臺灣博碩士論文加值系統

迴路式熱對流聚合酶連鎖反應（Capillary Loop Convective Polymerase Chain Reaction，CLCPCR），此方法在一迴路式載具底部利用單一熱源加熱使載具內部的流體產生溫度差，熱對流循環會帶動內部試劑循環流動完成PCR所需的溫度要求，相對於傳統機台耗時的熱傳導升降溫過程，不僅省時間、成本、能量，本實驗室進行PCR相關研究已行之有年，但最終目標是朝向逆轉錄聚合酶連鎖反應(Reverse Transcription Polymerase Chain Reaction)發展，因為現今地球上造成人類得到傳染病的病毒，其遺傳物質為RNA，因此針對RNA擴增技術的研究，能有效幫助我們對抗病毒的傳染。
本研究使用新型玻璃迴路式載具，其使用超音波加工機在玻璃片上銑削加工，依照設計的流道形狀銑出立體凹槽，再黏上蓋玻片進行封裝，後續實驗皆以此載具為設計出發點，進行機台開發以完成RT-PCR擴增研究。確認玻璃載具內試劑繞行一圈時間及內部溫度分佈，為擴增實驗是否成功的一大主因，實驗上以兩種不同尺寸的玻璃載具，相互比較參數差異，找出能讓RNA成功擴增的載具尺寸，擁有穩定的加熱源，能使RT-PCR試劑在經歷溫度循環時，能更有效率的進行擴增，因此實驗採用電子控制板作為控制器進行PID溫度控制，以達到加熱速度快、穩定的目標溫度等特性。
實驗結果證明，機台可成功擴增目標模板，此新型機台亦可在室溫以外的環境溫度之間成功擴增，由於此機台組裝上簡易且攜帶方便，且控制程式碼將會開源於網路上，未來希望能將此擴增機台普及化，讓醫學檢驗技術更加快速且普及於民眾之間。

Capillary Loop Convective Polymerase Chain Reaction (CLCPCR) is a new application of convective PCR. It can complete the temperature cycle of PCR with a single heater. By heating the bottom of the loop container, the fluid would form a Rayleigh–Bénard convection cell and generate a circulating flow continuously. This new method can economize more time,energy,cost than traditional PCR machine.
Our lab has researched the topic of PCR for many years.But the final purpose is to develop Reverse Transcription PCR,because genetic material of virus is composed of RNA.If we can learn more detail about RNA,it will help us to fight infectious diseases
We cooperate with SPINTECH company to develop the novel container.A glass substrate is fabricated with groove of loop-shaped with an ultrasonic machining approach.A fabricated glass substrate will be covered with another glass to form the test piece.The novel container will be the starting point of this thesis,the following experimental design,development will base on this novel container.
Ensuring the fluid field and the temperature distribution in the container is the main factor of PCR experiment.We compare two different container of size in order to find the more suitabler container. With the stable heater will let the fluid field stably cycle,the yield of experimental result will increase.The RT-PCR experiment will be more efficienter than before.With these benefit,we will use PID temperature controller to create faster,stabler temperature controller.
The experimental results show that the HEK293T template with the lowest initial concentration 100ng/μL can be amplified. This RT-PCR machine can be operated between 12℃~33℃.The code of machine will upload on the internet.In the future,we hope that more faster medical laboratory tests can popularize in the world.

口試委員會審定書 i
誌謝 ii
中文摘要 iii
ABSTRACT iv
目錄 vi
附圖目錄 ix
附表目錄 xii
符號說明 xiii
第 1 章 緒論 1
1.1 前言 1
1.2 研究動機與目的 2
1.3 文獻回顧 3
1.3.1 聚合酶連鎖反應 3
1.3.2 Arduino控制器 8
1.4 論文架構 9
第 2 章 研究原理與方法 25
2.1 聚合酶連鎖反應 25
2.1.1 基本原理介紹 25
2.1.2 影響聚合酶連鎖反應的因素 26
2.2 即時定量聚合酶連鎖反應 29
2.2.1 基本原理介紹 29
2.3 熱對流聚合酶連鎖反應 31
2.3.1 基本原理介紹 31
2.3.2 迴路式熱對流聚合酶連鎖反應 31
2.4 凝膠電泳 32
2.5 超音波加工 33
第 3 章 實驗設備與步驟 37
3.1 實驗試劑與化學藥品 37
3.1.1 逆轉錄聚合酶連鎖反應 37
3.1.2 洋菜凝膠電泳檢測 38
3.2 實驗儀器 39
3.2.1 溫控系統 39
3.2.2 超音波加工系統 41
3.2.3 溫度量測 41
3.2.4 聚合酶連鎖反應 42
3.2.5 凝膠電泳 43
3.3 CLCRT-PCR實驗步驟 44
第 4 章 實驗結果與討論 56
4.1 玻璃載具尺寸與探討 56
4.2 玻璃迴路式載具溫度量測與流場觀測結果 57
4.2.1 玻璃迴路式載具流場觀測結果 57
4.2.2 玻璃迴路式載具溫度量測結果 58
4.3 溫度控制器參數校正及調整 59
4.3.1 設定溫度校正 59
4.3.2 控制電熱片加熱結果 59
4.4 CLCRT-PCR定性實驗結果 60
4.4.1 定性實驗測試──刪去Hot-Start步驟 60
4.5 迴路式載具靈敏度與環境溫度影響 61
4.5.1 新型CLCRT-PCR機台之靈敏度 61
4.5.2 環境溫度對新型CLCRT-PCR機台之適用性 61
第 5 章 結論與未來展望 70
5.1 結論 70
5.2 未來展望 71
參考文獻 72

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