迴路式熱對流聚合酶連鎖反應的即時螢光分析機台開發

The development of a real-time fluorescence analysis PCR machine base on the Capillary Loop Convective Polymerase Chain Reaction

許宗峻(Zong-Jyun Hsu)

指導教授 ： 陳炳煇

國立臺灣大學/工學院/機械工程學研究所/碩士(2017年)

https://doi.org/10.6342/NTU201702023

摘要

此論文基於迴路式熱對流聚合酶連鎖反應(Capillary Loop Convective Polymerase Chain Reaction ,CLCPCR)技術開發俱備即時影像分析的核酸擴增機台，並欲透過改變前研究的熱啟動方式提升試劑靈敏度，相對於原先研究使用的PCR反應液，嘗試使用他種PCR反應液降低試劑成本、檢驗平台通用性。迴路式熱對流聚合酶連鎖反應(CLCPCR)為新型的核酸擴增技術，只需單一底部溫控加上環境散熱的搭配，能夠產生符合核酸擴增的流場溫度並持續不停地循環，減低往復升降溫的時間，本機台最快能夠於15分鐘將65 copies/tube初始模板濃度專一地將DNA擴增成功。
本研究機台建設初期建立機台的整體外型與內部構造，並使用溫度控制器與撓性超薄電熱片進行溫控，加熱塊設計上，使用非對稱式加熱銅塊以固定流體轉向，為了提供良好光遮蔽性，機台近乎為封閉性結構，因此內部需要搭配可調節式散熱，使用Arduino開發板整合於溫度控制器進行風扇流量的調控。在即時影像分析部分，基於原先溫度控制程式，添加即時影像分析、控制風扇強度的功能，前者能進行影像顯示、螢光分析與數據曲線擬合，節省以往分析所需的時間，後者控制機台內部散熱以追求較適合本平台核酸增生的環境溫度。
架設好軟硬體整合後的機台，開始進行核酸擴增定性、定量實驗。使用前研究的引子對、PCR反應液與新式熱啟動方式，實驗結果證實能將最低初始模板濃度為6.5 copies/tube擴增成功，本研究也進行起始模板濃度為6.5×107、6.5×105、6.5×103、65 copies/tube的定量實驗，實驗結果的標準曲線趨近於線性，證實本系統有潛力作為精準的即時定量核酸擴增平台。在他牌PCR反應液定量結果方面，進行6.5×107、6.5×105、6.5×103 copies/tube三種起始模板濃度定量實驗，結果顯示趨近於線性的標準曲線，證實能使用此新PCR反應液於CLCPCR定量實驗而節省試劑成本。
此CLCPCR機台減低以往分析所需要的時間，軟硬體結合的架構能在使用上更為方便且具有可發展性，未來若能解決載具的穩定性並搭配此機台，相信此時將能夠實現精準的即時定量核酸擴增，能為台灣醫療檢測技術帶來一個低成本、快速的核酸擴增技術。

關鍵字

熱對流 ； 聚合酶連鎖反應 ； 即時影像分析 ； PCR機台

並列摘要

This study is based on technology of Capillary loop convective polymerase chain reaction(CLCPCR) to develop a real-time image analysis PCR machine. Compare to the previous related study, a new hot start PCR instrument is constructed to enhance the PCR sensitivity. To decrease the PCR reagent’s cost and test the performance of the CLCPCR machine, instead of using original PCR kit, a new PCR kit is tested in this research. CLCPCR is a new DNA amplification technology. The glass loop contains a stable circulation and suitable temperature distribution to excute PCR by using single temperature controller under the reagent container. This new method of PCR can help the whole process benefits from saving time to control the reagent temperature between heating and cooling. Within 15 minutes, the CLCPCR machine in this thesis can sussfully excute PCR with initial DNA copies of 65 copies/tube.
In the initial stage of this research, the structure of CLCPCR machine was created to fit the glass loop PCR system, and a PID controller with electric heater was used as temperature control system. Moreover, an asymmetric heating copper block was designed to produce a fixed direction of circulation in the glass loop. To prevent PCR process from interference of external light, the overall structure was desigined as a nearly closed system. Hence, a heat dissipation strategy is needed. This study combines an Arduino microcontroller, which is designed as an adjustable fan intensity system, in the PID controller. Furthermore, apart from incorporating the fan controller function in PID temperature software, this study adds real-time image analysis as well. The foregoing two tools cam provide image display, fluorescence analysis, data curve fitting, and fan intensity control. The user can have advantages of saving time for analzing data and regulating the themal convection in the PCR machine.
For next step, the CLCPCR machine conducted the qualitative tests and quantitative experiments. With the previous study’s designed primer, PCR kit, and this study’s new hot start PCR instrument, this CLCPCR machine can have a good PCR sensitivity and specificity in experiment of using 6.5 copies/tube as initial DNA concentration. Moreover, this study carried out the quantitative experiments in the following four initial copies 6.5×107, 6.5×105, 6.5×103 and 65 copies/tube. From the result of the corresponded linear standard curve, it can prove this CLCPCR machine has the potential to be a precise qPCR commertial machine. Aslo, this study used another PCR kit to excute the quantitative experiments in the following three initial copies 6.5×107, 6.5×105 and 6.5×103 copies/tube. The outcome has a great linear corresponded standard curve. This can verify the CLCPCR machine can use other PCR kit and have a potential to obtain a good performance of quantitative PCR.
This CLCPCR machine can shorten the fluorescent analysis time and is convenient for user to research PCR with the combination of whole software and hardware components. In the future, if the deviation of dimensions from the glass loop can be reduced, an accurate real-time quantitative PCR can be obtained, and Taiwan medical device can definitely have a low cost and fast detection of qPCR techonology.

並列關鍵字

thermal convection ； Polymerase Chain Reaction ； Real-time image analysis ； PCR machine

參考文獻

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