不同的光學機制對即時定量聚合酶連鎖反應檢測之影響

Abstract

本論文以開發新式聚合酶連鎖反應（Polymerase Chain Reaction，PCR）之機台為基礎，進行光學感測器的選用與數據分析，達到低成本，高準確度之目標，並希望未來能將此機台普及至一般診所進行即時的疾病檢測。 傳統PCR必須經過約四十次95℃、60℃、72℃三種溫度循環達到核酸擴增。本機台結合毛細管熱對流聚合酶連鎖反應（Capillary Convective Polymerase Chain Reaction，CCPCR）與即時定量聚合酶連鎖反應（Real-time Polymerase Chain Reaction，Real-time PCR）。僅透過底部95℃單一溫控進行加熱，在試管中試劑經由自然對流達到所需的三種溫度，並隨時間觀測試管內螢光強度變化。即時顯示擴增情形，使核酸擴增約在30分鐘完成。 首先更換試劑容器，以單支25NTD之玻璃毛細管改為低於1NTD之塑膠毛細管，大幅降低耗材成本。但塑膠材質透光度比起玻璃減少42.7%，故需針對光學感測器進行改善。本機台使用鎳鉻絲加熱，紅銅塊傳導熱使試管底部維持在95℃左右，並且溫控準確，標準差約在0.2℃。光學機制採取一藍色發光二極體（Light-Emitting Diode，LED）從下方往上打光，再從側面收取試管內被激發之螢光。 本研究因更換毛細管材質降低透光度，故選用三種光學感測器進行實驗，分別為color sensor、silicon detector與webcam影像分析。首先進行靈敏度實驗，可得50copies/tube的B型肝炎核酸能成功擴增。再選用三種核酸濃度進行重複性實驗，分別為5×105copies/tube、5×103copies/tube和5×101copies/tube，螢光擴增結果可經由曲線擬合公式，繪製出各感測器之標準曲線。作為本機台初始濃度之定量分析。

A new polymerase chain reaction (PCR) machine is developed for discussing the optical sensor selection and analyzing the fluorescence data in this study. The goal is to achieve low cost and high accuracy. In the future, we hope to popularize this machine at clinics for immediate disease detection. General PCR nucleic acid amplification test must be controlled to the three temperature, which are 95℃, 60℃and72℃. This machine combines two technologies that are capillary convective polymerase chain reaction (CCPCR)and Real-time polymerase chain reaction(Real-time PCR). By single temperature control, the capillary will be heated to 95℃. Then, observing the change of fluorescence intensity in a capillary continuously when the reagent in the tube reaches the desired three temperatures by natural convection. Finally, Nucleic acid amplification completed within 30 minutes. At the first, this study replace the reagent container from a glass capillary cost 25NTD to a plastic capillary cost lower than 1NTD. This change greatly reduce the cost of tubes. However, compared to plastic and glass, the transmittance decreased by approximately 40%, so the optical sensors must be improved. This research used a nickel-chromium wire as a heater for CCPCR. The copper block is heated to temperature 95℃ fixedly by the nickel-chromium wire at the bottom of the capillary and the temperature standard deviation is 0.2℃. About the optical mechanism, blue light-emitting diodes (LED) is used to light the capillary from below and collect the fluorescence signal from the side. This study selects three kinds of optical sensors to experiment, namely color sensor, silicon detector and webcam. Sensitivity test with nucleic acid of HBV initial concentration of 50 copies/tube was amplified successfully. Repeatability test with three kinds of concentration of 5×105copies/tube, 5×103copies/tube and 5×101copies/tube was plot fluorescence curves. By curve fitting, we can draw the standard curve of this CCPCR machine to find the initial concentration of virus.