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標題: | 以即時聚合連鎖反應快速鑑定鮪魚種類 Rapid identification of tuna species by Real-Time PCR |
作者: | Meng-I Chen 陳孟宜 |
指導教授: | 蕭仁傑 |
關鍵字: | 鮪魚,物種鑑定,即時聚合連鎖反應, tuna,species identification,real-time PCR, |
出版年 : | 2010 |
學位: | 碩士 |
摘要: | 鮪魚是高經濟性魚種,不同種鮪魚的價格差異非常大,經由魚片的方式進行貿易與販售的鮪魚,已無法從外觀特徵辨識種類,因此只能依賴基因鑑種。過去利用基因鑑定物種是以DNA定序鑑種技術最為可靠,但需時長達數天到一週。本研究的目的在於利用Real-time PCR技術,發展快速即時鑑定鮪魚物種的方法。此研究目標是以設計具有物種專一性的引子和雜合探針(hybridization probe)用於偵測到單一鹼基多型性(Single Nucleotide Polymorphism, SNP)的變異,藉以區別物種。在此研究中,我們已成功在cytochrome b和control region基因中,針對南方黑鮪(Thunnus maccoyii)、大目鮪(T. obesus)、太平洋黑鮪(T. orientalis)和黃鰭鮪(T. albacares)的SNP設計專一性探針,以及針對大目鮪的物種設計專一性引子,並利用MCA(Melting Curve Analysis)的方法利用Tm值大小將太平洋黑鮪和長鰭鮪與其他物種作區隔。探針和引子皆具高度的鑑別力;探針的準確率皆高達75%以上,南方黑鮪和太平洋黑鮪的探針更高達100%。以觀察員依外型鑑定的大目鮪、黃鰭鮪和長鰭鮪作探針鑑種,其結果大致與型態一致。針對不同濃度的DNA模板(500, 250, 50, 25, 5, 2.5, 0.5, 0.25 ng μl -1)進行與個別探針表現的穩定性和再現性做測試;其結果顯示探針偵測DNA模板的最低濃度為0.25 ng μl -1,且DNA濃度與Ct (Cycle threshold)值呈反比。將探針以旗魚科、劍旗魚科和鯊魚的樣本做物種鑑定測試,四個探針的測試結果皆可判讀為非目標物種,表示其引子和探針具有相當的鑑別度。此方法從DNA萃取到以Real-Time PCR技術鑑定物種只需半個工作天,並且可避免傳統PCR與DNA定序鑑定法可能發生的汙染。 Tuna is one of highly valuable seafood, and usually distributed as filleted, broiled and canned products. There are 8 species in Thunnus genus, and each species has very different commercial values, therefore it is important to authenticate tuna species when the morphology characters were removed. DNA based techniques has been extensively used recent years for validation of the species of tuna products. Real-time PCR is one of bio-molecular approaches to identify the quality and quantity of target genes, and can be completed within 2 hours compared to DNA sequencing method, which would take 3 days at least. In this research, we developed a rapid method for species identification by using TaqMan probes and species specific primer to detect single-base variation of the target gene. According to our results, two species specific primers were successfully used to distinguish T. obesus from others by analyzing Ct values and to differentiate T. alalunga and T. orientalis from other species by melting curve analysis. Moreover, we successfully identified T. maccoyii , T. obesus, T. orientalis and T. albacares from other species by SNPs (single nucleotide polymorphisms) probe, designed from mtDNA cytochrome b and control region gene. The correction rates of these probes were higer than 75% and up to 100%. Furthermore, we tested the probes on the samples which were morphologically identified and canned tuna products. The revealed Most results are consistent with morphology. Also, the four probes are tested in sensitivity with eight dilutions of DNA template (500, 250, 50, 25, 5, 2.5, 0.5, 0.25 ng μl -1). The results of sensitivity test suggested that the detection limit was 0.25 ng μl -1, and the Ct value is inversely proportional to the concentration of DNA template. Moreover, we applied tuna specific probes to detection non-Thunnus species, which are Istiophoridae, Xiphiidae, Carcharhiniformes and Lamniformes. The result shows that the primers of the probe systems can only amplify Thunnus genus species, indicating the good species-specificity of the probes. These assays are very useful to rapidly discriminate tuna species in a short time without post-PCR contamination. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/47077 |
全文授權: | 有償授權 |
顯示於系所單位: | 海洋研究所 |
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