以薄膜側向流免疫層析法對HLA-A3101基因序列之快速偵測

Rapid Detection of Gene Sequence HLA-A3101 Using Lateral-flow Immunochromatographic Assay

黃煜勛(Yu-Shiun Huang)

指導教授 ： 吳瑞璋

中原大學/工學院/化學工程研究所/碩士(2017年)

https://doi.org/10.6840/cycu201700403

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。於DNA前端引子修飾digoxigenin及後端引子修飾biotin，對HLA-A3101基因序列做專一性偵測。使用金銅合金試管進行PCR反應，在三程序(分別為變性反應、引子黏合反應及延伸反應)為(1,1,1秒)且循環25次的條件下，可以縮短45%之總反應時間。在相同的條件下使用金銅合金試管，進行PCR反應所得之HLA-3101基因序列，在側向流薄膜試條的訊號強度遠遠高於使用塑膠試管所得之訊號。而且金銅合金試管內樣品的殘留量非常低，僅需一次清洗後即可重複使用，並不影響下次實驗。奈米金抗體複合物在pH= 8.5以及抗體濃度為10µg/ml為其最適化條件，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，本次實驗的肉眼偵測極限為樣品4.9ng，而經由分析為0.49ng。實驗中另外對引子進行驗證，證明前後端引子對於結果具有專一性。試條再現性部份，使用Intra assay及Inter assay得到變異係數分別為4.6-20.6%與6.5-38.9%，雖然 Inter assay的變異係數較大，但仍然不影響肉眼之判讀結果。

關鍵字

側向流 ； 快速偵測 ； HLA-A3101

並列摘要

In this study, sequence-specified DNA segments were detected on membrane-based lateral-flow strips, incorporated with immune reactions and color readouts developed by gold nanoparticles. The Human leukocyte antigen A-3101, which modified with digoxigenin and biotin labels was detected on the membrane-based lateral-flow strips. The PCR procedure using the gold-copper alloy tube shortened the total reaction time up to 45% in 3 seconds/cycle by 25 cycles. Manipulated in the same condition, the signal intensity of HLA-3101 gene sequence was stronger than that performed by a plastic tube. The gold-copper alloy tube was also verified reusable because of its negligible carried-over contamination. To avoid the complex from aggregation, the optimal conjugation condition of mouse anti-digoxigenin antibody with colloidal-gold nanoparticles was determined as pH=8.5 and antibody concentration at least 10µg/ml. The signal was observed as low as 0.01 μl of 491ng/μl analyte by naked eyes, and the detection limit was 0.001 μl of 491ng/μl by instrumental analysis. The specificity of primers were also investigated for their validation. The coefficient of variation values of signal intensity in intra assay and inter assay were 4.6-20.6% and 6.5-38.9%, respectively.

並列關鍵字

Rapid Detection ； Lateral Flow ； HLA-A3101

參考文獻

世基生醫. 世基生物醫學股份有限公司

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