臺灣博碩士論文加值系統

摘要
使用聚合酵素鏈鎖反應 (Polymerase Chain Reaction, PCR) 方法檢測大豆原料及其加工產品中基因改造原料，是目前的趨勢，然加工過程中的高溫會造成DNA的斷裂，而DNA的完整性為決定是否可使用PCR檢測的重要因素之一，本研究發現當DNA經過100 ℃ 180分鐘或121 ℃ 60分鐘之熱處理時，使用PCR產物在444 bp以上之引子對 (primer pair) ，會隨熱處理程度的劇烈而逐漸無法檢測，318 bp以下之引子對則皆可順利檢測。使用本研究室設計之兩組分別針對大豆特有Lectin基因之LeB引子對 (PCR產物為219 bp) ，與針對抗嘉磷賽除草劑大豆 (Roundup ReadyTM soybean, RRS) 轉殖基因CTP-EPSPS之CTEP引子對 (PCR產物為318 bp) ，進行大豆原料及加工製品中之RRS檢測，在大豆原料方面，其靈敏度可達0.01 %，在加工產品上則達0.1 %。若同時使用上述兩組引子對，以複式聚合酵素鏈鎖反應 (multiplex PCR) 取代一般PCR進行檢測，雖然其靈敏度在大豆原料上由0.01%降至0.1%，在加工產品上由0.1%降至1%，但卻具有快速、減少人為誤差、避免發生偽陰性結果的優點。以LightCycler之SYBR Green Ⅰ方法對RRS原料進行定量分析，5 % RRS之定量結果分別為5.3 %、5.3 %、5.7 %，C.V.值為4.2 %低於歐盟定量結果的25 %。對RRS自製加工產品方面，雖然Lectin基因與CTP-EPSPS基因之定量結果會隨加工過程而降低，但在RRS含量方面，5 % RRS 原料為5.6 %、5 % RRS豆漿為5.7 %、5 % RRS豆皮為4.9 %、5 % RRS素雞為4.6 %，結果不受加工過程的影響。因此，本實驗建立之定量方法不但可應用於大豆原料之RRS定量，也可直接使用於大豆加工製品上。

Abstract
Currently, PCR is the primary method for analyzing genetically modified organism (GMO) in soybean ingredients and its processed food. However, The degradation of DNA by heat is a crucial factor in PCR analysis. This experiment showed that when DNA were heated at 100 ℃ for 180 minutes or at 121 ℃ for 60 minutes, PCR products fragment which above 444 bp would become undetectable due to above treatments, whereas PCR product under 318 bp can be detected. This experiment identified RRS by targeting at two genes in soybean ingredient and its processed food: one of which is the soybean-specific LeB primer in the Lectin gene (PCR product fragment 219 bp), and the other is CTEP primer in RRS transgenic gene CTP-EPSPS (PCR product fragment 318 bp). The result showed that soybean ingredient has a sensitivity of 0.01 %, whereas the processed food showed 0.1 % sensitivity. As general PCR is replaced by multiplex PCR for testing, though the sensitivity of multiplex PCR on soybean ingredient decreased from 0.01 % to 0.1 %, and processed food decreased from 0.1 % to 1 %, it offers a wide range of benefits such as reduction of human error, prevention of false negative, and acceleration of the analysis process. SYBR Green I method of LightCycler was used for RRS quantification. The result of 5 % RRS quantification were found to be 5.3 %, 5.3 %, 5.7 %, with the C.V. values of 4.2 % far lower than the EU quantified value of 25 %. As regards to RRS self-processed food, while the quantification results of Lectin gene and CTP-EPSPS gene would drop along with processing, their RRS amount were conserved (5 % RRS in soybean was 5.6 %, 5 % RRS in soymilk was 5.7 %, 5 % RRS in topi was 4.9 %, and 5 % RRS in vegetable chicken was 4.6 %). Hence, the quantification method established in this study can be both applied to RRS quantification in soybean ingredient and soybean processed food directly.

目錄
目錄---------------------------------------------------------------------------------v
圖次-------------------------------------------------------------------------------vii
表次--------------------------------------------------------------------------------x
壹、 前言-------------------------------------------------------------------------1
貳、 前人研究-------------------------------------------------------------------3
一、 大豆之簡介-----------------------------------------------------------3
(一)、大豆之歷史---------------------------------------------------3
(二)、大豆之用途---------------------------------------------------3
二、 基因改造作物之介紹-----------------------------------------------5
三、 抗蟲與抗殺草劑兩種主要基因改造作物之介紹--------------7
(一)、抗蟲之基因轉殖植物---------------------------------------7
(二)、抗殺草劑之基因轉殖植物---------------------------------9
四、 基因改造食品之檢測方法----------------------------------------17
(一)、定性方法-----------------------------------------------------17
(二)、定量方法-----------------------------------------------------20
五、 基因改造食品之管理與標示措施-------------------------------22
參、 實驗設計------------------------------------------------------------------27
肆、 材料與方法---------------------------------------------------------------28
一、 材料-------------------------------------------------------------------28
二、 藥品-------------------------------------------------------------------28
三、 儀器設備-------------------------------------------------------------29
四、 實驗方法-------------------------------------------------------------30
伍、 結果與討論---------------------------------------------------------------39
一、 大豆及其加工產品之DNA抽取--------------------------------39
二、 DNA熱破壞程度與PCR產物長度之關係--------------------40
三、 PCR引子對之選擇與設計----------------------------------------49
四、 大豆加工過程對LeC與CTEP檢測之影響-------------------50
五、 以LeC與CTEP檢測不同Roundup ReadyTM soybean
含量之大豆原料DNA--------------------------------------------55
六、 以LeC與CTEP檢測不同Roundup ReadyTM soybean
比例之豆漿、豆皮與素雞DNA---------------------------------58
七、 以LeC與CTEP檢測市售大豆、豆漿、豆皮、素雞、
素肉、大豆蛋白與冷凍毛豆--------------------------------------59
八、 以multiplex PCR取代一般PCR檢測---------------------------69
九、 以LightCycler進行定量分析------------------------------------77
陸、 結論------------------------------------------------------------------------88
柒、 參考文獻------------------------------------------------------------------90
圖次
圖一、大豆加工食品之種類----------------------------------------------------4
圖二、嘉磷塞異丙胺之結構--------------------------------------------------12
圖三、Shikimic acid代謝路徑之細節--------------------------------------13
圖四、環狀胺基酸與組胺酸之生合成圖示--------------------------------14
圖五、質體PV-GMGT04與抗殺草劑大豆所插入DNA片段--------15
圖六、CP4 EPSPS的作用機制之圖示------------------------------------16
圖七、聚合酵素鏈反應之圖示-----------------------------------------------24
圖八、以SYBR Green Ⅰ進行定量之示意圖---------------------------25
圖九、歐盟檢測GMO之流程-----------------------------------------------36
圖十、PCR引子對在 (a) Lectin基因與 (b) 轉殖基因上之位置
示意圖-------------------------------------------------------------------37
圖十一、不同熱破壞程度DNA之電泳圖--------------------------------43
圖十二、以LeA檢測不同熱破壞程度大豆DNA-----------------------44
圖十三、以LeB檢測不同熱破壞程度大豆DNA-----------------------45
圖十四、以LeC檢測不同熱破壞程度大豆DNA-----------------------46
圖十五、以LeD檢測不同熱破壞程度大豆DNA-----------------------47
圖十六、以LeE檢測不同熱破壞程度大豆DNA-----------------------48
圖十七、大豆及其加工產品所抽取DNA之電泳圖--------------------52
圖十八、以LeC檢測0 %、5 %與100 %Roundup ReadyTM
soybean DNA含量之大豆與其加工產品--------------------53
圖十九、以CTEP檢測0 %、5 %與100 % Roundup ReadyTM soybean DNA含量之大豆與其自製加工產品---------------54
圖二十、以LeC檢測不同含量Roundup ReadyTM soybean
DNA-------------------------------------------------------------------56
圖二十一、以CTEP檢測不同含量Roundup ReadyTM
soybean DNA----------------------------------------------------57
圖二十二、以LeC檢測不同含量Roundup ReadyTM soybean
之豆漿DNA------------------------------------------------------61
圖二十三、以CTEP檢測不同含量Roundup ReadyTM soybean
之豆漿DNA------------------------------------------------------62
圖二十四、以LeC檢測不同含量Roundup ReadyTM soybean
之豆皮DNA------------------------------------------------------63
圖二十五、以CTEP檢測不同含量Roundup ReadyTM soybean
之豆皮DNA------------------------------------------------------64
圖二十六、以LeC檢測不同含量Roundup ReadyTM soybean
之素雞DNA------------------------------------------------------65
圖二十七、以CTEP檢測不同含量Roundup ReadyTM soybean
之素雞DNA------------------------------------------------------66
圖二十八、以LeC檢測市售大豆及其加工產品------------------------67
圖二十九、以CTEP檢測市售大豆及其加工產品----------------------68
圖三十、以multiplex PCR檢測0 %、5 %與100 %Roundup
ReadyTM soybean DNA含量之大豆與其自製加工產品---71
圖三十一、以multiplex PCR檢測不同含量Roundup ReadyTM soybean DNA----------------------------------------------------72
圖三十二、以multiplex PCR檢測不同含量Roundup ReadyTM soybean之豆漿DNA------------------------------------------73
圖三十三、以multiplex PCR檢測不同含量Roundup ReadyTM soybean之豆皮DNA------------------------------------------74
圖三十四、以multiplex PCR檢測不同含量Roundup ReadyTM soybean之素雞DNA------------------------------------------75
圖三十五、以multiplex PCR檢測市售大豆及其加工產品-----------76
圖三十六、LeB PCR產物之melting point測定--------------------------80
圖三十七、EPSP PCR產物之melting point測定------------------------81
圖三十八、不同濃度標準品與樣品之LeB PCR產物定量結果------82
圖三十九、不同濃度標準品與樣品之EPSP PCR產物定量結果---83
圖四十、以LeB定量檢測5 % RRS標準品之標準曲線--------------84
圖四十一、以EPSP定量檢測5 % RRS標準品之標準曲線-------85
表次
表一、歐盟2001年之GMO標示項目-----------------------------------26
表二、本實驗所使用PCR引子對之簡介--------------------------------38
表三、各種大豆及其加工產品之DNA抽取結果-----------------------42
表四、大豆原料之定量結果-------------------------------------------------86
表五、自製大豆加工製品之定量結果-------------------------------------87

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