Research On The Key Technology Of Fully Automatic Fluorescent Quantitative PCR And Its System Development

Fluorescence quantitative PCR is the highest-level test item for molecular diagnostics,which is widely used in emergency,heredity,genetic screening,cancer and other fields.At present,there are some shortcomings such as manual sample addition,long sample preparation time,and slow temperature drop of the instrument in real-time fluorescence quantitative PCR detection.Furthermore,compared with the foreign PCR instruments,domestic PCR instruments exhibit poor performances with lower market share.Therefore,this project proposes a new idea for the nucleic acid amplification cycle based on the magnetic-power switching constant temperature zone for the extraction-free reagents,builds an integrated hardware test platform for automatic switching of constant-temperature zone heat transfer and weak fluorescence detection,and to verifies and tests the system by fluorescence quantitative PCR reagent.This has important research significance for improving the detection cycle and the performance of domestic instruments,and breaking the monopoly of foreign high-end PCR detection technology.This article analyzed the mathematical quantitative method of fluorescent quantitative PCR instrument,the principle of chemical fluorescent probe method,chemical fluorescent dye method,and studied the key system components such as the temperature control system and optical system of the fluorescent quantitative PCR instrument.In addition,this paper discussed the advantages and disadvantages of PCR process temperature requirement,temperature control and fluorescence detection.In this paper,a temperature control system based on magnetomotive switching constant temperature area is put forward to achieve high precision constant temperature control with a temperature control accuracy of ±0.13°C.We conducted heat transfer analysis on the thermodynamic principle of the reaction liquid and use the COMSOL Multiphysics software to simulate the heat transfer model of the reaction liquid to get the actual temperature information of the reaction liquid.By using 515 nm laser excitation and photodiode to detect the peak wavelength of 580 nm,we set up a fluorescence confocal optical detection system,which integrates the excitation and detection of the optical path into the detection point.This project carried out the design of the mechanical platform by using SOLIDWORKS,also built the hardware system of high precision temperature control,motion control platform and fluorescent confocal optical detection.Using expert PID algorithm and state machine algorithm of high precision constant temperature and accurate positioning of motion control,the weak fluorescence signal to realize continuous dynamic acquisition of eight channels,and the use of Visual Studio prepared man-machine interface.After the completion of the platform construction,we finished the test for temperature control,motion control,optical system and used the clinical PCR reagent for the whole machine test,which can detect the positive quality control product and get the real-time fluorescence standard S curve.The high precision constant temperature control up to 0.13?,the optical linear fitting is equal to 0.992,and the precision is less than 2%.