標定奈米金之核酸滾動循環擴增技術應用於側向流免疫層析法對禽流感DNA之偵測

Application of Nano-Au-labeled Rolling Circle Amplification on Membrane-Based Lateral-Flow Detection of Avian-Influenza DNA

鄭丹蘋(Tan-Ping Cheng)

指導教授 ： 吳瑞璋

中原大學/工學院/化學工程研究所/碩士(2011年)

https://doi.org/10.6840/cycu201100708

摘要

本研究利用人類禽流感病毒H5N1中的H5片段358mer序列作為目標物DNA (Target)，再設計一段末端與其互補的RCA 環狀模版探針，使探針與其目標物雜合後，在固相硝化纖維膜上加入RCA材料進行側向流動及恆溫DNA擴增，經由膠體金作為顯色劑後，可判讀出是否含有禽流感之DNA序列並達成偵測訊號放大之目的。本研究驗證可在固相硝化纖維膜上擴增RCA反應訊號，並將其應用在偵測H5序列之專一性測試，也詳細討論到在改變時間及接金抗體使用量等條件變化下，偵測極限會隨之變化，將硝化纖維膜浸泡於接金抗體的時間以10分鐘為佳，而使用1μM/μl配置出的接金抗體混合buffer的比例須1:1就可以在不浪費藥品的原則下得到清楚的訊號值。於提升H5標的物濃度進行RCA反應時發現，要到0.8pmol時訊號才可被明顯觀察到。

關鍵字

側向流免疫層析法 ； 核酸滾動循環擴增技術 ； 奈米金

並列摘要

This study designed an H5 primer of which sequence could hybridize a RCA circular template and its DNA target.The sequence specificity of the target DNA was verified on a nitrocellulose membrane based lateral-flow detection device using a DNA probe to hybridize the DNA Target. The DNA probe is modified with a digoxigenin ligand which can be recognized by its corresponding anti-body at the end of RCA reaction. This study verified the feasibility of RCA reaction on the lateral-flow membrane phase and also optimized the adding amount of RCA template, the adding amount of DNA probe, and the immersion time of membrane in nano-gold particle solution for obtainingthe best detection signal on the membrane. This study also tested the detection limit of the H5 DNA in various conditions. Problems encountered and possible solutions were marked in this study.

並列關鍵字

Rolling Circle Amplification ； nanogold ； Lateral-Flow Detection ； Avian-Influenza DNA

參考文獻

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