利用基因序列分析及PCR-RFLP技術鑑定四種鮪魚（Thunnus）魚種之探討   全文下載

Identification of Four Thunnus Tuna Species Using Mitochondrial Cytochrome b Gene Sequence and PCR-RFLP Analysis

本實驗利用基因序列分析及聚合?連鎖反應法-限制?作用片段長度多型性分析﹙polymerase chain reaction – restriction fragment length polymorphism；PcR-RFLP﹚技術，來鑑定4種台灣常見的鮪魚魚種，包括黑鮪﹙Thunnus thynnus；bluefin tuna﹚、長鰭鮪﹙Thunnus alalunga；albacore﹚、黃鰭鮪﹙Thunnus albacares；yellowfin tuna﹚及大目鮪﹙Thunnus obesus；bigeye tuna﹚。四魚種採樣皆來自北太平洋、北大西洋及印度洋，分析其粒線體細胞色素b基因之部分376 bp片段差異。實驗結果顯示，在此基因片段中大目鮪與其他三魚種差異最大（4.235%），而黑鮪與長鰭鮪種間相似度則最高（98.687~99.674%）。接著以限制?圖譜分析此376 bp序列，找出3種限制?Bsp 1286I、Hinc II及Rsa I，實際測試應用於片段多形性分析，以DNA電泳判讀其差異。最後，利用此PcR-RFLP技術，可成功鑑定12件市售鮪魚生魚片樣品之魚種，證實此技術可精確且快速地辨別此4種生鮮鮪魚。

Sequence analysis and polymerase chain reaction – restriction fragment length polymorphism (PCR-RFLP) technique were used to identify the species of Thunnus thynnus, T. alalunga, T. obesus and T. albacares. Genetic variation of 376 bp fragments in mitochondrial cytochrome b gene of 4 Thunnus species captured from northern Pacific Ocean, northern Atlantic Ocean and Indian Ocean was analyzed. The restriction enzymes and restriction fragment length pattern in each Thunnus species were obtained. It was found that the 376 bp fragment of cytochrome b gene in T. obesus showed the obvious divergence of 4.235% different from other 3 species. The highest interspecific similarity ranged 98.687～99.674% between T. thynnus and T. alalunga. PCR-RFLP by using 3 specific restriction enzymes Bsp1286, Hinc II and Rsa I could precisely and quickly identify the species of the 4 common Thunnus species. Furthermore, this technique successfully detected the species of 12 samples of commercial tuna fillets (sashimi).