A real-time convective PCR machine in a capillary tube instrumented with a CCD-based fluorometer

Yi-Fan Hsieh; Da-Sheng Lee; Ping-Hei Chen; Shao-Kai Liao; Shiou-Hwei Yeh; Pei-Jer Chen; An-Shik Yang

doi:10.1016/j.snb.2013.04.003

A real-time convective PCR machine in a capillary tube instrumented with a CCD-based fluorometer

Sens Actuators B Chem. 2013 Jul 5:183:434-440. doi: 10.1016/j.snb.2013.04.003. Epub 2013 Apr 11.

Authors

Yi-Fan Hsieh¹, Da-Sheng Lee², Ping-Hei Chen¹, Shao-Kai Liao¹, Shiou-Hwei Yeh³, Pei-Jer Chen⁴, An-Shik Yang²

Affiliations

¹ Department of Mechanical Engineering, National Taiwan University, Taipei 106, Taiwan.
² Department of Energy and Refrigerating Air-Conditioning Engineering, National Taipei University of Technology, Taipei 106, Taiwan.
³ Graduate Institute of Microbiology, National Taiwan University, Taipei 106, Taiwan.
⁴ Graduate Institute of Clinical Medicine, National Taiwan University, Taipei 106, Taiwan.

Abstract

This research reports the design, analysis, integration, and test of a prototype of a real-time convective polymerase chain reaction (RT-cPCR) machine that uses a color charged coupled device (CCD) for detecting the emission of fluorescence intensity from an RT-cPCR mix in a microliter volume glass capillary. Because of its simple mechanism, DNA amplification involves employing the cPCR technique with no need for thermocycling control. The flow pattern and temperature distribution can greatly affect the cPCR process in the capillary tube, a computational fluid dynamics (CFD) simulation was conducted in this study for the first time to estimate the required period of an RT-cPCR cycle. This study also tested the PCR mix containing hepatitis B virus (HBV) plasmid samples by using SYBR Green I fluorescence labeling dye to assess the prototype performance. The measured results from the image-processing scheme indicate that the RT-cPCR prototype with a CCD-based fluorometer can achieve similar DNA quantification reproducibility compared to commercial machines, even when the initial DNA concentration in the test PCR mix is reduced to 10 copies/μL.

Keywords: CCD; CFD.; RT-cPCR; cPCR.