以雜交層析法及奈米金粒子偵測人類乳突腫瘤病毒16型之研究

A Study of HPV16 DNA Detection Using Nanogold Particles and Hybrid Chromatography

李蕙君(Hui-Chun Li)

指導教授 ： 侯劭毅

國立臺北科技大學/工程學院/生物科技研究所/碩士(2009年)

摘要

子宮頸癌是全球婦女排名第二的常見癌症，目前已有足夠證據顯示人類乳突腫瘤病毒感染是導致子宮頸上皮細胞內層腫瘤以及侵犯性子宮頸癌最重要的原因，幾乎所有的子宮頸癌都肇因於人類乳突腫瘤病毒的感染，其中以第16、18型最主要。本研究主要目的是以點漬分析法和雜交層析法偵測人類乳突腫瘤病毒第16型DNA。
本研究分為兩步驟，首先以HMY09和BMY11當引子進行聚合酶鏈鎖反應，將SiHa細胞染色體的晚期基因構築至大腸桿菌質體上。然後利用streptavidin和biotin與anti-streptavidin antibody的強結合力，設計一段互補部分晚期基因的biotin-DNA序列，能和固定在硝化纖維膜上的streptavidin或anti-streptavidin antibody結合；設計另一股部分互補晚期基因的硫醇-DNA，將末端修飾硫醇基的DNA夠固定在奈米金粒子的表面上。由於biotin-DNA和硫醇-DNA能和目標DNA雜交結合，利用點漬分析法和雜交層析法進而可以判讀DNA約略濃度。實驗結果證實，以點漬分析法在單股偵測可到100 amol，進一步銀還原能夠偵測到100 zmol；以雜交層析法在單股偵測至少能到160 zmol。而在雙股的構築質體偵測上，點漬分析法可偵測到100 amol，銀還原後可偵測到100zmol，雜交層析法可偵測到100 amol。

關鍵字

人類乳突腫瘤病毒 ； 奈米金粒子 ； 雜交層析法

並列摘要

Human papillomavirus can lead to cancer of the cervix, vulva, vagina, and anus in women. High risk HPV type 16 and 18 infections have been commonly identified in cervical cancer. The purpose of this study is to detect DNA of HPV16 using gold nanoparticales dot blot test and hybrid chromatography test.
The assay could be implemented via three steps: First, we constructed an E.coli plasmid, pHC, containing SiHa cell chromosome HPV16 L1 region. Second, we designed biotin-DNA and SH-DNA probes as capture and detect probes, each complementary with L1 region. The former probe would associate with streptavidin and anti-streptavidin antibody on the nitrocellulose membrane, while the latter probe would associate with gold nanoparticles using covalent bond. Third, with hybridization, dot blot and chromatography methods were used to detect HPV. Base on the results, 100 amol of ssDNA and 100 amol of dsDNA were detected using the dot blot method. Furthermore, 160 zmol of ssDNA and 100 amol of dsDNA were detected using the chromatography method.

並列關鍵字

gold nanoparticles ； HPV 16 ； Hybrid Chromatography

參考文獻

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