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研究生： 林志韋
Lin, Chih-Wei
論文名稱： tmigd1基因單核苷酸多型性之乾式生物檢測試紙系統
Dry-Reagent Strip Biosensor for Single Nucleotide Polymorphism in tmigd1 Gene
指導教授： 郭瑞昭
Kuo, Jui-Chao
共同指導教授: 黃秀琳
Huang, Hsiu-Lin
學位類別： 碩士
Master
系所名稱： 工學院 - 材料科學及工程學系
Department of Materials Science and Engineering
論文出版年： 2016
畢業學年度： 104
語文別： 中文
論文頁數： 178
中文關鍵詞： 免疫層析法 、奈米金 、單核苷酸多型性 、菜鴨
外文關鍵詞： lateral-flow immunoassay, gold nanoparticle, single nucleotide polymorphism, Tsaiya duck
相關次數： 點閱：101  下載：1 
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    • 免疫層析法是一種操作簡便、低成本、時間需求低的檢測技術，並已被廣泛的應用在各種的生物檢測應用上。近年來，此種方式已由原始之抗體-抗原檢測方式成功應用至基因檢測、甚至是Single Nucleotide Polymorphism (SNP)偵測。本論文探討免疫層析試紙之開發與相關製程條件之參數，並將成品應用於菜鴨之tmigd1基因之SNP檢驗上。

    本論文選定奈米金粒子作為免疫層析試紙之顯色單元，並由試驗結果選定0.67M NaCl作為奈米金粒子表面單股核酸探針修飾之最終鹽度設定。在後續的試紙先行測試中，Streptavidin使用量在0.25μg下時將不會有肉眼可辨識之假陽性訊號產生，因此後續的試驗中將以此數值作為試紙參數調整之起始數值。

    本論文中使用Primer Extension作為SNP辨識之核心機制，利用特定DNA聚合酶不具有3’-5’外切性之特性，只有目標SNP存在時才能發生聚合反應並將Biotin-16-dUTP嵌入產物中。產物將因帶有Biotin而能夠被試紙上之Streptavidin所捕捉。在此反應中Mg2+離子濃度為影響試紙訊號特異性最主要因素，次之為試紙上Streptavidin之使用量。本論文選定1mM Mg2+搭配0.12μg Streptavidin作為最終的標準設定。且由試驗結果可知增加PCR產物使用量與Primer Extension反應循環次數均可有效得提升G/A genotype之信號強度，同時G/G與A/A genotype之信號特異性不會受到顯著的影響。本研究再額外使用6組DNA樣品以7個Primer Extension循環進行再現性驗證，由試驗結果可發現6組樣品均可得到預期中的訊噪對比，但特異性訊號強度會受到PCR產物濃度影響。最後根據計算，本檢驗方式之時間需求約為40分鐘、單位成本為38.5元新台幣。

    In this research, we developed a lateral-flow immunoassay combined with primer extension (PEXT) method for SNP genotyping of tmigd1 gene in Tsaiya duck (Anas platyrhynchos), and discussed the effects of experiment parameters on both signal specificity and intensity.

    According to our result, the optimal setup is 1mM Mg2+ with 0.12μg streptavidin. Increasing the amount of PCR product or PEXT reaction cycle number can enhance the signal without observable change of signal specificity. To validate the reproducibility, we perform the test with additional 6 DNA samples. We were able to obtain specific signals from all the DNA samples, but the signal intensities were be affected by the concentration of PCR prducts.

    According to our calculation, time requirement of this method is about 40 minutes, and the cost for 1 strip test is about 38.5 NTD.

    中文摘要 I Extended Abstract III 誌謝 XII 目錄 XIV 表目錄 XX 圖目錄 XXI 專有名詞對照表 XXXIV 第一章　前言 1 第二章　文獻回顧 5 2.1　去氧核醣核酸的性質與相關基本技術 5 2.1.1　去氧核醣核酸的基本性質 5 2.1.2　核酸片段的人工合成 13 2.1.3　聚合酶鏈鎖反應 21 2.1.2　核酸鹼基序列的定序 24 2.2　單核苷酸多型性與相關檢測技術 28 2.2.1　單核苷酸多型性簡介 28 2.2.2　單核苷酸多型性之檢測技術 33 2.3　免疫層析法 40 2.3.1　免疫層析法簡介 40 2.3.2　免疫層析法的SNP檢測應用 46 2.4　奈米金粒子的光學性質 51 2.4.1　簡介 51 2.4.2　局部表面電漿 57 2.4.3　電漿子的耦合 65 第三章　試驗材料與方法 71 3.1　試驗流程與試驗材料 71 3.2　PCR-RFLP 73 3.2.1　PCR 73 3.2.2　RFLP 78 3.2.3　瓊脂糖凝膠電泳 79 3.2.4　數位電泳影像分析 83 3.3　PEXT反應與檢測 88 3.3.1　PEXT反應 88 3.3.2　PEXT反應產物之試紙檢驗 93 3.4　PEXT產物檢測用之免疫層析試紙 95 3.4.1　單股核酸探針修飾奈米金粒子 95 3.4.2　試紙組裝與試驗溶液配置 102 第四章　試驗結果 104 4.1　PCR-RFLP 104 4.2　SNP試紙檢驗 109 4.2.1　先行測試 109 4.2.2　試紙信號特異性 111 4.2.2.1　黏合溫度 112 4.2.2.2　Streptavidin 115 4.2.2.3　鎂離子濃度 121 4.2.3　試紙信號強度 125 4.2.3.1　PEXT Primer用量 126 4.2.3.2　PCR產物用量 128 4.2.3.3　PEXT反應循環次數 131 4.2.4　試紙檢驗的再現性 134 第五章　討論 139 5.1　SDS對金粒子表面修飾之影響 139 5.2　PEXT試驗參數對試紙訊號特異性之影響 140 5.2.1　Streptavidin使用量 140 5.2.2　鎂離子濃度 143 5.2.3　參數比較 147 5.2.4　其餘影響信號特異性之因素 151 5.3　PEXT試驗參數對試紙信號強度之影響 152 5.3.1　PEXT引子用量 152 5.3.2　PCR產物溶液用量 153 5.3.3　PEXT循環次數 156 5.3.4　參數比較 159 5.4　影響再現性試驗所得信號強度的因素 163 5.5　試驗時間需求與成本估算 165 5.5.1　試驗時間需求估算 165 5.5.2　試驗成本估算 167 第六章　結論 169 參考文獻 171

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