新型複式即時定量聚合酶連鎖反應檢測機台開發與研究

Development of a Novel Multiplexing Real-Time PCR Machine for DNA Quantification

吳孟勳(Meng-Hsun Wu)

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(2004年)

https://doi.org/10.6342/NTU.2004.00963

摘要

本論文主要是設計一種新式的複式即時定量聚合酶連鎖反應檢測儀器，儀器內獨特設計之光學檢測系統是以全波段氙氣燈為激發光源，以高感度光譜儀為螢光擷取系統，搭配準確控制的溫控系統，完成複式即時定量聚合酶連鎖反應(Multiplexing Real-Time PCR)。相較於一般市售的Real-Time PCR機器使用離散式螢光檢測，本論文中設計之螢光擷取並使用連續式波段螢光檢測系統，可同時檢測5個通道以上的樣本。實驗結果顯示，在進行複式即時定量聚合酶連鎖反應時可同時檢測到試劑中多種不同螢光，且以本論文設計之系統實驗結果將與商用機型的實驗結果比較，有較好之DNA增生結果。本系統之PCR實驗結果由標準曲線圖（Standard Curve）可以看出本系統在PCR實驗時，隨著不同的起始濃度在PCR增生後所得到之Cp值呈現線性的關係，因此在不同的起始濃度下PCR都有很穩定的增生效率，當起始濃度每增加10倍時，PCR反應之Cp值減少約3.9，經過計算本系統PCR反應時每次的溫度循環增生效率約為1.8倍。本系統之PCR實驗結果由變異係數（CV值）的比較可以看出本系統在PCR實驗中有良好的重現性（Reproducibility），論文中將與商用機型詳細比較。

關鍵字

全波段氙氣燈 ； 高感度光譜儀 ； 連續式波段螢光檢測系統 ； 連鎖反應 ； 複式即時定量聚合酶

並列摘要

This study sets up a prototype of Multiplexing Real-Time polymerase chain reaction (PCR) machine that employs a xenon light source for exciting fluorescence and a miniature spectrometer for detecting the emission fluorescence intensity from Real-Time PCR mix in a ml volume glass capillary. Compared with the traditional Real-Time PCR machine with discrete channels fluorescence wavelength detection, the prototype can provide continuous wavelength detection and can be employed for multiplex DNA quantification. To compare the DNA quantification accuracy and reproducibility of the present system with those of the commercial system, we use a HBV DNA template with LC-Red 640 dye and an Internal Control template with LC-Red 705 dye in this study. The results of this experiment reveal that the Real-Time PCR prototype yields more amplification quantity than the commercial system. As the initial copies of template DNA increase every ten times, Cp decreases by a value of 3.9. Therefore, the number of template DNA increases by 1.8 times each thermal cycle. The results also reveal that this prototype has the same accuracy for DNA quantification and the reproducibility within five intra assay samples as the commercial system.

並列關鍵字

spectrometer ； xenon light source ； continuous wavelength fluorescence detection chann ； Multiplexing Real-Time polymerase chain reaction (

參考文獻

Belgrader, P., Young, S., Yuan, B., Primeau, M., Christel, L. A., Pourahmadi, F., and Northrup, M. A., 2001, “A battery-powered notebook thermal cycler for rapid multiplex real-time PCR analysis,” Anal. Chem., Vol. 73, pp. 286-289.

Blömeke, B., Sieben, S., Spötter, D., Landt, O., Merk, H., F., 1999, “Identification of N-Acetyltransferase 2 genotypes by continuous monitoring of fluorogenic hybridization probes,” Analytical Biochemistry, Vol. 275, pp. 93–97.

Frank, M. S., Edward, S. F., 1997, “Absorption spectrum (340 -640 nm) of pure water. I. Photothermal measurements,” Applied Optics, Vol. 36, Issue 33, pp. 8699-8709.

Khandurina, J., McKnight, T. E., Jacobson, S. C., Waters, L. C., Foote, R. S., and Ramsey, J. M., 2000, “Integrated system for rapid PCR-based DNA analysis in microfluidic devices,” Anal. Chem., Vol. 72, pp. 2995-3000.

Lee, D.-S., Wu, M.-H., Ramesh, U., Lin, C.-W., Lee, T.-M., Chen, P.-H., 2004, “A novel real time PCR machine with a miniature spectrometer for fluorescence sensing in a micro liter volume glass capillary,” Sensors and Actuators B: Chemical, Vol. 100, Issue 3, pp. 409-418.