease which is caused by the feed organism effectively.

中文摘要 I
英文摘要 II
致 謝 III
表目次 VII
圖目次 VIII
一、前言 1
二、文獻整理 3
魚類神經壞死病毒之介紹 3
2.1 魚類神經壞死症的病史與病癥 3
2.2 神經壞死病毒的分類 4
2.3 神經壞死病毒的特性 4
2.4 神經壞死病毒宿主與地理分佈 5
2.5 神經壞死病毒的傳染途徑 7
2.6 神經壞死病毒不同環境因素下的特性與檢測方式 8
三、材料與方法 16
3.1 檢體來源 16
3.1.1 橈足類來源 16
3.1.2 橈足類汙染魚類神經壞死病毒 17
3.1.3 野生魚類檢體來源 17
3.1.4 檢體處理歸檔 18
3.2魚類神經壞死病毒之檢測 18
3.2.1 RNA的萃取 20
3.2.2 RT-PCR及Nested-RT-PCR 21
3.2.3 組織病理 23
3.2.4 原位雜交 26
3.3橈足類汙染魚類神經壞死病毒後原位雜交之觀察 30
3.3.1橈足類組織切片及染色 30
3.3.2橈足類組織切片之原位雜交 30
四、結果 32
4.1 RT-PCR和Nested-RT-PCR靈敏度之比較 32
4.2 核酸探針靈敏度之比較 32
4.3 檢體鑑定與分類整理 33
4.4 野生生物神經壞死病毒之分子檢測結果 34
4.5 橈足類汙染魚類神經壞死病毒之分子檢測結果 34
4.6 原位雜交之檢測結果 35
五、討論 36
5.1 RT-PCR與Nested-RT-PCR靈敏度之比較 36
5.2 核酸探針靈敏度之比較 36
5.3 橈足類帶原魚類神經壞死病毒之分子檢測 37
5.4 原位雜交之檢測結果 38
5.5 下雜魚的分子檢測 38
六、參考文獻 40
表…… 49
圖…… 50
附錄… 63

台灣魚類資料庫 http://fishdb.sinica.edu.tw/

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