即時定量熱對流聚合酶連鎖反應平台定量方法之研究與開發

Abstract

本論文主要是針對動態的熱對流聚合酶連鎖反應(cPCR)定量檢測，建立出模型化公式並開發出可偵測核酸初始濃度之即時同步定量毛細管熱對流聚合酶連鎖反應平台以驗證之。cPCR相較於商用機台有著輕便小型化、簡單、反應時間短及價格低廉等優點，因此讓cPCR成為市場上另一個有潛力的選擇。針對目前cPCR並無適合的模型化公式去探討分析，因cPCR並無循環的概念且變性作用、引子煉合及延伸作用同時發生，其原理與傳統PCR不同。故本論文針對連續時間的cPCR建立出一套模組化公式來定量核酸初始濃度，利用實驗建立出標準化曲線分析及進行單管定量。此外，本論文也建立即時定量熱對流聚合酶連鎖反應平台來驗證模組化公式，其主要組成元件為480nm的LED為激發光和裝置530nm濾光片偵測螢光的感光耦合元件(CCD)。實驗結果顯示針對B型肝炎(HBV)及使用可嵌入鹼基對的螢光染劑(SYBR Green I)可成功擴增及靈敏度可達5 copies/tube和相關係數(R2)為0.985以上。期望此即時定量熱對流聚合酶連鎖反應平台可以在準確度，反應時間，便攜性和成本方面提供良好的性能，進而顯著提升生物醫療的品質。

This thesis developed a phenomenal model for convective quantitative polymerase chain reaction and designed prototype platforms to demonstrate the validness of the model. Although the compactness, simplicity, fast reaction time, and low cost of the platform let convective PCR (cPCR) be a potential alternative of commercial PCR, lacking of a suitable model for quantitative detection of DNA amplification limits its applications in many fields. The current models for quantitative PCR (qPCR) based on thermocycling, however, are not applicable to cPCR since three reactions of denaturation, annealing, and extension occur simultaneously in the capillary and the concept of thermocycling is irrelevant to cPCR. Considering the dynamics of DNA reactions of cPCR, this thesis developed a phenomenal model in time domain to determine the initial DNA concentration quantitatively. The model shows that cPCR possess a standard line of inflection points (SLIP), similar to the standard curve for conventional PCR. Moreover, the model indicates cPCR can give the quantitative method for single assay test. To test the validness of the model, this thesis designed the fluorescence detection systems of cPCR to verify the model. It mainly consists of a 480-nm LED as an excitation light source, a CCD camera, and an optical filter to provide a fluorescence detection channel at a wavelength of 530 nm for real-time cPCR DNA quantification. The experimental results show that real-time quantitative cPCR can give the sensitivity as low as 5 copies and R2 larger than 0.985 for the PCR mix containing hepatitis B virus (HBV) plasmid samples, by using SYBR Green I fluorescence labeling dye to assess the prototype performance. The experimental results expect that the capillary convective qPCR can provide good performance from the aspect of accuracy, time, portability, and cost. This result has the potential to enhance significantly the medical care of life.