一種新式核酸擴增系統之研究-毛細管熱對流聚合酶連鎖反應

Abstract

聚合酶連鎖反應(Polymerase Chain reaction, PCR) 需三個不同溫度的反應步驟，分別為雙股裂解、引子黏合和引子延伸，大部分傳PCR統機台利用金屬塊加熱試管以進行溫度迴圈，升降溫過程所耗的時間導致傳統PCR完成45個迴圈需要2小時左右，耗時與價格不斐的溫控裝置讓PCR儀器無法成為居家檢測之儀器。本篇論文提出一新型的核酸擴增技術-「毛細管熱對流聚合酶連鎖反應」，此技術僅需一個乾浴槽（dry bath），其為每個實驗室中必備的設備，加熱試管底部並利用環境溫度冷卻試管，管內的試劑因上下試劑之密度不同而形成一自然對流之流體迴路，讓試劑進行PCR三大步驟。此方法讓試劑液體自發性的流動而變溫，縮短機台因加熱和冷卻所需要花耗的時間。如此無需昂貴的熱循環儀，每個實驗室即可進行核酸的檢測。而如何讓液體試劑在反應管內穩定循環且進行有效率的PCR反應是本技術成功擴增的兩大關鍵。因此，本篇論文著重於兩大參數的探討：第一、反應管的直徑與高度比參數，決定是否能在試劑底部至頂部建立足夠的溫差已達到PCR三個反應溫度且維持一穩定環流；第二、熱對流循環的PCR試劑設計準則，本技術在反應過程中為持續經過不同溫度梯度的對流循環，故必須有與傳統PCR不同的試劑設計才能提高其擴增成功率。本篇論文成功研究出所需之物理参數與特殊試劑設計準則，利用本技術擴增不同病毒的質體DNA，在25 分鐘內皆可擴增完畢；用真實檢體測試下靈敏度達 30 copies/tube。除此也針對不同的病毒設計熱對流PCR專屬試劑，如新型流感病毒(H1N1)、蝦白斑病毒(WSSV)與人類BK多瘤病毒(BKV)，其測試後的結果與傳統PCR機台一致。為了未來簡易檢測之需求，本論文也探討含鎖核酸（Locked Nucleic Acids, LNA）的引子可否避免不必要的擴增副產物。本論文的未來方向是盼望架構於本技術下發展出便宜、便利、非專業人員即可現場操作檢測技術，可對人類和動植物傳染性疾病的防治有一定的助益。

A typical polymerase chain reaction (PCR) is a process containing three temperature steps for denaturation, annealing and elongation. In a traditional thermal cycler, metal block is used to heat or cool the reaction tubes, and more than two hours are needed for 45 cycles of the repeat heating and cooling steps. However, expensive thermal controller and long time-consuming made thermal cyclers are not suitable in self-test for personal use. In this study we describe a new method for DNA amplification using a principle of convection. In this platform, only a dry bath is required for heating the bottom of the reaction tube. Then the tube is cooled by the surrounding air by which the reagent in the tube forming convection repeatly. Thus, reaction mixture in the tube should undergo the three steps of PCR in the convection. This kind of convection in the tube is just like the phenomenon during water is boiled and it makes the reagents in the tube should fluid spontaneously to achieve different temperatures. This technique can shorten the amplification time prominently and solve the traditional problem for long time-consuming process during heating and cooling. By this way, no expensive equipment is needed and it makes this new method feasible to be used in almost all the laboratories. The key point of successful working of this platform is how to make the fluid reagent mixture of PCR reaction in the tube to form a stable and efficient convection. Here we focus on two important parameters. One is the ratio between height and diameter of the reaction tube, which determines whether the temperature gradient from the bottom to top can fulfill the three-step temperatures needed in the PCR reaction. The other is the criterions of the reagents designed in convective PCR. Because reagents in convective PCR reaction undergo a continued temperature gradient which is different from traditional PCR, it requires particular criterions for reagents in order to improve the level of yield and the rate of success. In this study, we also provide the physical parameters and design criterions of particular reagents for successful convective PCR. Using this newly established method, DNA amplification could be completed within 25 minutes and the sensitivity was measured to reach 30 copies/per tube. Besides, specific reagents were also designed for various virues, including new influenza virus H1N1, white spot syndrome virus (WSSV), and polyomavirus (BKV). Moreover, we also explore whether primers conjugated with LNA should avoid side products in the amplification. We will focus on developing a cheaper and more convenient technique for examination using our established platform on site, where it could be operated by persons without well-training. We also believe this study will provide a new direction and will be helpful and useful in the future in controlling infectious disease for human, animal and plants.