利用恆溫式圈環形核酸增幅技術快速鑑定已被列入CITES之鯊魚物種

Fast identification of CITES-listed shark species by loop-mediated isothermal amplification (LAMP)

林子群(Tzu-Chun Lin)

指導教授 ： 蕭仁傑

國立臺灣大學/理學院/海洋研究所/碩士(2019年)

https://doi.org/10.6342/NTU201902814

摘要

對於無法從外觀判定種類的鯊魚樣本，使用生命條碼技術雖然能夠準確的鑑定物種，卻有耗時和需求精密儀器等缺點，為了縮短鑑種時間與現場實測的可能性，本研究使用恆溫式圈環形核酸增幅技術（LAMP）針對已被列入瀕臨絕種野生動植物國際貿易公約（CITES）的鯊魚物種作複合式LAMP檢測，個別針對三段粒線體基因，淺海狐鮫的NADH dehydrogenase subunit 2基因、深海狐鮫的Cytochrome c oxidase subunit I基因、紅肉丫髻鮫的Cytochrome B基因設計出物種專一的引子組，於實驗室模擬現場操作使用LAMP比色套件，以組織均質機（BioMasher® II）取得DNA於乾浴槽於65°C中穩定加熱，在1個小時內即可完成檢測，若DNA放大成功，反應溶液的外觀顏色會從粉紅色改變至橘黃色，利於肉眼上的辨認。在84個檢測樣本中，針對三個目標物種的複合式LAMP檢測達到非常好的結果，在靈敏度（sensitivity）、特異度（specificity）和準確度（accuracy）的值上皆達到100%，沒有偽陰性或偽陽性的發生，由於 LAMP這項技術的操作簡單、檢測時間短以及能用肉眼分辨目標物種等特性，因此我們認為適合漁業管理者作為現場初步的快速篩檢工具，以提升執法效率。

關鍵字

鯊魚 ； 物種鑑定 ； 粒線體基因 ； 分子生物檢測 ； 恆溫式圈環形核酸增幅技術

並列摘要

Identification of shark species at landing is difficult if the key diagnostic features have been removed on board. Although DNA barcoding technique can identify the species without any morphological characters, it takes longer time, requires sophisticated instruments and higher cost. To shorten the process of species identification and to perform it in the field, an alternative technique named loop-mediated isothermal amplification (LAMP) was applied to identify CITES-listed shark species. Species-specific primers were designed to target three mitochondrial genes (ND2, COI and CytB) for pelagic thresher shark (Alopias pelagicus), bigeye thresher shark (A. superciliosus) and scalloped hammerhead shark (Sphyrna lewini), respectively. A homogenizer (BioMasher® II) was used to release DNA from the shark tissues, then DNA amplicons were reproducibly generated for 30 min in the incubator at 65°C. The result of the multiplex LAMP assay demonstrated a high degree of sensitivity (100%), specificity (100%), and accuracy (100%) in 84 samples. The LAMP assay described here provided a fast, reliable and on-site method to distinguish 3 CITES-listed shark species from the other non-target species.

並列關鍵字

Shark ； Species identification ； Mitochondrial gene ； Molecular assay ； Loop-mediated isothermal amplification (LAMP)

參考文獻

Abramowitz S (1996) Towards inexpensive DNA diagnostics. Trends in Biotechnology, 14(10): 397-401.

Baum JK, Kehler D, Myers RA (2005) Robust estimates of decline for pelagic shark populations in the northwest Atlantic and Gulf of Mexico. Fisheries, 30(10): 27-29.

Baum JK and Myers RA (2004) Shifting baselines and the decline of pelagic sharks in the Gulf of Mexico. Ecology Letters, 7(2): 135-145.

Baum JK and Worm B (2009) Cascading top‐down effects of changing oceanic predator abundances. Journal of Animal Ecology, 78(4): 699-714.

Bonfil R (1994) Overview of world elasmobranch fisheries. Food & Agriculture Org.