Research On A Photoelectric Detection System For Real Time PCR

This thesis includes two parts. We design a real-time PCR photoelectric detection system in this paper. On the other hand, we put forward a preparation method which applies to the droplet of digital PCR and complete successfully preparation of the droplets of diameter of 10-100μm, as well as we make the preliminary design of digital PCR photoelectric detection system.The research of photoelectric detection system of Real-time PCR includes four parts which are theoretical calculation and simulation, principle model construction, design optical-electro-mechanical integration and system testing. In the part of theoretical calculation and simulation, we analyze the light source, optical transmission, coupling and the photoelectric conversion efficiency. Thereinto, a high-power LED is used as the light source, so we test its luminous intensity, luminous angle and linearity. The results show that its luminous intensity distribution is in accord with the Lambert law, its light emitting angle is 120° and the characterization of linearity of linear fitting R2 are 0.9866,0.9982,0.9982 when light angle are 0°,30°,60°, respectively. A Planoconvex lens set is used as a coupling element between the light source and the optical fiber with the coupling efficiency is 97.21%. A Y-type quartz optical fiber is adopted to transfer excitation light source and emission fluorescence. The system has advantages of simple structure, low cost, easy to implement. Moreover, the transmission loss is reduced to below 1dB/km. We choose the high sensitivity and low noise photoelectric multiplier tube as the photoelectric detector which enable the detection dynamic range of system is about 105 and the output noise is about 0.38 mV. In the optical-electromechanical integration, we design a fluorescent quantization detecting system based on rotational scan. The system consists of a fluorescence detection device, a light source excitation device, a thermal cycling module device and an optical signal transmission device. In the system, we use a motor to drives a strobe piece to rotate, opening one optical path in turn, to realize multi-channel detection. The optical device is fixed with no need for repositioning, which can shorten the detection time and save space. So the detection system is high precision, high efficient, low-cost operation. In application research, this system can detect the fluorescence signal intensity variety in a HIV-1 quantification reagent during PCR cycles. In summary, we have successfully designed the Real-time PCR photoelectric detection system with simple construction, small volume and suitable detection range, which accomplish the excitation and detection for weak fluorescence signals.In this paper, we use the droplet technique to form the mono-disperse water in oil droplets, which diameter is between 10 to 100μm. These droplets can be used as micro-reactor vessels for a lot of biochemical reaction. The droplets have the advantage of stable thermodynamic and chemical performance in the PCR amplification reaction temperature interval, so they can be used in the PCR for rare disease scenes. By means of putting water into the oil to form the PCR reaction reagent, the method greatly reduces the non-specificity of the PCR reaction. We apply the CCD as the core device to design and construct optical imaging system which resolution can reach 7.5μm to image the droplets array.