Design And Optimization Of Large Field Of View Rapid Nucleic Acid Concentration Detection System For Digital PCR Chip

Digital polymerase chain reaction(digital polymerase chain reaction,d PCR)is an absolute quantitative nucleic acid detection technology,which plays an important role in the recent nucleic acid detection of Corona Virus Disease 2019(COVID-19).The main steps of using d PCR chip to detect COVID-19 virus include: nucleic acid extraction,reagent mixing,reagent sampling,PCR amplification,and fluorescence image collection and analysis for d PCR chip.In the field of d PCR chip fluorescence image acquisition,the detection of deoxyribonucleic acid(DNA),which can simultaneously achieve highresolution and large-field imaging,has always been one of the main research directions in the field of detection.Achieve rapid acquisition,high resolution and large field of view one-time imaging,which can improve nucleic acid detection efficiency and detection accuracy.The key to this problem is to design a fluorescence microscope with a large field of view that can meet the size and resolution requirements of the d PCR chip.In order to solve this problem,fill the gap in the detection field,and improve the efficiency and accuracy of nucleic acid detection,this paper independently designed a large field of view,high-throughput rapid nucleic acid concentration detection system for d PCR chips.The system is mainly composed of a large field of view CMOS camera,a self-designed large field of view fluorescent compact objective lens,a compound eye illumination system,a filter module,an ultra-thin dichroic mirror and an excitation light source.The system can complete one-time imaging of the four channels(FAM,HEX,ROX and Cy5)of the d PCR chip,and the single-channel imaging and analysis time is less than 10 s.This work has achieved a higher level of nucleic acid detection technology applied to d PCR gene chips in medical technology,and has the advantages of simple production,high practicability,complete functions,high cost efficiency,high detection accuracy and simple operation,and has a wide range of applications.prospect.The main work of the thesis is as follows:1.A lighting system that meets the excitation wavelength of the fluorescent dyes in the four channels of the d PCR chip is designed.Compared with traditional d PCR excitation light sources such as mercury lamps,xenon lamps,LED lamps and halogen lamps,the lighting system uses a combination of a white laser light source and a blue LED array light source as the excitation light source.The use of the light source not only greatly reduces the cost of the equipment,and reduces the volume of the equipment,but also enables the equipment to meet the requirements of the excitation wavelength of the four-channel fluorescent dye at the same time.There is no need to purchase an expensive variable wavelength laser light source,and individually design a wavelength-compliant LED array for each channel to provide excitation light for each channel.In addition,compared with the high-pressure mercury lamp light source,the white laser light source has a higher excitation efficiency,and can fully excite the fluorescent dyes in the d PCR chip in a short time.In the process of fluorescence image collection,the camera’s exposure time is shortened from 10 s to 100 ms,which provides the possibility for faster nucleic acid detection.2.Optimization of the entire set of large field of view high-throughput fluorescence microscopy imaging system.In order to make the fluorescent dye in the test agent in the d PCR chip be uniformly and fully excited,thereby further improving the detection efficiency of the system,this paper proposes to use a double-row compound eye illumination system as the excitation light path of the system excitation light source,and design it through the simulation software Light Tools The lighting module,experiments show that the lighting area of the system can reach 33mm×22mm,enough to cover a28mm×18mm d PCR chip,and by calculating the uniformity of illumination can reach more than 90%,it can be well uniform and fully excited on the d PCR chip Fluorescent fuel in each droplet.At the same time,in order to reduce the impact of the filter system in the non-parallel optical path,a 0.21 mm ultra-thin dichroic mirror was customized.Through experimental verification,a 0.21 mm ultra-thin dichroic mirror was used.The actual resolution of the system It can reach 6.96μm.3.In order to complete the rapid and accurate analysis of the collected fluorescence images of the d PCR chip,this paper proposes an image analysis algorithm that applies image processing and deep learning.Analyze and solve the problems that may occur in the analysis process one by one,and verify the accuracy of the algorithm analysis through the d PCR chip containing different concentrations of reagents.4.In order to verify the detection performance of the system,this article selects EGFR gene L858 R,19del mutant lung cancer virus and simulated COVID-19 virus as samples for detection.Mutant lung cancer This article is based on the detection results of ROX channel and simulated COVID-19 virus.This article is based on the detection results of HEX channel.The concentration of the initial solution of the selected DNA template is 1.0×105 copies/μL,and it is diluted down by a 10-fold gradient.The d PCR fluorescence image collection result of this system is compared with the result of a commercial d PCR detector(Bio Digital·Green).The result shows that the system can quickly complete the detection of different concentrations of reagents in the d PCR chip without splicing,and the detection is accurate.The rate can meet the requirements of the second-class medical device license,and the correlation coefficient between the detected concentration and the expected concentration can reach 99%.In this paper,through the analysis of the size and microstructure of the d PCR chip,the simulation design and experiment of the large field of view high-throughput microscope objective and the processing of the d PCR fluorescence image,the system can complete the detection of a d PCR chip within 10 s.The detection efficiency is improved,and the detection time is effectively shortened,which has important reference significance for the detection field of the application of d PCR chips.