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研究生:林子群
研究生(外文):Tzu-Chun Lin
論文名稱: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
論文名稱(外文):Fast identification of CITES-listed shark species by loop-mediated isothermal amplification (LAMP)
指導教授:蕭仁傑蕭仁傑引用關係
口試委員:謝幸媚劉光明周銘翊
口試日期:2019-07-23
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:海洋研究所
學門:自然科學學門
學類:海洋科學學類
論文種類:學術論文
論文出版年:2019
畢業學年度:107
語文別:中文
論文頁數:98
中文關鍵詞: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
DOI:10.6342/NTU201902814
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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。
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.
摘要 i
Abstract ii
目錄 iii
表目錄 v
圖目錄 vii
附錄目錄 xi
壹、前言 1
1.1研究對象 1
1.2國際保育政策 2
1.3傳統分子鑑種技術用於鯊魚漁業管理 4
1.4快速篩檢技術用於鯊魚漁業管理的可行性 5
1.5研究目的 6
貳、材料與方法 8
2.1鯊魚樣本蒐集與研究流程 8
2.2樣本物種鑑定 8
2.2.1 DNA萃取 8
2.2.2聚合酶鏈鎖反應(Polymerase Chain Reaction, PCR) 8
2.2.3 PCR產物之純化 9
2.2.4核酸序列分析:DNA毛細管自動電泳儀定序(automated DNA sequencing) 9
2.2.5線上資料庫比對 10
2.3恆溫式圈環形核酸增幅技術(LAMP)之物種專一引子組設計條件 10
2.4 DNA品質之測試 11
2.5恆溫式圈環形核酸增幅技術(LAMP)之反應條件 12
2.6 LAMP偵測極限濃度測試條件 13
2.7複合恆溫式圈環形核酸增幅技術(Multiplex LAMP)之檢測條件 13
2.8自行調配之LAMP比色測試條件 14
2.9靈敏度(Sensitivity)、特異度(Specificity)和準確度(Accuracy)之定義與計算 14
叁、結果 16
3.1樣本採集狀況以及DNA序列資料 16
3.2 LAMP物種專一引子組設計 16
3.3 LAMP反應測試 17
3.4 LAMP偵測極限濃度測試 19
3.5 Multiplex LAMP之檢測 20
3.6自行調配之LAMP比色測試 21
3.7 LAMP檢驗之潛力評估 22
肆、討論 23
4.1 LAMP引子的設計與檢測 23
4.2不同品質的鯊魚物種DNA萃取方法對檢驗的影響 24
4.3 LAMP試劑的測試與比較 24
4.4 LAMP檢測的現場操作評估 26
4.5 LAMP檢測與現行檢測方式的比較 26
伍、結論 29
陸、參考文獻 30

表目錄
表 1、本論文使用的檢體物種之名稱,樣本數(N)與族群量狀態。 38
表 2、引子組Sl_ND2之序列。 39
表 3、引子組Sl_ND2之物種專一引子各黏合區變異點數,以紅肉丫髻鮫為比較依據。 40
表 4、引子組Sl_CytB之序列。 41
表 5、引子組Sl_CytB之物種專一引子各黏合區變異點數,以紅肉丫髻鮫為比較依據。 42
表 6、引子組Ap_ND2之序列。 43
表 7、引子組Ap_ND2之物種專一引子各黏合區變異點數,以淺海狐鮫為比較依據。 44
表 8、引子組As_COI之序列。 45
表 9、引子組As_COI之物種專一引子各黏合區變異點數,以深海狐鮫為比較依據。 46
表 10、引子組Sl_ND2檢驗中的結果與訊號比例,以紅肉丫髻鮫為比較依據。 47
表 11、引子組Ap_ND2檢驗中的結果與訊號比例,以淺海狐鮫為比較依據。 48
表 12、引子組組合Ap_ND2/As_COI檢驗中的結果與訊號比例,以淺海狐鮫與深海狐鮫為比較依據。 49
表 13、引子組組合Sl_CytB/Ap_ND2/As_COI檢驗中的結果與訊號比例,以淺海狐鮫、深海狐鮫與紅肉丫髻鮫為比較依據。 50
表 14、引子組Sl_ND2檢驗之潛力評估,以紅肉丫髻鮫為比較依據。 51
表 15、引子組Ap_ND2檢驗之潛力評估,以淺海狐鮫為比較依據。 52
表 16、引子組組合Ap_ND2/As_COI檢驗之潛力評估,以淺海狐鮫與深海狐鮫為比較依據。 53
表 17、引子組組合Sl_CytB/Ap_ND2/As_COI檢驗之潛力評估,以淺海狐鮫、深海狐鮫與紅肉丫髻鮫為比較依據。 54


圖目錄
圖 1、研究流程。 55
圖 2、針對紅肉丫髻鮫(Scalloped hammerhead shark, S. lewini)所設計之物種專一性引子組Sl_ND2序列。 56
圖 3、針對紅肉丫髻鮫(Scalloped hammerhead shark, S. lewini)所設計之物種專一性引子組Sl_CytB序列。 57
圖 4、針對淺海狐鮫(Pelagic thresher shark, A. pelagicus)所設計之物種專一性引子組Ap_ND2序列。 58
圖 5、針對深海狐鮫(Bigeye thresher shark, A. superciliosus)所設計之物種專一性引子組As_COI序列。 59
圖 6、以引子組Sl_ND2搭配WarmStart® LAMP Kit進行LAMP反應之螢光結果1。 60
圖 7、以引子組Sl_ND2搭配WarmStart® LAMP Kit進行LAMP反應之螢光結果2。 61
圖 8、以引子組Sl_ND2搭配WarmStart® LAMP Kit在Real-time PCR儀器中進行LAMP反應,紅肉丫髻鮫之螢光訊號結果1。 62
圖 9、以引子組Sl_ND2搭配WarmStart® LAMP Kit在Real-time PCR儀器中進行LAMP反應,非目標物種與控制組之螢光訊號結果1。 63
圖 10、以引子組Sl_ND2搭配WarmStart® LAMP Kit在Real-time PCR儀器中進行LAMP反應,以毛細管電泳分析紅肉丫髻鮫、非目標物種與控制組之結果1。 64
圖 11、以引子組Sl_ND2搭配WarmStart® LAMP Kit在Real-time PCR儀器中進行LAMP反應,紅肉丫髻鮫之螢光訊號結果2。 65
圖 12、以引子組Sl_ND2搭配WarmStart® LAMP Kit在Real-time PCR儀器中進行LAMP反應,紅肉丫髻鮫之毛細管電泳結果2。 66
圖 13、以引子組Sl_ND2搭配WarmStart® LAMP Kit在Real-time PCR儀器中進行LAMP反應,紅肉丫髻鮫之螢光訊號結果3。 67
圖 14、以引子組Sl_ND2搭配WarmStart® LAMP Kit在Real-time PCR儀器中進行LAMP反應,非目標物種與控制組之螢光訊號結果3。 68
圖 15、以引子組Sl_ND2搭配WarmStart® LAMP Kit在Real-time PCR儀器中進行LAMP反應,以毛細管電泳分析紅肉丫髻鮫、非目標物種與控制組之結果3。 69
圖 16、以引子組Sl_ND2搭配WarmStart® Colormetric LAMP Kit進行LAMP反應,紅肉丫髻鮫、非目標物種與控制組之變色結果1。 70
圖 17、以引子組Sl_ND2搭配WarmStart® Colormetric LAMP Kit進行LAMP反應,以毛細管電泳分析紅肉丫髻鮫、非目標物種與控制組之結果1。 71
圖 18、以引子組Sl_ND2搭配WarmStart® Colormetric LAMP Kit進行LAMP反應,紅肉丫髻鮫、非目標物種與控制組之變色結果2。 72
圖 19、以引子組Sl_ND2搭配WarmStart® Colormetric LAMP Kit進行LAMP反應,以毛細管電泳分析紅肉丫髻鮫、非目標物種與控制組之結果2。 73
圖 20、以引子組Sl_ND2搭配WarmStart® Colormetric LAMP Kit進行LAMP反應,紅肉丫髻鮫、非目標物種與控制組之變色結果3。 74
圖 21、以引子組Sl_ND2搭配WarmStart® Colormetric LAMP Kit進行LAMP反應,紅肉丫髻鮫之魚肉、乾魚翅、非目標物種與控制組之變色結果。 75
圖 22、以引子組Sl_ND2搭配WarmStart® Colormetric LAMP Kit進行LAMP反應,以毛細管電泳分析紅肉丫髻鮫、非目標物種與控制組之結果3。 76
圖 23、以引子組Sl_ND2搭配WarmStart® Colormetric LAMP Kit進行LAMP反應,以毛細管電泳分析紅肉丫髻鮫之魚肉、乾魚翅、非目標物種與控制組之結果。 77
圖 24、以引子組Sl_CytB搭配WarmStart® Colormetric LAMP Kit進行LAMP反應,紅肉丫髻鮫、非目標物種與控制組之變色結果。 78
圖 25、以引子組Sl_CytB搭配WarmStart® Colormetric LAMP Kit進行LAMP反應,以毛細管電泳分析紅肉丫髻鮫、非目標物種與控制組之結果。 79
圖 26、以引子組Ap_ND2搭配WarmStart® Colormetric LAMP Kit進行LAMP反應,淺海狐鮫、非目標物種與控制組之變色結果。 80
圖 27、以引子組Ap_ND2搭配WarmStart® Colormetric LAMP Kit進行LAMP反應,以毛細管電泳分析淺海狐鮫、非目標物種與控制組之結果。 81
圖 28、以引子組As_COI搭配WarmStart® Colormetric LAMP Kit進行LAMP反應,深海狐鮫、非目標物種與控制組之變色結果。 82
圖 29、以引子組As_COI搭配WarmStart® Colormetric LAMP Kit進行LAMP反應,以毛細管電泳分析深海狐鮫、非目標物種與控制組之結果。 83
圖 30、以引子組Sl_ND2搭配WarmStart® Colormetric LAMP Kit進行LAMP極限濃度測試之變色結果1。 84
圖 31、以引子組Sl_ND2搭配WarmStart® Colormetric LAMP Kit進行LAMP極限濃度測試之毛細管電泳結果1。 85
圖 32、紅肉丫髻鮫之魚肉與乾魚翅樣本。 86
圖 33、以引子組Sl_ND2搭配WarmStart® Colormetric LAMP Kit進行LAMP極限濃度測試之變色結果2。 87
圖 34、以引子組Sl_ND2搭配WarmStart® Colormetric LAMP Kit進行LAMP極限濃度測試之毛細管電泳結果2。 88
圖 35、以引子組組合Ap_ND2/As_COI搭配WarmStart® Colormetric LAMP Kit進行Mutiplex LAMP反應,淺海狐鮫、深海狐鮫、非目標物種與控制組之變色結果。 89
圖 36、以引子組組合Ap_ND2/As_COI搭配WarmStart® Colormetric LAMP Kit進行Mutiplex LAMP反應,以毛細管電泳分析淺海狐鮫、深海狐鮫、非目標物種與控制組之結果。 90
圖 37、以引子組組合Sl_CytB/Ap_ND2/As_COI搭配WarmStart® Colormetric LAMP Kit進行Mutiplex LAMP反應,淺海狐鮫、深海狐鮫、紅肉丫髻鮫、非目標物種與控制組之變色結果。 91
圖 38、以引子組組合Sl_CytB/Ap_ND2/As_COI搭配WarmStart® Colormetric LAMP Kit進行Mutiplex LAMP反應,以毛細管電泳分析淺海狐鮫、深海狐鮫、紅肉丫髻鮫、非目標物種與控制組之結果。 92
圖 39、自行調配藥劑以引子組Sl_CytB搭配酚紅指示劑進行LAMP反應,紅肉丫髻鮫、非目標物種與控制組之變色結果。 93
圖 40、自行調配藥劑以引子組Sl_CytB搭配酚紅指示劑進行LAMP反應,以毛細管電泳分析紅肉丫髻鮫、非目標物種與控制組之結果。 94
圖 41、自行調配藥劑以引子組組合Sl_CytB/Ap_ND2/As_COI和引子組Sl_CytB搭配金屬指示劑(HNB)進行LAMP反應,淺海狐鮫、深海狐鮫、紅肉丫髻鮫、非目標物種與控制組之變色結果。 95
圖 42、自行調配藥劑以引子組組合Sl_CytB/Ap_ND2/As_COI和引子組Sl_CytB搭配金屬指示劑(HNB)進行LAMP反應,以毛細管電泳分析淺海狐鮫、深海狐鮫、紅肉丫髻鮫、非目標物種與控制組之結果。 96


附錄目錄
附錄一、LAMP 反應機制(Notomi et al., 2000)。 97
附錄二、Fish DNA Barcoding 引子表(Holmes et al., 2009)。 98

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