Research On Optical System And Fluorescence Detection Method Of Quantitative PCR

Polymerase chain reaction,also known as PCR technology,is mainly used for in vitro amplification of nucleic acids to achieve the detection of designated genes.This article mainly carried out the following tasks.One is to design and build the optical path structure and detection device for detection based on the principle of real-time quantitative fluorescent PCR,and to select and test the optical components to realize the weak light detection of signal fluorescence;It is to write the program for the image processing requirements required in the fluorescence detection process and test it in the experiment;the third is to design the hardware structure of the entire instrument according to the software and hardware requirements of the entire detection system,and build it in SolidWorks Mold,and process the prototype for testing.In terms of optical path structure,this research design adopts a confocal detection structure,and CMOS is used as an image sensor for image acquisition.At the same time,this research uses a new type of flat panel OLED as the fluorescent excitation light source based on the common confocal detection structure,which can reduce the volume of the light source and overcome some of the defects of the traditional light source,and cooperate with the design of the rectangular paraboloid in non-geometric optics.The reflector cup realizes the shaping and uniform distribution of light from the light source.In addition,a field lens is designed in the optical path to improve the collection of weak fluorescent signals,improve the accuracy of detection,and verify the effect of the device through simulation and experiments.As the CMOS industrial camera is used as the image acquisition device,it is necessary to use image processing technology to extract and detect the fluorescence signal in the processing of the fluorescence signal.This study uses the OpenCV open source library to write fluorescence image processing algorithms under the C++ framework,and finally realizes the detection of fluorescence signals and stores them in the corresponding matrix.In terms of the structure of the whole machine,it is designed in accordance with the requirements of miniaturization and integration as much as possible in the design of the entire optical structure and the use of the instrument,and a physical prototype is built for testing and experimentation to ensure the reliability and accuracy of the entire device.In the verification experiment,different concentrations of fluorescent dyes were used to replace the DNA amplification process in the cyclic reaction process to verify the fluorescence excitation effect of the detection system and the collection of fluorescence signals,and to judge the fluorescence threshold that can be detected by the CMOS sensor.At the same time,the fluorescence intensity excited by different concentrations is used to establish a corresponding relationship with the corresponding image gray value during the image processing process,so as to better output the fluorescence change curve during the reaction process.