核酸滾動循環擴增技術應用於加電場之 側向流免疫層析法對禽流感H5基因序列 之偵測

Application of Rolling Circle Amplification Technology on An Electric-Field Applied Lateral-Flow Membrane to Detect the Avian Influenza H5 Sequence

鄭雅云(Ya-Yun Zheng)

指導教授 ： 吳瑞璋

中原大學/工學院/化學工程研究所/碩士(2013年)

https://doi.org/10.6840/cycu201300836

摘要

本研究以人類禽流感病毒H5基因序列中的358mer片段作為標的物DNA (Target)，設計一段末端與其互補之環狀模版探針來檢測。此環狀模版探針更於側向流薄膜上進行滾動循環擴增(RCA)技術以放大H5標的物之偵測訊號。當H5標的物橫向流過試片時，會被膜上印製之測試線所捕捉，而後隨即進行滾動循環擴增反應以放大其偵測訊號；另於加入偵測探針之步驟前，先潤濕薄膜並施予電場。本研究之實驗結果顯示，於硝化纖維膜上之RCA反應時間以1小時為最適化，其訊號強度為未進行RCA反應之2.9倍。再於RCA反應後之膜上加以12伏特電壓，使用於RCA反應後擴增出之單鏈，與其雜合之探針，其訊號強度提升2.3倍，此強度之增幅比使用與環型結構互補之探針，還大的多，證實電場能有效的使纏繞的DNA舒展開來，進而有更多互補位可被探針捕捉，達到訊號放大之目的。在RCA反應一小時，通以12伏特電壓之條件下，H5標的物之偵測極限可達到0.1pmol，表示電場能提升8倍的訊號強度。

關鍵字

滾動循環擴增技術 ； 側向流檢測 ； 禽流感病毒 ； 直流電場

並列摘要

In this study, a circular DNA probe with a tail was design to detect its complementary target DNA, the Avian-Influenza virus H5 sequence. The circular DNA probe was latter conducted the Rolling-Circle- Amplification (RCA) reaction to amplify the detection signal. The detection procedure was performed on membrane-based lateral-flow strips. As the H5 DNA target flow laterally through the strip, it was captured on the test line on the membrane. A RCA reaction was then proceeded to amplify signals. Also, before adding the detective probe, the strip was wetted, and an electrical field was given. The experimental results indicated that the optimal reaction time on NC membrane was 1 hour. Under that condition, the signal was enhanced for about three times. Also, by applying a 12-voltage field on membrane, the RCA reaction signal was enhanced for 2.3 times. This result revealed that a direct electrical field could strengthen a DNA molecule, so that more complementary sites could be exposed to the detective probe. In this study, a detection limit of 0.1pmol of H5 DNA was achieved when the RCA reaction time was controlled at 1 hour under a 12-voltage field. An electrical field was also measured to enhance the detection limit in about eight times.

並列關鍵字

rolling circle amplification ； lateral-flow detect ； Avian Influenza H5 Sequence ； electric-field

參考文獻

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