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標題: | 利用即時聚合酶連鎖反應鑑定旗魚種類 Identification of billfish (Xiphiidae, Istiophoridae) species by a real-time PCR technique |
作者: | Hung-An Chang 張宏安 |
指導教授: | 蕭仁傑 |
關鍵字: | 旗魚,物種鑑定,即時聚合?連鎖反應, billfish,species identification,real-time PCR, |
出版年 : | 2015 |
學位: | 碩士 |
摘要: | 旗魚為世界上許多國家重要的經濟性魚種,可加工製成各式產品,如生魚片、魚鬆、魚筋、魚醬等。這些產品在臺灣市面上販售時,成分皆僅標註為旗魚,因此對於旗魚製品進行物種的鑑定,將有助於保育與漁業資源管理。在本研究中,我們發展了一套利用即時聚合酶連鎖反應的方法鑑定六個旗魚物種,分別為劍旗魚 (Xiphias gladius)、雨傘旗魚 (Istiophorus platypterus)、立翅旗魚 (Istiompax
indica)、紅肉旗魚 (Kajikia audax)、黑皮旗魚 (Makaira nigricans)以及小吻四鰭旗魚 (Tetrapturus angustirostris)。我們共設計了14 條引子,包含12 條物種專一性引子及2 條能區分六種旗魚與非旗魚物種的universal primer。結果顯示12 條引子對六種旗魚具有良好的鑑別能力,目標物種的ΔCt value 皆介於 -2.23 ~ 3.93 之間,極限濃度可達0.4 ng/μL。此外,為節省檢驗的成本與步驟,我們進一步將多組引子於同一個PCR 反應中進行測試,結果顯示六種旗魚能根據MCA 來鑑別,每個物種皆具有專一性的Tm value:X. gladius:Tm1 = 75.0 ± 0.0 °C, Tm2 = 83.5 ± 0.0 °C;I. platypterus:Tm1 = 75.2 ± 0.3 °C, Tm2 = 80.5 ± 0.2 °C;I. indica:Tm1 = 77.5 ± 0.0 °C, Tm2 = 80.5 ± 0.0 °C;K. audax:Tm = 80.5 ± 0.0 °C;M. nigricans:Tm1 = 76.0 ± 0.0 °C, Tm2 = 80.5 ± 0.0 °C;T. angustirostris:Tm = 77.0 ± 0.0 °C。根據我們的研究結果可建立出一套鑑定六個旗魚物種的流程。此方法具有高效率、高靈敏度,進行高通量檢測時成本較低,我們預期此方法將有助於掌握漁業資源與保障消費者權益。 Billfish is an important fishery resource all over the world, and is usually marketed as sashimi, filleted, fish floss and sauce. Most billfish products in Taiwan were only labeled with “billfish” in their ingredients. Therefore, species identification of the billfish products is necessary and will benefit the conservation and regulation of these fishery resourcs. In this study, we developed a real-time PCR approach to identify the six billfish species:Swordfish (Xiphias gladius), Sailfish (Istiophorus platypterus), Black marlin (Istiompax indica), Striped marlin (Kajikia audax), Blue marlin (Makaira nigricans) and Shortbill spearfish (Tetrapturus angustirostris). The whole system contained 14 primers, including 12 species-specific primers and two universal primers designed for discriminating the six billfish from non-billfish species. The 12 species-specific primers showed clear discrimination among the six billfish species, with ΔCt values of the target species ranging between -2.23 ~ 3.93 and the limit of detection is 0.4 ng/μL. In order to minimize the cost and time of whole identification system, we also tested the applicability of the multiplex qPCR by combining several species-specific primers in a single reaction to identify the species. The result showed that the six species can be identified according to the melting curve pattern and Tm values for X. gladius: Tm1 = 75.0 ± 0.0 °C, Tm2 = 83.5 ± 0.0 °C, I. platypterus:Tm1 = 75.2 ± 0.3 °C, Tm2 = 80.5 ± 0.2 °C, I. indica:Tm1 = 77.5 ± 0.0 °C, Tm2 = 80.5 ± 0.0 °C, K. audax:Tm = 80.5 ± 0.0 °C, M. nigricans:Tm1 = 76.0 ± 0.0 °C, Tm2 = 80.5 ± 0.0 °C, T. angustirostris:Tm = 77.0 ± 0.0 °C. An identification protocol for the six billfish species was established in this study. The methods is efficient, sensitive, and cheaper than other conventional methods, which will be helpful for fishery management and protecting the consumer’s rights. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/54176 |
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顯示於系所單位: | 海洋研究所 |
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