Research On Thermal-Cvcler And Fluorescence Detection Method Of Quantitative PCR

Polymerase chain reaction (PCR) is a technique used for rapid amplification of DNA in vitro. Quantitative PCR technology enables the accurate quantified calculation of DNAs in the reactant. High performance of thermal-cycler and fluorescence detection system is the basis of correct DNA amplification and quantitative. This dissertation carries on thermal-cycler and fluorescence detection method of a quantitative PCR system.The thermal-cycler is a instrument which can implement the polymerase chain reaction, a PCR thermal-cycler was designed based on thermoelectric cooling technology and embedded technology. In order to get the accurate parameters of components in the thermal-cycler, the equivalent circuit model of thermal-cycler based on thermoelectric coolers was established according to the thermal-electric analogies and was applied to study the static and dynamic thermal performance of thermal-cycler in one-dimension. The results show that the method can accurately evaluate the thermal performance of PCR thermal-cycler in one-dimension and determine the parameters of components in the thermal-cycler. The system temperature can achieve excellent performances both in static and dynamic state.PCR require precise reaction temperature. The temperature control method of the thermal-cycler system was studied in order to improve the control accuracy.fo the system temperature. According to the temperature characteristics of the thermal-cycler, a multi-modal segmented control strategy was studied. A fuzzy control method was used in the early stages of heating and cooling to get the fastest heating and cooling rate. An adaptive fuzzy-PID control method was used in the later period of heating and cooling to improve the static temperature precision. Experiment results show that the maxim heating rate of the system is greater than℃/s and the maxim cooling rate is greater than2℃/s, the static accuracy can be controlled at less than0.2℃. In order to ensure sufficient reaction times for the PCR samples, the relationship between hold-time and temperature was experimental studied in heating and cooling modeThe system temperature uniformity is a key factor to ensure the DNAs in various tubes can be amplified with equal efficiency. The factors affecting the system temperature uniformity were stuied by using finite element analysis method. The heat exchange between the surface of sample block and surrounding environment, and the matching error of thermoelectric cools are the main reasons for the poor temperature uniformity of the thermal-cycler. Experiment results show that the system temperature uniformity can be controlled in0.4℃by matching the thermoelectric cools with an error less than0.5%and implementing appropriate thermal insulation or compensation. The standard PCR samples were efficiently amplified in the thermal-cycler.The DNAs in the samples are quantified by detecting the fluorescent intensity of fluorophores in each sample. A novel confocal fluorescence detection system of quantitative PCR was designed according to the principle of fluorescence analysis and the fluorescent spectra characteristics of fluorophores in the quantitative PCR process. Fluorescent signals with different wavelength can be detected by using a photomultiplier tube and different narrowband filters. Noise sources, denoise performance and the fluorescence signal multiplier effect of this fluorescence detection system were tested and analyzed. The peaks of the fluorescence signals of each sample were calculated on computer by segment-selecting and digital denoising the data acquired from the fluorescence detection system. Experiment results show that the system can detect the fluorescence signal (center wavelength of610nm) of rhodamine B solution with a lowest concentration of0.005ug/ul and the fluorescence signal (center wavelength of520nm) of SYBR Green I added to the cucumber genomic DNA solution with a lowest concentration of0.78ng/ul. The calculated peaks have linear relationship with the concentration and the system can detect the fluorescent intensity in the Exponential phase of quantitative PCR.