臺灣博碩士論文加值系統

中文摘要

鯙科魚類在台灣俗稱為薯鰻或錢鰻，廣泛分佈於世界熱帶及亞熱帶海域，是珊瑚礁區重要的肉食性魚類，也是近海經濟性食用魚種。然而食用鯙類常有發生食物中毒之事件，而且爪哇裸胸鯙鰻 (Gymnothorax javanicus)亦被分離出神經毒熱帶性海魚毒ciguatoxin，所以鯙類ㄧ般被視為是含有熱帶性海魚毒之有毒魚種。在鯙類，熱帶性海魚毒之毒化過程係由草食性魚類攝食有毒渦鞭毛藻(Gambierdiscus toxicus) 後，再經食物鏈毒由肉食性鯙類攝食而來。若再經由人類攝食後，便會產生中毒現象。在2004 年 4 月在台灣淡水富基漁港發生ㄧ件食物中毒案例，47歲男性病患，因食用鯙類後，產生四肢無力、嘴麻、頭暈等中毒症狀，經分析其內臟毒性，顯示為0.12 MU/g，且小鼠會產生與熱帶性海魚毒中毒之相同症狀。
為建立上述發生食物中毒之鯙類種屬，乃首先利用引子(L newest /H 15149)增幅中毒檢體和4種台灣常見鯙類之粒線體cytochrome b gene 之部分基因片段376 bp，定序後比對顯示中毒檢體與有毒種爪哇裸胸鯙之鹼基間距離差異度最低，再以不同熱處理條件(100℃加熱30 min；100℃加熱60 min；121℃加熱15 min；121℃加熱30 min)進行實驗，結果顯示所有加熱處理方式所得魚肉之粗萃 DNA皆可順利進行PCR增幅，但除121℃加熱30 min 之PCR增幅訊號較微弱。另外，利用限制酶TaqⅠ、Sau96Ⅰ兩步驟可區分四種常見鯙類。其次，以另一對之引子(L 14724 /H 15149)增幅其它 6種台灣常見鯙類部分cytochrome b gene片段，其片段長度為486 bp，再以此引子針對不同熱處理條件進行實驗，顯示所增幅之PCR產物在121℃加熱15 min和 121℃加熱30 min增幅訊號隨之減弱。進一步以限制酶SspⅠ、NdeⅠ、Nla Ⅳ切割後可區分上述6種鯙類。顯示利用上述兩組引子及限制酶切位的差異，可以有效鑑定10種不同鯙類之種別。其次，10種鯙類之17個體，經檢測毒性得知除中毒檢體外，其他個體並未檢出毒性。
綜合上述，本研究建立了10種鯙類Mitochondria中cytochrome b gene之片段序列，及利用特異限制酶切位之差異，可有效的辨別新鮮和加工鯙類之魚種。同時，對10種鯙類魚體之毒性，經檢測結果顯示，僅中毒檢體有毒，其它檢體並皆無毒性。

Abstract

Moray eels are the commercially important fish species in Taiwan. These fish distribute in the tropical and subtropical coral reef district in the world extensively and are carnivorous and edible important species. Otherwise, the food poison incident occasionally occurred when people ate them. Therefore, these fish are commonly recognized as ciguateric species. Recently, one of ciguateric toxins, ciguatoxin, has been isolated from moray eel Gymnothorax javanicus. Ciguatoxin is produced by Gambierdiscus toxicus that is usually attached on coral. The toxin and related compound are fist accumulated by herbivorous fish and then transferred into carnivorous fish via food chain. When humans consume these toxic herbivorous or carnivorous fish, they may be intoxicated. A food poisoning incident has occurred due to ingestion of moray eel meat on April 2004 at Fugil Fishing Port, Tamsui. The patient of 47-year-old showed the symptoms of vertigo and paralysis. After toxicity analysis, it showed that the toxicity of viscera was 0.12 MU/g and the ciguateric symptoms were observed in all experimental mice.
To identify the species of toxic moray eel implicated into this food poisoning, 376 bp fragment sequence of cytochrome b gene on 4 species of fresh moray eel meats by using a pair of primers (L newest /H 15149) was successfully obtained. It was found that the species of toxic moray eel implicated into food poisoning might be judged as G. javanicus based on sequence analysis. After treated with different heating processes (100℃ for 30 min；100℃ for 60 min；121℃ for 15 min；121℃ for 30 min), it indicated that the 376 bp fragment of cytochrome b gene in all processed products could be amplified and differentiated the species. But DNA was degraded seriously by 121℃ heating for 30 min. In addition, using restriction enzyme analysis, including TaqⅠand Sau96Ⅰ, could distinguish these 4 species of moray eel and poisoning sample rapidly. On the other hand, the other pair of primers (L 14724 /H 15149) could successfully amplify other 6 moray eels. The length of this partial gene region was 486 bp, and the PCR products amplified by this pair of primers could distinguish the species by gene sequence and analysis of restriction enzyme SspⅠ, NdeⅠand Nla Ⅳ. The toxicity of 17 specimens of 10 moray eel species has determined and found to be not detected. It indicated all tested samples were nontoxic.
Over all, this study provided different patterns of partial mitochondrial cytochrome b gene of 10 moray eel species. After sequencing, it was found that no variation in sequences was detected among individuals within each species. The species of moray eel implicated into food poisoning was identified as Gymnothorax javanicus. It is the first molecule-based study of moray eels diversity and sheds light on the evolution and taxonomy of this major moray eel family. Although the sample implicated into food poisoning was found to be toxic, the toxicity of the other tested moray eels was not detacted.

目 錄
中文摘要………………………………………………………………….Ⅴ
英文摘要………………………………………………………………….Ⅶ
第一章、文獻整理
一、鯙類之特徵及分佈………………………………………………2
二、熱帶性海魚毒之分佈……………………………………………9
三、熱帶性海魚毒中毒之症狀……………………………………..10
四、熱帶性海魚之毒成份…………………………………………..11
五、熱帶性海魚毒中毒機制………………………………………..15
六、一般鑑定魚種方法……………………………………………..16
七、以mtDNA來鑑定魚種之特性與優點………………………...20
八、應用mtDNA做為水產加工品魚種之鑑定……………….......24
第二章、引起食物中毒鯙類之毒性及魚種探討
一、前言……………………………………………………………..30
二、實驗材料…………………………………………………….....31
三、實驗方法………………………………………………………..31
1.毒成分之抽出……………………………………………….......31
2. DNA萃取………………………………………………............32
3. 引子(primer)選擇…………………………………………......33
4. PCR增幅反應………………………………………………....33
5. 電泳分析……………………………………………………...34
6. PCR產物之純化………………………………………………34
7. PCR產物定序…………………………………………………35
8. 限制酶分析PCR產物………………………………………..35
四、結果……………………………………………………...........36
五、討論…………………………………………………………...39
圖表..……………………………………………………………….41
第三章、建立6種台灣常見鯙類之毒性與基因魚種鑑定法之探討
一、前言…………………………………………………………....53
二、實驗材料……………………………………………………....54
三、實驗方法……………………………………………………....54
1. 毒成分之抽出………………………………………………...54
2. DNA之萃取…………………………………………………..54
3. 基因引子 (primer)之設計…………………………………....55
4. PCR增幅反應…………………………………………………55
5. 電泳分析………………………………………………….......55
6. PCR產物之純化……………………………………………....55
7. PCR產物定序……………………………………………........55
8. 限制酶分析PCR產物………………………………………..55
四、結……………………………………………………............56
五、討論…………………………………………………………....59
圖表..………………………………………………………………..61
綜合結果與討論……………...…………………………………………73
參考文獻………………………………………………………………...74

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