Disposable Static Chip Based Real-time Fluorescence Quantiative Polymerase Chain Reaction

Real-time fluorescence quantitative PCR (Real-time FQ-PCR) is a quantitative DNA analysis method that has found wide applications in molecular biology and clinical medicine research as well as in daily diagnosis due to its specificity and sensitivity to target DNA sequences. Desktop systems need to be improved in term of reagent consumption and throughput to meet the increased test demands. In addition, point of care test requires portable systems. Thus miniaturization and high throughput of real-time FQ-PCR become an important issue.In first chapter the status of real-time fluorescence quantitative PCR and PCR chip is reviewed, and real-time FQ-PCR based an ITO thermostat chip was proposed.In second chapter, the reliability of the static ITO thermostat chip was improved by introducing a copper foil on the ITO layer. The temperature control and calibration were studied.In third chapter, a fluorescence detection system for real-time fluorescence detection was established based on an inverted microscope. Blue LED was used as the light source and a PMT for fluorescence detection. Good results were obtained with a 25x objective lens,300 mA constant current drive for LED and 600 V high voltage for the PMT.In the last chapter, real-time FQ-PCR was carried out with the ITO glass chip for the first time as demonstrated by the measurement of aλDNA template using SYBR Green I as the fluorescence probe. High throughput was achieved by parallel array operation, and cross contamination was avoided by one time use of the reaction vials. Linear calibration curve with correlation coefficient of 0.9989 was obtained in the range of 3.8×105～3.8×108 copies with a slope of -4.9 and an intercept of 52.5. The relative standard deviation of the Ct was less than 5%.