Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/90053
標題: Development of a Novel Platform Integrating Rolling Circle Amplification and Dipping Strip Technology for the Detection of Viral Nucleic Acids
新型檢測病毒核酸技術平台之研發
作者: 陳信宏
Hsin-Hung Chen
關鍵字: 無;NO
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摘要: 
目前已有多種檢測蛋白質目標物的快速檢測技術,但以核酸為檢測目標物的快速檢測技術仍然極為少見,且在靈敏度與檢驗速度方面,仍存有進步的空間。因此 本研究即以引起嚴重經濟損失的番茄黃化捲葉病毒,(Tomato yellow leaf curlvirus, TYLCV ) 之 核 酸 作 為 測 試 目 標 , 結 合 滾 環 式 擴 增 法 ( Rolling circleamplification, RCA)合成探針、與浸泡式測試條(Dipping strip, DS)技術,以快速核酸雜合的方式,研發新型核酸檢測技術平台。此研究策略是將 Digoxigenin混合在 RCA 反應中,由於逢機引子與 DNA polymerase 的作用,可增幅出多股多套體探針。並結合新開發的 DS 技術,點印 TYLCV 外鞘蛋白基因 DNA 於測試條的尼龍膜上,配合新設計的反應槽與垂直式核酸雜合反應器反應後,最後呈色以肉眼判讀。目前已成功地自番茄植株中檢測出 TYLCV 的存在。本研究開發的探針變性溫度只需 68℃,遠低於傳統方法合成探針所需溫度。核酸雜合溫度可以降到 38℃,適用於一般實驗室生長箱溫度,不需另購專用的核酸雜合箱。配合 DS 技術平台測試後,核酸雜合反應可大幅縮短至 4 小時完成。其檢測靈敏度可達 10 pg,並能以肉眼區分感染 TYLCV 病株與健康植株,而不需昂貴的儀器判讀。RCA 探針與 DS 技術平台的結合,不僅能夠增加探針與被測物的碰撞機會與增強檢測信號強度,並能大幅減少操作時間。未來可適用於農業、環境及臨床檢測方面,提供研究人員更簡易、快速的核酸雜合檢測方法。

Many point-of-care rapid detection technologies for protein targets have been well-developed and widely used. However, such technologies for nucleic acid targets were still underdeveloped. In this study, a novel platform for the detection of viral nucleic acids was developed by integrating the high sensitivity and specificity of probes synthesized by rolling circle amplification (RCA) and the simplicity of dipping strip (DS) technique. The strategy involves the synthesis of Digoxigenin (DIG)-labeled multimeric probes by RCA, which utilizes random hexameric primers to initiate the reaction and circumvents the limitation of specificity. In combination with the newly designed reaction vessel, vertical hybridization mechanism, and blotting material, the DS technique facilitates simple and rapid detection of viral nucleic acids, with results easily diagnosed by naked eyes. Currently, the novel platform has been applied in the detection of Tomato yellow leaf curl virus in tomato seedlings and plants. The optimal quantity of template and denaturing temperature were determined to be 1 µg (for 10 pg of target DNA) and 68 ℃, respectively. Hybridization is performed at 38 ℃, which is much lower than the traditional procedure, without the need for specialized hybridization ovens. The whole operation could be completed within 4 hours, with the sensitivity reaching 10 pg. The combination of probes synthesized by RCA and DS techniques increased the interactions between targets and probes, and thereby enhanced the intensity of the detection signal and reduced the operation time. It is expected that the novel platform could provide simple and rapid virus detection tools for front-line workers and contribute to agricultural, environmental, and clinical studies.
URI: http://hdl.handle.net/11455/90053
Rights: 同意授權瀏覽/列印電子全文服務,2018-07-16起公開。
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