A novel real-time PCR machine with a miniature spectrometer for fluorescence sensing in a micro liter volume glass capillary

Abstract

This study sets up a prototype of real-time polymerase chain reaction (RT-PCR) machine that employs a miniature spectrometer for detecting the emission of fluorescence intensity from RT-PCR mix in a micro liter volume glass capillary. The RT-PCR machine is one of the major instruments for SARS virus test during the outbreak in Asia in early 2003. Comparing with traditional RT-PCR machine with discrete channels fluorescence wavelength detection, the prototype can provide continuous wavelength detection and can be employed for multiplex DNA quantification. However, only one HBV SC 11 DNA template with the SYBR Green I labeling dye were used in this study to compare DNA quantification accuracy and reproducibility of the present system and the commercial system. The two machines have different optical engine arrangement and so two separate analytical models were proposed to predict the fluorescence intensity from the RT-PCR mix during thermal cycling for the machines. Predicted results agree well with the measured data for both machines. From the predicted results, different approaches should be adopted to determine the initial DNA concentration for quantification from time recorded history of the fluorescence intensity. Measured results illustrate that the RT-PCR prototype has the same accuracy for DNA quantification and reproducibility within five intra-assay samples as compared with the commercial machine.