- 學位論文
迴路式毛細管熱對流聚合酶連鎖反應平台的開發與研究
The Development of a Capillary Loop Convective Polymerase Chain Reaction Platform
摘要
迴路式熱對流聚合酶連鎖反應(Capillary Loop Convective Polymerase Chain Reaction，CLCPCR）為一種新式核酸擴增技術，僅以單一底部加熱源使迴路式毛細管內部試劑產生穩定的循環流動，無須繁複的升降溫過程即能使核酸在PCR所需之溫度間不斷增生。本論文目的為針對迴路式毛細管聚合酶連鎖反應開發一簡單穩定之擴增平台，驗證此系統之可行性並蒐集實驗相關參數。 實驗初期須確認管內熱流情形合乎聚合酶連鎖反應之需求。本論文藉由將微小粒子加入迴路式毛細管中觀察流場運動情形，確認管內流體在底部單一加熱情況下有循環流動的現象發生後，再量測管內流體溫度，在環境溫度25~26℃以及底部加熱溫度為135℃的情況下，管內流體之最高溫及最低溫皆符合聚合酶連鎖反應之需求。 本實驗接著建立一簡單CLCPCR定量檢測平台，並先進行擴增定性測試。為增加本系統擴增之穩定性，我們找尋並設計出適合迴路式毛細管擴增環境之引子對，並測試其穩定性及靈敏度，經實驗證明，本系統在高(6×106copies/tube)、中(6×104copies/tube)以及低(6×102copies/tube)三種初始模板濃度下皆能在25分鐘內擴增成功。定性測試成功後進而進行CLCPCR定量測試，確認光學機構可以成功偵測到螢光強度之變化，再使用影像分析軟體將拍攝到之PCR過程螢光變化趨勢數值化，並定義一Ct值繪製對應的標準曲線。由結果顯示標準曲線相當趨於線性，因此證明了此系統之可行性及發展性。 CLCPCR平台相較於傳統PCR平台縮短了擴增過程的時間，且單一溫控系統也降低了機台製造成本，未來若能將整體系統設計最佳化提高檢測精準度，相信必能投入市場為台灣的醫療產業盡一份心力。
並列摘要
Capillary loop convective polymerase chain reaction (CLCPCR) is a new DNA amplification technology. It can replicate DNA simply by using a single temperature controller to heat the bottom of the loop solely to generate stable circulation. The purpose of this thesis is to develop a solid amplified platform to examine the possibility of CLCPCR and collect parameters of the experiments. The initial stage of the experiments must ensure whether the temperature in the loops meets the requirement of the PCR. In this thesis, first, the researcher added tiny particles in the loops to observe the movement so as to make sure circular phenomena occurred when heating the bottom of the loops solely. Then, the researcher measured the fluid temperature and found that the maximum and minimum fluid temperature in the loops meet the PCR requirements in the condition of 25~26℃ surrounding temperature with 135℃ bottom heated temperature. For next step, we built a detective CLCPCR platform and conducted qualitative test. In order to increase the stability and sensitivity of the platform, we sought and designed the appropriate CLCPCR primer. The experiments have proved that the platform can amplify in 25 minutes in the following three initial copies 6×106copies/tube (high),6×104copies/tube (medium) and 6×102copies/tube (low). After qualitative test, the researcher then carried out the quantitative test to ensure optical mechanism can detect the fluorescent varieties. The researcher then uses the image analysis software to digitalize the recorded fluorescent varieties and define the Ct value and then draw the corresponded standard curve. The results indicated that the standard curve tend to be linear so it proved the feasibility and expansibility of the platform. Compared to traditional PCR platform, the CLCPCR platform can both shorten the amplified time and reduce the cost of manufacturing machines because of the single temperature controller. As a result, if the platform can be optimized to enhance the detective accuracy in the future, it can definitely contribute to the Taiwanese medical device industry and bring commercial value into the market.