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標題: | 微型玻片式PCR加熱模組之研究 The Study of Slide-based PCR Thermal Cycler |
作者: | Chung-Che Huang 黃崇哲 |
指導教授: | 陳林祈 |
關鍵字: | 即時定量聚合?鏈鎖反應,矽膠加熱片,PCR晶片,H1N1, quantitative polymerase chain reaction,flexible heater,PCR chip,H1N1, |
出版年 : | 2013 |
學位: | 碩士 |
摘要: | 即時定量聚合酶鏈鎖反應(real-time quantitative polymerase chain reaction, real-time qPCR)是結合DNA擴增與螢光感測來對病原體檢測方法。在實驗室的高靈敏度、低偵測極限儀器體積通常較龐大,因此想發展可攜式的即時定量PCR感測器。本研究針對PCR加熱模組微小化,以輕薄的矽膠加熱片作為加熱單元,並利用PDMS做成PCR晶片。PCR加熱模組以壓克力為基材,利用CNC銑床進行加工為所需形狀,將加熱單元、風扇、散熱鋁塊以及熱電偶等元件安置其中。其升降溫速率分別為1.1與0.7°C/s,溫度振幅可小於0.1°C。PCR晶片為配合暗場式螢光模組的訊號量測,利用鋁翻模製作反應槽高度為1mm,反應槽所容納反應試劑體積為10μl。為避免聚合酶吸附在PDMS表面上,添加2.5% PVP於反應試劑中也不會對聚合酶有抑制現象。在自製加熱模組驗證中,使用H1N1的互補DNA序列作為標的,當黏合溫度為50°C 時有較高的產率。分別以自製加熱模組與市售儀器進行核酸擴增,而後利用洋菜膠電泳分析。自製加熱模組PCR擴增曲線以sigmoid model進行曲線配適,可得到擴增效率為0.97(市售)與0.70(自製)。日後若能整合暗場式螢光感測模組,成為可攜式即時定量PCR儀器,對防疫現場實測或是居家醫療的概念實現,會有很大幫助。 Real-time quantitative polymerases chain reaction (real-time qPCR) is combined DNA amplification and fluorescent sensing for pathogen detection. Because laboratory instruments with high sensitivity and low detection are usually bulky, the concept of portable real-time qPCR is developed. This research is focused on the miniaturization of PCR thermal module. We select thin silicone film as heating unit and used PDMS as PCR chip. This PMMA-based device is constructed out by CNC machining, and it is modularized compactly in order to accommodate silicone film, fan, heat sink and thermocouple. Heating and cooling rate of the heating module is 1.1 and 0.7°C/s separately and the temperature amplitude is smaller than 0.1°C. In order to suit the dark-field fluorescent sensing module, the height of reaction chamber is 1mm and the chamber contains 10μl per reaction. Taq polymerase is apt to absorb on the surface of PDMS, 2.5% PVP is added in PCR mixture to avoid absorption and it doesn’t inhibit polymerase activity. We use the cDNA sequence of H1N1 as target template. In order to improve PCR efficiency, we choose 50 °C as annealing temperature. In home-made heating module authentication, commercial PCR instrument is a control group. The fluorescent signal is analyzed by sigmoid model fitting to the amplified efficiency, and the efficiency were found to be 0.97 (commercial instrument) and 0.70 (homemade). If we can integrate dark-field fluorescent sensing module, a portable real-time qPCR instrument will achieve the concepts of on-site detection or point-of-care. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/16918 |
全文授權: | 未授權 |
顯示於系所單位: | 生物機電工程學系 |
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