以側向流層析法對HLA-Cw1602基因序列之快速偵測

Rapid Detection of Gene sequence HLA-Cw1602 Using Lateral-flow Immunochromatographic Assay

林冠廷(Guan-Ting Lin)

指導教授 ： 吳瑞璋

中原大學/工學院/化學工程研究所/碩士(2016年)

https://doi.org/10.6840/cycu201600443

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，進行側向流薄膜試條偵測，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FITC，對與貝賽特氏症有強相關性之HLA-Cw1602雙股DNA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，可與奈米金粒子達最佳的結合效果。由側向流薄膜試條偵測結果發現，透過將鋪放抗體接金之結合墊置於26℃乾燥箱中進行乾燥，相較於37℃烘箱乾燥法之未純化PCR產物偵測極限由0.1µl(5ng)下降至0.004µl (0.2ng)。實驗中另外對PCR反應專一性進行探討與驗證，證明前後端引子對於結果呈現具專一性；側向流薄膜試條之再現性，使用Intra-assay及Inter-assay得到變異係數分別為2.7-9.8%與9.9-14.9%，可證實薄膜試條間性能差異不大。本研究提出之條碼型側向流試條，利用兩者修飾特殊配體不同，可同時偵測HLA-Cw1602與HLA-B*5801，由獨立性偵測結果，訊號變異係數皆低於11%。由這些結果顯示出側向流薄膜試條於遺傳性疾病之快速診斷有相當大的潛力。

關鍵字

側向流；HLA-Cw1602；快速偵測

並列摘要

In this study, sequence-specified DNA segments were detected on membrane-based lateral flow strips, incorporated with an immune reaction and color readouts developed by gold nanoparticles. The Human leukocyte antigen Cw1602 DNA was detected on the membrane-based lateral-flow strip. The optimal condition of mouse anti-digoxigenin antibody conjugated with colloidal-gold nanoparticles was determined as pH=8.5 and antibody concentration at least 10µg/ml, to avoid the complex of gold-antibody from aggregation. Compared with the traditional 37℃ oven-drying method, drying in a dry cabinet enhanced the detection limits, from 0.1µl (5ng) to 0.004µl (0.2ng) for PCR unpurified samples. The specificity of polymerase chain reaction was also investigated. The coefficient of variation values of intra-assay and inter-assay were 2.7-9.8% and 9.9-14.9%, respectively, revealing the test procedure had a good repeatability. The barcode-style lateral flow strip was designed to detect HLA-Cw1602 and HLA-B*5801 DNA on the same strips. The membrane-based lateral flow strips show a great promise for rapid medical diagnosis of multiple genetic diseases. They also showed good performance in the independent study, with CV% lower than 11%.

並列關鍵字

Rapid Detection；HLA-Cw1602；Lateral Flow

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