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研究生:張冠霖
研究生(外文):Kuan-Lin Chang
論文名稱:台灣常見貝類及其加工品基因鑑定技術開發之探討
論文名稱(外文):Studies on identification gene assay for shellfish and related products in Taiwan
指導教授:謝宥諒
指導教授(外文):You-Laing Hsieh
口試委員:謝承紘葉彥宏謝宥諒
口試委員(外文):Cheng-Hong HsiehYen-Hung YehYou-Laing Hsieh
口試日期:2014-07-24
學位類別:碩士
校院名稱:亞洲大學
系所名稱:保健營養生技學系
學門:農業科學學門
學類:食品科學類
論文種類:學術論文
論文出版年:2014
畢業學年度:102
語文別:中文
論文頁數:91
中文關鍵詞:貝類
外文關鍵詞:shellfish
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貝類是台灣重要的漁獲資源,在利用時,多經過去除外殼等外部特徵,在經過一連串後續加工程序或貯藏,如:冷藏、冷凍與加熱等步驟,由於可供辨識的外部特徵消失,因此需利用其他方法來鑑定貝類物種。由於加工過程劇烈,造成蛋白質裂解,因此,蛋白質分析方法較難應用在加工品貝類物種鑑定上,故本實驗選用基因技術來鑑定貝類物種,而儲藏與加工時間及程度的差異皆會影響基因序列的完整性,進而影響到基因鑑定貝類物種的成功與否,故鑑定加工之物種時需先觀察其DNA裂解情形,選用合適的長度的目標基因進行引子設計,再搭配相關的基因方法,以辨別貝類物種;台灣沿海養殖及捕獲的貝類包括有馬蹄蛤(Geloina erosa)、黃金蜆(Corbicula fluminea)、文蛤(Meretrix lusoria)、環文蛤(Cyclina sinensis)、蝦夷扇貝(Mizuhopecten yessoensis)、毛蟶(Sinonovacula constricta)、菲律賓簾蛤(Ruditapes philippinarum)、綠殼菜蛤(Perna viridis)、海瓜子(Ruditapes philippinarum)、蚵仔(Crassostrea gigas)、花蛤(Gomphina aequilatera)等。
本研究首先建立11種貝類完整之COI基因組序列和9種貝類完整之Cyt b完整基因序列,其片段分別為1,740 bp、1,445 bp,再利用所建立之資料搜尋貝類物種合適之基因片段,作為物種鑑定引子設計之參考依據。
再者,利用所建立COI基因資料設計一組共同且具專一性之引子COI-F/R,其所增幅之片段長度為460 bp,利用此組引子進行生鮮貝類與加工貝類產品進行PCR-RFLP物種鑑定技術開發,利用限制酶BsaJI、HaeIII、BmrI、MslI、NlaIII共五種限制酶,可成功區別十一種貝類。
由於市售產品之DNA會因加熱高溫烹煮、混雜其它之貝類會影響DNA之完整性。針對仿製蜆精、馬蹄蛤精和蠔精本實驗開發multiplex-PCR利用引子Geloina-F/R、Gigas-F/R、Corbicula-F/R分別成功增幅出片段長度為183 bp、122 bp、256 bp;仿製干貝醬的部分利用Cyt b基因組設計特異性引子yessoensis-F/R可成功增幅出片段長度為320 bp。
根據上述仿製實驗檢測,證明開發之PCR-RFLP與multiplex-PCR技術方法的可用性,實際採集台灣北、中、南、東四個地區之市售貝類樣品:蠔蜆精(錠)33件、干貝醬24件。經基因檢測原料結果顯示蠔蜆精(錠)於中部有1件不符合標示之產品,而干貝醬的部分檢出率則為29.1%。
Shellfish is one of the most important fisheries resources in Taiwan. Before utilizing, most times they’re shucked, processed (freezing, or heating, etc.) and stored. Hence, as a result of losing distinguishable exterior characteristics, other identify approaches are needed for shellfish species forensics.
Due to the severe processed procedure, protein is cleaved. Thus, analytic tools of proteins are not applicable to processed shellfish species. Therefore, the study adopts genetic technologies for identification. The level and differences of the storage and processing time will affect the completeness of the genome sequencing, and lead to the result of the identification. For this reason, firstly the research observes the DNA cleavage status of the sample, choosing suitable length of the target genome with related genetic technology for shellfish species identification.
In Taiwan, popular fisheries and aquaculture of shellfish species include Geloina erosa, Corbicula fluminea, Meretrix lusoria, Cyclina sinensis, Mizuhopecten yessoensis, Sinonovacula constricta, Ruditapes philippinarum, Perna viridis, Tegillarca granosa, and Crassostrea gigas.
The study establishes these 11 shellfishes’ complete COI genome sequencing and 9 Cyt b complete genome sequencing, which the base pairs are 1740 bp, 1,445 bp respectively. With the suitable genome base pairs data of shellfish species, the study sets it as the primer design of the species identification.
Moreover, by utilizing the established COI genome, the study then designs the common but solely primer: COI-F/R, which the base pair is 460 bp. After adopting the primer with PCR-RFLP technology of 5 restriction enzymes: BsaJI、HaeIII、BmrI、MslI、NlaIII, the research successfully identifies 11 unprocessed and processed shellfish species.
Since the products are often processed under circumstances like high temperature or blended with other shellfish species, the DNA completeness is influenced. The study establishes multiplex-PCR primers Geloina-F/R, Gigas-F/R, and Corbicula-F / R which the base pairs are 183 bp, 122 bp, and 256 bp respectively to identify fake clams, Geloina erosa clams and oysters clams. As for fake scallop sauce, the study utilize Cyt b genome to design the specific primer yessoensis-F/R and successfully increase the base pairs to 320 bp.
According to the above testing results, the research approves the applicability of PCR-RFLP and multiplex-PCR. By collecting shellfish related samples all over Taiwan, including 33 oyster clam tab and 24 scallop sauce products, the testing results indicate that there is one unqualified product of oyster clam tab whereas the identification possibility of scallop sauce is 29.1%.

目錄 i
誌謝 iii
中文摘要 1
Abstract 3
前言 5
研究背景與目的 6
文獻整理 8
一、 原料貝類之特徵、分布 8
二、 一般鑑別貝類物種之方法 17
三、 Cyt b與COI序列之介紹 23
四、 以基因分析加工品之原料檢測 25
第一章、台灣常見貝類物種 mtDNA 基因組 Cytochrome b gene與Cytochrome c oxidase I gene序列之建立 29
一、前言 29
二、材料與方法 30
三、結果 34
四、討論 36
五、圖表 38
第二章、貝類及其加工品之基因鑑定技術應用 59
一、前言 59
二、材料與方法 60
三、結果 63
四、討論 65
五、圖表 66
第三章、市售貝類加工產品原料基因鑑種技術之開發 77
一、前言 77
二、材料與方法 78
三、結果 80
四、討論 81
五、圖表 83
總結論 86
參考文獻 87

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