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  • 學位論文

以紫外-可見光光譜及側向流免疫層析法結合磁珠分離技術檢測HLA-A3101之單股DNA

Detection of the Single-stranded DNA of HLA-A3101 Using UV-Visible Spectrum and Lateral-flow Immunoassay with the Separation Technology of Magnetic Beads

指導教授 : 吳瑞璋
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摘要


人類白細胞抗原(human leukocyte antigen, HLA)作為人體最複雜的基因系統,同時也是多態性最為豐富的區域,與人體的免疫系統息息相關,因此與HLA之相關疾病都以免疫反應失調為主。本研究使用之基因HLA-A3101,與止痛用藥卡巴氮平(Carbamazepine)過敏反應具有高度的相關性。 HLA-A3101前置引子修飾digoxigenin,反置引子修飾biotin,藉由PCR複製後與經Streptavidin修飾的微米磁珠結合反應。透過分光光度計檢測吸光度,在6倍稀釋濃度之PCR產物與6 ul之磁珠使用量下,磁珠上之DNA含量具有最大值27.3 ng。磁珠與PCR產物反應後,透過NaOH溶液打斷DNA雙股之間的氫鍵,使脫離之單股DNA混合於溶液中。在最適化條件下,實驗選擇PCR產物與磁珠室溫下反應30 mins,在單次反應下使用濃度0.05 M 之NaOH分離出單股DNA,並在薄膜試條上進行側向流檢測。由於PH值過高影響膠體金顯色,實驗添加0.2 M之HCl中和溶液,並將單股DNA與前置引子進行預混合。結果顯示,雖然在控制線上成功使膠體金正常顯色,在測試線上即使以預混合的方式反應,也無法經由肉眼可見判讀的訊號。由以上結論可知,單股DNA溶液濃度不足或是在預混合反應下是否復性為雙股結構,兩者皆為側向流檢測需考量的關鍵因素。

並列摘要


Human leukocyte antigen (HLA), as the most complicated gene system and polymorphic region, is closely related to the human immune system. Its related diseases are thus mainly dominated by immune-response disorders. This research aims HLA-A3101 as the target analyte due to its high correlation with allergic reactions to the analgesic drug, Carbamazepine. In the experiment, a forward primer of HLA-A3101 is modified by ligand digoxigenin and a reverse primer by biotin. After amplification, its PCR product interacts with the micron-size magnetic beads modified by streptavidin on the surface. When the PCR product was diluted its concentration in 1/6 times and interacted with 6ul of magnetic beads at room temperature for 30mins, the maximum content of dsDNA on the magnetic beads, 27.3 ng, was obtained by interpreting the absorbance of UV-visible spectrum. A 0.05M NaOH solution was afterwards added to break the hydrogen bonds between dsDNA’s and receive ssDNA’s from the solution. Under those optimal conditions, ssDNA’s are obtained and then detected on lateral-flow immunoassay (LFIA) strips. The strip signals were found absent due to the effects of pH values. A 0.2M HCl solution was used to adjust the pH value back to the range good for color appearing of the colloidal gold. As the result, the signals of colloidal gold were successfully developed on the control line; however, the test line were still absent in signals. Tests for trying premixing ssDNA’s with the forward primer was carried out prior to dropping ssDNA samples on the LFIA strips, but in fail. To the conclusion, the concentration of ssDNA could be insufficient by NaOH-conducted denaturing procedure. Approaches for increasing the concentration of ssDNA were suggested to develop in the future.

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