| Nucleic acid quantitative technology,the basis of precision medicine,is an important means of disease diagnosis.DDPCR(digital PCR),as one of the nucleic acid quantitative detection methods,has ultra-sensitive,absolute quantitative characteristics and a large dynamic range.Compared with q PCR(Quantitative Real-time PCR),it has the advantages of no need to rely on external standards,absolute quantification,strong reproducibility,and higher tolerance to inhibitors.It is widely used in various fields of biomedicine.ddPCR generates emulsion droplets from DNA and fluorescent substances,and then uses each droplet as an independent reaction system for amplification.The nucleic acid content is judged by detecting whether each droplet has a fluorescent signal.The emulsion droplet chip is a chip used to carry droplets used for PCR,and the emulsion droplet chip scanning imaging analyzer is used to perform fluorescence excitation,photographing,identification,and analysis of the droplets on the chip to determine the quality of droplet generation or Nucleic acid quantification equipment.This article summarizes the research and application of nucleic acid detection by the predecessors,and proposes his own research plan;designed and built a droplet chip imaging analysis device based on image analysis,and conducted in-depth research on image processing algorithms,and designed and optimized the emulsion The droplet chip scans the upper computer software of the imaging analyzer and evaluates its functions.Finally,the device is applied to the detection of EGFR T790 M mutation.The design of the device can be divided into hardware design and software design.The hardware part uses STM32 as the main control chip,and indirectly controls each module through it’s driver.From the mechanical structure,the hardware can be divided into a fluorescence excitation module,a fluorescence imaging module,a camera movement control module,a filter conversion module,a dark box,and a supporting structure.In this paper,HEX and FAM dyes are selected as fluorescent markers,and a fluorescent excitation light path that can emit uniform light beams and an imaging light path that can transmit fluorescent images to the camera at equal magnification are designed.The upper computer software has the functions of lower computer communication,image processing,data analysis and storage,and the upper computer program is written by Lab VIEW.The upper computer communicates with the lower computer through the serial port,and the instructions are placed in the drop-down list,which facilitates the identification of the instructions and avoids sending errors.Image processing is performed based on morphology,the Hough gradient method is used to identify droplets in the image,and the misidentified droplets are screened out according to the characteristics of the fluorescent droplet image.Manually thresholding,K-means clustering,and grayscale histogram are used to classify the fluorescent droplets.Due to the instability of the latter two methods,the manual threshold setting is chosen as the method of droplet clustering in this paper.The molecular copy number was calculated according to the principle of Poisson distribution.Finally,the device was used to detect the droplets generated by the droplet generator of Changchun Jite Biotechnology Co.,Ltd.In the bright field,the final recognition rate of droplets with a radius of 7-15 pixels reaches 99.89%,and the recognition accuracy rate reaches 99.96%.The device is used to detect mutations in the T790 M gene,and the Bio Rad instrument is used as a control to verify the feasibility of the procedure and the practicability of the device. |
