Development of a CCD-based fluorimeter for real-time PCR machine

Abstract A monochromic charged coupled device (CCD) is employed to obtain time evolved fluorescence images that are reflected from PCR mix in a 20 μL capillary during thermal cycling in the real-time polymerase chain reaction (real-time PCR). This fluorescence detection system consists of a 470 nm light emitting diode (LED) as an excitation light source, a CCD camera, and a rotating disk with six optical filters to provide six fluorescence detection channels at 530, 560, 610, 640, 670 and 710 nm for multiplex real-time PCR DNA quantification. In this study, the PCR mix that contains HBV SC 11 DNA template with SYBR Green I fluorescence labeling dye is tested to verify the prototype performance. An adaptive image processing scheme based on a lower–upper–middle (LUM) filter algorithm is developed to eliminate noise while keeping the necessary information from recorded fluorescence images. The measured results obtained by the proposed image processing scheme illustrate that the real-time PCR prototype with CCD-based fluorimeter can achieve similar DNA quantification reproducibility as the commercial machine even when the initial DNA copies, X , in the tested PCR mix is down to 10 3 copies/mL (0.01 fM).