兩種新型毛細管熱對流聚合酶連鎖反應機台的開發與研究

Development of two Novel PCR Machines base on the Capillary Convective Polymerase Chain Reaction

10.6342/NTU.2011.02786

張維真

熱對流 ； 聚合酶連鎖反應 ； 核酸檢測 ； 可攜性 ； 即時定量核酸檢測 ； Convection ； Polymerase chain reaction ； Nucleic acid detection ； Portable ； Real-time nucleic acid detection

國立臺灣大學機械工程學系學位論文

2011年

碩士

陳炳煇

繁體中文

本論文主要為針對毛細管熱對流聚核酶連鎖反應（CCPCR）技術開發設計兩種新型機台：現場檢測型與即時定量型。毛細管熱對流聚核酶連鎖反應（Capillary Convective Polymerase Chain Reaction，CCPCR）為新式單一溫控熱對流聚合酶連鎖反應機制，僅需使用單一底部溫控加熱，溫度維持在95℃左右，上方藉由環境溫度散熱，而與底部溫控形成上下溫度梯度，使得試劑內部流場產生自然對流中的環流現象，可於30分鐘後，將標的DNA成功且專一地擴增出來。 第一部分，本論文開發設計之現場檢測型CCPCR機台，提出以正溫度係數熱敏電阻（PTC Thermistor）做為底部發熱元件，其平坦的表面與特殊的自發熱特性，提供了CCPCR反應所需之單一穩定溫控，具備體積小、操作簡單、成本低、耗電量低等特性，並可使用鋰電池供電來達成其可攜性的目的，經實際核酸擴增測試後，整體具備高靈敏度之核酸擴增能力，讓疾病檢測更為迅速方便。 第二部份，本論文開發設計之即時定量型CCPCR機台，採用下打激發光源、上收螢光訊號的設計，搭配穩定底部溫控，可以準確完成CCPCR擴增過程之即時螢光偵測與DNA定量（Real-time CCPCR），並可同時進行4件檢體核酸擴增。而有鑒於CCPCR技術僅需一穩定底部加熱，無需反覆的升降循環溫控，因此有別於一般Real-time PCR常使用之門閥循環值（Cycle threshold，Ct），本論文定義一Ct（Crossing time）值來做為定量分析的依據，根據實驗結果所繪製之標準曲線（Standard curve）可觀察出，起始模板濃度的對數值與螢光擴增曲線所得到之Ct值呈現相當地線性關係，將可做為Real-time CCPCR定量分析的根據。整體機台只需配置一底部加熱溫控器結合一螢光偵測設備，相較需要複雜溫控的商用即時定量機台來說，成本相當便宜。 若能依據本論文實驗結果，將此機台趨向最佳化、產品化及人性化角度來進行改良設計，進而推廣到家庭及個人照護，相信對於未來醫療檢測會有極大的貢獻。

In this study, we developed two novel prototypes of platform base on the capillary convective polymerase chain reaction（CCPCR）technology, including onsite detection type and real-time detection type. CCPCR is a novel technology for polymerase chain reaction. It only needs one single temperature controller under the bottom of glass capillary tube in which the sample is contained, and the bottom temperature is maintained at about 95℃. Thus, the temperature gradient between the bottom and the top will drive a thermal convection loop that can successfully execute PCR within 30 minutes. For the onsite detection type platform, we applied the positive temperature coefficient thermistor (PTC Thermistor) as the bottom heating components. PTC thermistor provides a flat surface and a steady temperature control for the CCPCR due to its special self-heating characteristic. Overall, the advantages of the detection device are compact, simple, low cost, portable, low power consumption, powered by a lithium battery, and perform the high sensitivity of nucleic acid detection to make disease detection more quickly and easily. In addition, for the real-time CCPCR platform design, we set up the excitation light source on the bottom of the glass capillary and the sensor on the top for fluorescence detection. The optical system and the accurate temperature control will be able to quantitatively identify the initial concentration of the template DNA. Four samples can be simultaneously execute CCPCR. For the quantitative analysis, we define a Ct (Crossing time) value. According to the results, the logarithm of the initial DNA concentration and the Ct value shows linear correlation which can be taken as a calibration curve in real-time CCPCR. Compared to the commercial real-time PCR platform, the cost of ours platform is quite low. Based on the results of this study, we can promote this technology for the family and personal care after doing the improved design to become optimized, commercialized and humanized. We believe that these platforms will have a great contribution in medical detection in the future.

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