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研究生:邱雅琦
研究生(外文):Ya-Chi Chiu
論文名稱:應用生物快速篩選方法測定魚類樣品中戴奧辛含量之探討
論文名稱(外文):Studies on CALUX bioassay for dioxin in fish products
指導教授:黃登福黃登福引用關係周薰修周薰修引用關係
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:119
中文關鍵詞:生物快速篩選法氣相層析高解析度質譜儀戴奧辛魚類
外文關鍵詞:CALUX bioassayGC-HRMSDioxinfish products
相關次數:
  • 被引用被引用:1
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傳統檢測戴奧辛是利用氣相層析高解析度質譜儀(gas chromatography-high resolution mass spectrometer, GC-HRMS)之方法,其優點為可確認29種戴奧辛類化合物及分別檢出其濃度,數據準確,但缺點為成本昂貴、檢驗耗時,因此無法解決突發事件所激增大量檢體之篩檢。由於戴奧辛含量可藉由其對生物毒性的反應而推估,因此國際上已研發戴奧辛生物快速篩檢技術(Chemically Activated LUciferase eXpression bioassay, CALUX bioassay),其為細胞毒性檢測方法,藉由冷光之強弱與2,3,7,8-四氯戴奧辛(2,3,7,8-tetrachloro-p-dibenzodioxin, 2,3,7,8-TCDD)毒性產生之冷光強度比較即可推算出戴奧辛之含量,可做為食品中戴奧辛定性及濃度定量之篩檢工作。
本研究以生物快速篩檢技術檢測市售魚、貝、蝦及蟹類之戴奧辛含量,其標準曲線具有良好的線性關係(相關係數0.999以上)。同日內及異日間之重複性及再現性分析,變異係數分別小於7.8%及9.4%,顯示本系統有好的再現性及穩定性。空白樣品分析管制圖,可確保樣品在檢驗過程中均未遭受污染。DR-CALUX®組套之檢測極限(limit of detection, LOD)為0.21 pM 2,3,7,8-TCDD/well,定量極限(limit of quantitation, LOQ)為0.64 pM 2,3,7,8-TCDD/well。添加回收試驗之回收率在高脂魚、低脂魚、蝦、貝及蟹類中,分別為101.2%、92.1%、97.5%、94.5%及106.0%,變異係數在7.1% 以下。參考物質分析管制圖,顯示每批樣品於檢驗過程中之系統及隨機誤差均控制良好。魚、貝、蝦及蟹類共72件檢體之戴奧辛含量檢測結果,分別為0.58 pg DR-CALUX®-TEQ/g fresh weight、0.21 pg DR-CALUX®-TEQ/g fresh weight、0.15 pg DR-CALUX®-TEQ/g fresh weight及0.58 pg DR-CALUX®-TEQ/g fresh weight,除了1件鱈魚為7.6 pg DR-CALUX® -TEQ/g fresh weight及1件黑鮪魚為4.7 pg DR-CALUX®-TEQ/g fresh weight外,其餘70件之戴奧辛含量皆低於歐盟訂定的各類食品中戴奧辛/呋喃及戴奧辛類多氯聯苯最大限值標準(maximum level ):魚類及魚類產品中總戴奧辛毒性當量應為4 pg WHO-PCDD/F+PCB-TEQ/g fresh weight以下,及我國之限量標準4 pg WHO-PCDD/F-TEQ/g fresh weight以下。另外將72件檢體之CALUX生物快速篩檢法檢測結果與傳統化學分析方法(GC-HRMS法)之檢測結果做相關性之分析,魚、貝、蝦及蟹類之總戴奧辛含量(PCDD/F+ PCB)的相關係數分別為0.93、0.97、0.75及0.96,相關性良好,顯示CALUX 生物快速篩檢法為篩檢水產品中戴奧辛含量之有效方法。
目 錄

中文摘要 ------------------------------------------------- 1
英文摘要 ------------------------------------------------- 3
壹、前言 ------------------------------------------------- 5
貳、文獻回顧 --------------------------------------------- 7
ㄧ、戴奧辛類化合物之結構 --------------------------------- 7
二、戴奧辛類化合物之特性 --------------------------------- 8
三、戴奧辛類化合物之毒性 --------------------------------- 9
四、戴奧辛類化合物之毒性當量表示方式 -------------------- 13
五、台灣地區戴奧辛的暴露來源及進入人體的途徑 -------------15
(一)戴奧辛的暴露來源 ------------------------------------ 15
(二)進入人體的途徑 -------------------------------------- 16
(三)終生平均暴露劑量 -------------------------------------16
六、歐盟降低食品和飼料中戴奧辛之策略 -------------------- 18
七、魚類中戴奧辛含量之標準 ------------------------------ 19
(一)歐盟 ------------------------------------------------ 19
1.行動值( action level ) -------------------------------- 19
2.最大限值( maximum level ) ------------------------------19
3.戴奧辛及戴奧辛類多氯聯苯之最大限值 -------------------- 19
(二)台灣 ------------------------------------------------ 19
八、食品中戴奧辛及其類化合物之測定方法 ------------------ 21
(一)戴奧辛類化合物之毒性機制 ---------------------------- 21
(二) DR-CALUX® 細胞檢測方法 ---------------------------- 22
(三) DR-CALUX® 細胞檢測法之操作步驟 -------------------- 23
九、食品中戴奧辛背景值之情形 ---------------------------- 23
(一)食品中戴奧辛之含量 ---------------------------------- 23
(二)魚類產品中PCDD/F與PCB之濃度比例 --------------------- 25
(三)台灣歷年魚類產品中戴奧辛含量之情形 ------------------ 25
叁、材料與方法 ------------------------------------------ 27
ㄧ、檢體來源 -------------------------------------------- 27
二、材料及儀器設備 -------------------------------------- 27
(一)裝置 ------------------------------------------------ 27
(二)器具及材料 ------------------------------------------ 27
(三)試藥及試劑 ------------------------------------------ 28
三、實驗方法 -------------------------------------------- 29
(一)樣品萃取 -------------------------------------------- 29
(二)樣本淨化 -------------------------------------------- 30
1. 20%及33%硫酸矽膠之製備 ----------------------------- 30
2.無水硫酸鈉之配製 -------------------------------------- 30
3.沖提液(正己烷/乙醚 (97:3,v/v)溶液)之配製 ----------- 31
4.淨化管柱之配製 ---------------------------------------- 31
5.淨化方法 ---------------------------------------------- 31
(三)接種和暴露 ------------------------------------------ 32
1.細胞冷凍 ---------------------------------------------- 32
2.細胞解凍 ---------------------------------------------- 33
3.細胞繼代培養 ------------------------------------------ 34
4.細胞接種 ---------------------------------------------- 36
5.暴露 -------------------------------------------------- 36
6. 96孔培養盤之充填 ------------------------------------- 38
(四)細胞收成及分析測定 ---------------------------------- 38
1.試劑之配製 -------------------------------------------- 38
2.細胞收成(harvesting the cells)----------------------- 39
3.培養盤之測定(measurement of microtiterplates)-------- 40
4.空白試驗 ---------------------------------------------- 40
5.重複性及再現性試驗 ------------------------------------ 41
6.添加回收試驗 ------------------------------------------ 41
7.參考物質(reference material)試驗 -------------------- 41
8.市售魚類、貝類及蝦蟹類樣品之檢測 ---------------------- 42
9.品質管制 ---------------------------------------------- 42
肆、結果與討論 ------------------------------------------ 43
ㄧ、檢體來源 -------------------------------------------- 43
(一) 93年度檢體 ----------------------------------------- 43
(二) 94年度檢體 ----------------------------------------- 43
(三)採樣部位 -------------------------------------------- 44
二、檢體之脂肪含量 -------------------------------------- 44
三、冷光儀之性能測試 ------------------------------------ 45
四、標準曲線 -------------------------------------------- 45
五、DR-CALUX® 組套之檢測極限與定量極限 ----------------- 45
六、重複性及再現性試驗 ---------------------------------- 45
七、空白試驗 -------------------------------------------- 46
八、添加回收試驗 ---------------------------------------- 46
九、參與國際間共同試驗之結果 ---------------------------- 46
十、參考物質試驗 ---------------------------------------- 47
十一、生物快速篩選方法之探討 ---------------------------- 47
(ㄧ)以生物快速篩選方法檢測魚類產品之結果 ---------------- 47
(二)傳統分析方法與生物快速篩選方法之比較 ---------------- 48
(三)與其他國家之比較 ------------------------------------ 50
伍、結論 ------------------------------------------------ 51
陸、參考文獻 -------------------------------------------- 52
謝 辭 ------------------------------------------------- 106
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