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研究生:林欣緣
研究生(外文):Shin-Yuan Lin
論文名稱:利用粒線體16SrRNA基因以PCR-RFLP方法鑑定有毒螺類
論文名稱(外文):Application of PCR-RFLP Analysis of Mitochondrial 16S Ribosomal RNA Gene on Identification of Species of Toxic Marine Snail
指導教授:黃登福黃登福引用關係
指導教授(外文):Deng-Fwu Hwang
學位類別:碩士
校院名稱:國立臺灣海洋大學
系所名稱:食品科學系
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:2006
畢業學年度:94
語文別:中文
論文頁數:92
中文關鍵詞:螺類限制片段長度多型性聚合酶連鎖反應
外文關鍵詞:gastropodPCR-RFLP
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世界上分佈之腹足綱軟體動物大約有 88,000 種,然而在台灣曾檢測出有數種玉螺科、織紋螺科及榧螺科之螺類含有海洋生物毒並引發食物中毒案例。通常物種鑑定是經由螺類形態學上之特性作分類,但在缺乏外殼時往往難以鑑定。近年來新式的物種鑑定方法採用DNA 分析,特別是限制片段長度多型性聚合酵素連鎖反應 ( polymerase chain reaction-restriction fragment length polymorphism; PCR-RFLP )。
為了鑑定數種有毒螺類,以直接定序法和PCR-RFLP 技術分析新鮮、凍藏、水煮 ( 100℃, 30-60 min ) 及高壓殺菌 ( 121℃, 15-30 min ) 過之細紋玉螺、腰帶玉螺、大玉螺、白玉螺、橡子織紋螺、疣織紋螺、球織紋螺、正織紋螺、蠟燭榧螺和金唇榧螺等物種。
由研究結果得知,從新鮮、凍藏及水煮螺肉可抽取高分子之 DNA ( >1,000 bp ) ,然而透過高溫處理僅能取得低分子量之DNA。對於新鮮、凍藏及水煮之螺肉可利用 PCR 引子 16S L22/R22增幅出長度約383-396 bp之特異性片段;引子 16S LS22/RS 22增幅之244-257 bp小片段則適用於高壓滅菌處理之螺肉。
在分析引子 16S L22/R22 與 16S LS22/RS22 增幅之不同序列後,選出五種具有特異性切位之限制酶,包括Apo I、 Pac I、 Hinc II、 Acc I 和 Vsp I。限制酶 Apo I 可以區分細紋玉螺、腰帶玉螺、大玉螺和白玉螺。Pac I、 Hinc II 和 Acc I 能區分出橡子織紋螺、疣織紋螺、
球織紋螺和正織紋螺。Vsp I 則可以區分出蠟燭榧螺和金唇榧螺兩種。進一步透過DNA 膠電泳圖即可快速、確實的鑑別這些物種,且利用相同限制酶可以同時區分經生鮮、凍藏、水煮及高溫滅菌之螺類物種。
進一步利用上述之方法鑑定小琉球中毒案烹煮過之螺肉樣品,經由貝殼形態鑑定、序列比對及 PCR-RFLP 分析後,指出此螺類物種為疣織紋螺。因此,藉由此項技術可提供用來鑑定不同加熱溫度處理之螺類物種。
Approximately 88,000 species of gastropod mollusk distribute in the world. However, several species of Naticidae, Nassariidae and Olividae have been detected to contain marine toxins, and outbreaks of food poisoning cases in Taiwan occurred. Species is usually identified by morphological characteristics of marine snail. But it is difficult to identify without shell. Recently, species identification is developed based on DNA analysis, especially the polymerase chain reaction-restriction fragment length polymorphism ( PCR-RFLP ).
In order to identification of several toxic gastropod species in Taiwan, the direct sequencing and the PCR-RFLP technology were used to identify gastropod species in fresh, frozen, cooked ( 100℃, 30-60 min ) and steam sterilized ( 121℃, 15-30 min ) meat of Natica lineata, N. vitellus, Polinices didyma, P. tumidus,. Nassarius glans, N. papillosus, Niotha clathrata, Zeuxis scalaris, Oliva lignaria and O. reticulata.
Judging from the data of this study, high molecular weight DNA(>1,000 bp) was obtained from fresh, frozen and cooked meat, while relatively low molecular weight DNA was obtained from samples treated by high temperature. The PCR primers of 16S L22/R22 specifically amplify 383-396 bp of fragments from fresh, frozen and cooked meats; smaller fragments of 244-257 bp are used for steam sterilized meats amplified with primers of 16S LS22/RS22.
After analyzing the different sequences amplified with primers 16S L22/R22 and 16S LS22/RS22, five restriction enzymes with specific cutting sites, including Apo I, Pac I, Hinc II, Acc I and Vsp I, were used. The restriction enzyme Apo I could differentiate the species of Natica lineata, N. vitellus, Polinices didyma and P. tumidus. Pac I, Hinc II and Acc I could differentiate the species of Nassarius glans, N. papillosus, Niotha clathrata and Zeuxis scalaris. Vsp I could differentiate the species of Oliva lignaria and O. reticulata. The polymorphic pattern in the DNA electrophoretic gel could support to identify these species precisely and quickly. By the same restriction enzyme could differentiate species of fresh, frozen, cooked and sterilized meats simultaneously.
Furthermore, applying above method to identify the gastropod species in the residue of cooked gastropod meat from food poisoning incident in Hsiau Liouchiou Island, after shell identification, sequence comparing and PCR-RFLP analyzing, it indicated that the species of gastropod was Nassarius papillosus. Therefore, by this study could provide useful technique in identifying the gastropod species of different heating meat.
第一章 文獻整理………………………………………………………. 1
一、台灣歷年食用螺類引發之中毒事件…………………………….2
二、螺類之毒成分分析法…………………………………………….5
三、螺類之分類與實驗中有毒螺種簡介…………………………….8
四、利用 mtDNA鑑定物種之特性……………………………………15
五、分子生物技術鑑定物種…………………………………………18
第二章 有毒生鮮螺類 16S rRNA gene 序列分析及 PCR-RFLP 鑑
種技術之建立………………………………………………..23
一、前言………………………………………………………………24
二、材料與方法………………………………………………………25
三、結果………………………………………………………………30
四、討論………………………………………………………………33
附圖與附表……………………………………………………………..37
第三章 利用 PCR-RFLP 技術鑑定不同溫度處理之有毒螺類
物種…………………………………………………………..51
一、前言………………………………………………………………52
二、材料與方法………………………………………………………53
三、結果………………………………………………………………55
四、討論………………………………………………………………59
附圖與附表……………………………………………………………..64
綜合結果與討論………………………………………………………..79
參考文獻………………………………………………………………..81
附錄……………………………………………………………………..92
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