利用基因技術鑑定台灣珊瑚礁魚類笛鯛魚種之探討

Application of Genetic Techniques on Species Identification of Coral Reef fish Lutjanidae in Taiwan

黃耿琳(huang kêng lin)

指導教授 ： 謝承紘 ； 李明明

亞洲大學/資訊學院/生物資訊學系碩士班/碩士(2007年)

摘要

笛鯛科魚類廣泛分佈於全世界熱帶及亞熱帶海域，屬珊瑚礁區的肉食性魚類，亦為台灣重要之經濟性魚類，產地遍佈於台灣各沿海。全世界共計有 4 亞科 22 屬 129 種，而台灣產則有 4 亞科 10 屬 51種。由於所處海域之關係，某些笛鯛科魚類會因為經由食物鏈之途徑而攝食到有毒的渦鞭毛藻 Gambierdiscus toxicus 或捕食已遭到 G.toxicus 毒化的草食性魚類，而導致人類食物中毒事件之發生。1991年6 月台灣首次記錄熱帶性珊瑚礁海魚毒之中毒事件，台北縣一家五口因食用紅鱠肝臟後，發生類似熱帶性珊瑚礁海魚毒之食物中毒症狀。近年則是在 2006 年 2 月的時候，發生三位民眾因食用購自高雄前鎮漁港魚市場的類似笛鯛科魚類而引起疑似為熱帶性珊瑚礁海魚毒之中毒案例，其中以48 歲的男性及女性這兩位病患較為嚴重，在食入類似笛鯛科魚類後產生腹痛、腹瀉、四肢無力、盜汗、皮膚癢以及肌肉劇烈疼痛等症狀，而男性病患肌肉劇烈疼痛之症狀過了一個月依舊尚未改善。經生物毒性測試分析由高雄長庚紀念醫院及台南市衛生局所提供之檢體魚片各一片後，其內臟部位抽出液之毒性分別為2.45 MU/g 和 5.36 MU/g，且小鼠會產生與熱帶性珊瑚礁海魚毒中毒之相同症狀，而注射肌肉部位抽出液之小鼠雖有熱帶性珊瑚礁海魚毒之中毒症狀出現但並未死亡。同時為瞭解上述引起食物中毒之魚類魚
種，因此利用基因直接定序和聚合酶連鎖反應– 限制酶作用片段長度多型性分析技術(polymerase chain reaction – restriction fragmentlength polymorphism；PCR-RFLP)技術比較中毒檢體與其它六種台灣漁市場常見可能含有熱帶性珊瑚礁海魚毒魚類之基因體差異。本研究使用之引子為L14735/H15149，可自中毒檢體和其它已知採集六種魚種之檢體中增幅出部分cytochrome b gene 之DNA片段，其片段為475bp，經定序得知，兩個中毒案件的殘留魚片檢體之魚種同屬Lutjanu sbohar。另以限制酶Hae III 區分所有檢體，得知兩個中毒案件的殘留魚片檢體與已知L. bohar 檢體皆呈現相同切割片段，為183 bp 、151
bp 和134 bp，此切位片段圖譜顯著與其它已知魚體之PCR-RFLP 圖譜有異。故基因直接定序和PCR-RFLP 法均可順利確認產生中毒之魚片檢體之魚種為L. bohar。此外，其它從漁市場採集已知六種魚類個體並未檢出毒性。

關鍵字

基因技術鑑

並列摘要

Ciguatera is one of marine fish poisoning to eat tropical or subtropical coral reef fish, it is estimated to affect more than 25,000 persons per year globally. The distribution of ciguatera is confined to discrete regions of the Pacific Ocean, western Indian Ocean and Caribbean Sea. The ciguateric toxins are produced by the toxic dinoflagellate Gambierdiscus toxicus, and are accumulated firstly in herbivorous fish and subsequently in carnivorous fish through the food chain effect. While some people eat the toxic fish, it will cause food poisoning. The Genus Lutjanus fish is one of carnivorous coral reef fishes that distributes around the sea area of Taiwan, and it is also one of the commercially important fish species in Taiwan. A food poisoning incident has occurred in February 2006 due to intake of the Lutjanus–like fish meat purchased from Cianjhen fishing market in Kaohsiung. Two patients of 48 years old showed diarrhea, muscle weakness, vertigo and severe muscle pain. These two poisoning samples were provided by Kaohsiung Chang-Gung Memorial Hospital and Tainan City Health Bureau, respectively. The results showed that these two poisoning samples contained 2.45 MU/g and 5.36 MU/g of toxins in the viscera extracts, respectively, but no death of mice was found in the muscle extracts. The other samples were collected from different fish markets but did not show any ciguateric symptoms and death of mice. In order to identify the fish species of poisoning samples, gene sequencing and polymerase chain reaction – restriction fragment length polymorphism (PCR-RFLP) were used. The primer pair L14735/H15149 was used and a DNA fragment of 475 bp from cytochrome b gene was obtained. After gene sequencing, the species of both causative fish fillets was identified as Lutjanus bohar. Furthermore, restriction enzyme Hae III was found to differentiate all tested fish recollected from fish markets. Both causative fish fillets showed 3 fragments ( 183 bp, 151 bp and 134 bp ), which were the same as those of L. bohar in PCR-RFLP pattern. Therefore, the species of causative fish fillets was identified as L. bohar.

並列關鍵字

Genetic Techniques

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