以薄膜側向流層析法對HLA-B*5801基因序列之快速偵測

Rapid Detection to Gene HLA-B*5801 Using Membrane-based Lateral Flow

鍾岳哲(Yue-Zhe Zhong)

指導教授 ： 吳瑞璋

中原大學/工學院/化學工程研究所/碩士(2015年)

https://doi.org/10.6840/cycu201500463

摘要

本研究利用抗體-抗原免疫反應及經修飾配體(ligand)之DNA標的物，進行側向流薄膜偵測，並以膠體金顯色進行判讀。研究中於DNA前端引子修飾digoxigenin及後端引子修飾biotin，各別對HLA-B*5801及禽流感(avian influenza) H5N1中的H5序列做專一性偵測。奈米金抗體複合物在pH= 8.5及抗體濃度為10µg/ml之最適化條件下，可與膠體金顆粒達最佳的結合效果。由側向流感測器偵測結果發現，於PCR程序中，若添加Dig-11-dUTP所呈現的結果相較於一般PCR產物，其感測信號確實提升，有利於肉眼判讀。偵測結果分別為未純化1.2ng及純化1ng，對照於文獻一般PCR產物偵測極限2ng，確實增強其偵測極限。實驗中另外對配體-受體之免疫反應與選擇性進行探討，結果證明前後端引子對於結果呈現具有重要性及專一性。對於添加Dig-11-dUTP與未修飾前端引子進行測試，發現添加Dig-11-dUTP確實可以對於奈米金複合物進行結合，進而驗證此Dig-11-dUTP添加物可有效的增強信號結果。

關鍵字

側向流；HLA-B*5801；快速偵測

並列摘要

In this study, a sequence-specified DNA segments were detected on porous polymer surfaces, incorporated with an immune reaction and color development by nano-size gold particles. The Human leukocyte antigen B*5801 DNA’s and Avian-Influenza (AI) H5 DNA were individually detected on the membrane-based lateral-flow strips. The optimal conjugation condition of mouse anti-digoxigenin with colloidal-gold particles was determined as pH=8.5 and antibody concentration at least 10µg/ml, to avoid the complex of gold-antibody from aggregation. Compared with a general PCR product, signal intensity was enhanced after dig-labeled dUTP was added in the procedure. The detection limit was enhanced from 2 ng to 1.2ng for unpurified and 1ng for purified samples. The specification and selectivity of the ligand-receptor immune reaction were discussed. Addition of Dig-11-dUTP indeed able to effectively enhance the signal intensity.

並列關鍵字

Rapid Detection；HLA-B*5801；Lateral Flow

參考文獻

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