恆溫環形核酸增幅法在保育臺灣鮭魚的應用

Abstract

臺灣鮭魚、(Oncorhynchus formosanus)是臺灣知名的瀕危珍稀物種，估算有效族群數目、控管外來物種的入侵是保育臺灣鮭魚的關鍵工作。族群的性別比例屬於族群數量隨機因子的一種，會影響有效族群數目的估算，但由於臺灣鮭魚幼形成熟，缺乏性別雙型性，因此由外型辨識性別十分困難。又鑒於臺灣每年由國外進口數量可觀的鮭科魚類的發眼卵及全魚，有可能因為天災與人為疏失，使得外來物種或其夾帶之病原菌流逸，威脅臺灣鮭魚的生存。因此，我們以LAMP( Loop-mediated isothermal amplification)法為基礎，開發出經濟、可靠的臺灣鮭魚性別及物種鑑別技術(樣本數120尾，總反應體積5 ul，正確率100%)。其具有: (1)非侵入式: (2) 操作簡單快速(反應時間少於1小時)(3)專一性高;(4)肉眼可直接辨別結果等優點，除適用於臺灣鮭魚外，亦可應用於保育其它鮭科魚類。Species and sex identification are the most important parameters for conservation management. However, it is extremely difficult to perform such identification in Formosa landlocked salmon (Oncorhynchus formosanusi. Both sexual dimorphism in landlocked dwarf form Formosa landlocked salmon and morphological differences among cherry salmon complex are minimal. We developed a simple, rapid and noninvasive method to identify sex and species of this critically endangered species using a loop-mediated isothermal amplification (LAMP) technique . The LAMP assay shows the advantage of simple detection (evaluated by visual inspection), rapid reaction time «1 h), isothermal condition (less equipment required) and high efficiency (only 0.5-5 pg of DNA was required in the reaction mixture). Our method is economical and reliable (n=120, reaction system 5 ul, accuracy 100%). The LAMP assay can be easily performed in the field and is a valuable tool for detecting sex ratios in wild populations and identifying species in commercial imports. This is the first application of LAMP in identifying species and sex of salmonids as far as we know, and it clearly shows the potential application of LAMP in molecular ecology and conservation efforts.