Detection And Processing Of Fluorescence Signal In Droplet Digital PCR

Digital PCR detection technology(Digital PCR,d PCR)which can directly detect the copy number of DNA target sequence,is the most advanced PCR detection technology based on PCR technology and microfluidics technology.Compared with the traditional fluorescence Quantitative PCR detection technology,digital PCR detection technology has more excellent sensitivity,specificity and accuracy,especial in complex background and the expression of small differential identification.However,in the field of current digital PCR detection technology,there are only 4digital PCR instrument manufacturers in the world and all are foreign enterprises;For the purpose of reducing the cost of procurement,breaking the monopoly of foreign enterprises and improving the level of medical services,It is necessary to carry out the research of digital PCR technology.The detection and the processing of the micro-drop fluorescence signal in the digital PCR instrument in this thesis were studied.The main contents include:micro-droplet generation method and detection principle of fluorescence signal,micro-droplet fluorescence signal acquisition system hardware design and analysis,micro-droplet fluorescence signal peak detection method,micro-droplet fluorescence signal peak compensation algorithm.The main contents of this thesis are as follows:(1)First the flow of micro-droplet fluorescence signal detection is introduced.Then described the micro-droplet generating,advantages and disadvantages of the three methods.Finally,the working principle of optical signal detection system of micro-droplet was analyzed.(2)About micro-drop fluorescence signal acquisition system hardware design and analysis of the work carried out was: based on FPGA and USB2.0 data transmission technology,I has completed the design and analysis of hardware core circuit in micro-droplet fluorescence signal data acquisition system.(3)Based on the built-in micro-drop fluorescence signal detection platform,the micro-drop fluorescence digital signal peak detection research was studied.The mainresearch contents include: interception of effective data segment of fluorescence signal of micro-droplet,baseline calibration of fluorescence signal of micro-droplet,filtering of fluorescence signal of micro-droplet and effective peak extraction of fluorescent signal of micro-droplet.(4)To study the compensation algorithm of the peak data of micro-droplet fluorescence digital signal,I mainly compared two methods: general least-squares method and recursive least square method.To improve the detection accuracy of the digital PCR instrument,the peak data needs to be compensated for fluorescence.Owing to the cross interference of the fluorescence wavelength when fluorescent dyes are excited by fluorescence,the obtained peak data is skewed,so In order to improve the detection accuracy of the digital PCR instrument,it is necessary to perform fluorescence compensation on the peak data.