- 學位論文
利用光學模擬設計即時定量聚合酶連鎖反應的光學檢測機制
Using Optical Simulation to Design the Optical Detection System of Real-time Polymerase Chain Reaction
摘要
近年來，市面上有許多即時定量聚合酶連鎖反應（real-time polymerase chain reaction，簡稱real-time PCR）機台，但大多數都價格昂貴且體積龐大，無法普及到一般診所和家庭，因此，本論文將研究新的機台去改善上述問題。 本論文利用TracePro光學模擬軟體進行模擬。首先，我們建立一簡單的光學模型，包括毛細管、銅塊、發光二極體（light-emitting diode，簡寫LED）和感測器，其中毛細管內有試劑。我們考慮五種打收光方式，分別為下打光側收光、側打光下收光、側打光側收光、側打光上收光和上打光側收光。根據模擬結果和實際機台設計，我們選擇下打光側收光。 接著，我們利用熱電晶片作為機台的加熱源，加熱銅塊後，再由銅塊加熱毛細管底部，機台內部利用風扇降低環境溫度。毛細管下方的銅塊打洞，LED的光線可以到達毛細管，試劑被激發後釋放螢光，由側面的感測器擷取螢光訊號。溫度設定和螢光曲線在電腦軟體操作和顯示。 然後，機台進行四孔同時核酸擴增，50copies/tube的B型肝炎核酸在30分鐘內成功擴增。最後，我們進行不同核酸濃度的定量分析，核酸濃度分別為5×10^5copies/tube、5×10^4copies/tube、5×10^3copies/tube、5×10^2copies/tube和5×10^1copies/tube和negative control，擷取螢光訊號後畫出螢光擴增曲線，利用門檻值（threshold）和正規化（normalize）的方法，算出此機台的標準曲線，可以作為本機台定量分析的標準。
並列摘要
In recent years, many of real-time polymerase chain reaction (real-time PCR) machines are on the market. Few of real-time PCR machines are used in clinic and at home, because most of all are expensive and large. Thus, in this thesis, the new machine with capillary convective polymerase chain reaction (CCPCR) that is developed is investigated. First, this study built a simple optical model that contains a capillary tube with reagent, a light-emitting diode (LED) and a color sensor. Then, light traces with five arrangements of positions of LED and sensor were simulated by a software, TracePro. With the highest fluorescence energy reaching the sensor, this study choose the arrangement with the LED under capillary tube and the sensor on the side of capillary among five cases studied. The information of energy of fluorescence to different cases is a reference of other study associated. Then, we used a thermoelectric cooling (TEC) as a heater for CCPCR and its input power can be controlled by a computer. The TEC heats the copper block, and then the copper block heats the bottom of capillary tube. The capillary tube in the machine is cooled by three fans. Lights emitted from the LED pass through the hole of the copper block and the fluorescence markers in reagent are excited. If the amplicons are successfully amplified, the fluorescence intensity will increases with time. The sensor on the side catches fluorescence and the monitor shows the intensity of fluorescence. Finally, the test with nucleic acid of HBV of 50copies/tube was amplified successfully in four well and it takes thirty minutes. We got a group of fluorescence amplification curves of 5×10^5 copies/tube、5×10^4 copies/tube、5×10^3 copies/tube、5×10^2 copies/tube、5×10^1 copies/tube and negative control to analysis. The fluorescence curves are called the standard curves. That can be used to establish an empirical formula. One can apply such a formula to calculate unknown initial concentration of virus in the tested sample with the same reagent as those for obtaining standard curves.