A Rapid Nucleic Acid Detection Method Based On Microfluidic Technology

Since the discovery of the 2019-n Co V at the end of 2019,it is still spreading wildly in many countries.COVID-19 caused by it has had a very serious impact on people's lives.Since the virus is easy to spread and the symptoms are not obvious at the beginning of the illness,it is very likely that the virus carrier will cause the virus to spread further without knowing it.Therefore,it is necessary to expand the scope of screening for infected persons.Existing nucleic acid detection methods are complicated in operation and relatively time-consuming,which puts a certain degree of pressure on large-scale detection,and therefore requires a faster detection method.In order to improve the detection speed,this paper combines nucleic acid detection and microfluidic technology to design the detection method from both qualitative and quantitative perspectivesIn terms of qualitative detection,in order to achieve the goal of point-of-care testing,a microfluidic nucleic acid precipitation blocking qualitative detection method was designed.Explore the blocking mechanism,detection mechanism and experimental plan,design the microfluidic chip channel,build a microfiltration porous structure inside the channel,and block the porous structure by using the characteristics of ethanol precipitation of nucleic acids.The positive performance is blocked,and the negative performance is passed.Interpret the results qualitatively.After determining the feasibility of the scheme,set the judgment result time to 10 minutes,and conduct a large number of repeated experiments to verify the accuracy of the detection method.In addition to the macroscopic visual observation,the microscope and the high-speed camera are used to simultaneously monitor and shoot in the microscopic state,measure and count the size range of the flocculent precipitate during the experiment,and provide reference and comparison for the subsequent use of this method to detect other viruses.In terms of quantitative detection,for the droplet digital PCR technology,the existing detection and recognition steps are designed and improved,so that the detection speed of the detection section is accelerated,thereby effectively reducing the time required for the overall nucleic acid detection process.Firstly,according to the droplet generation mechanism,the droplet generation process of different channels was analyzed and compared through the experiment methods,and a large number of droplets were prepared by selecting suitable microchannels.Secondly,the experimental data was processed and the influence of the two-phase flow rate on the droplet generation size and frequency was obtained from its trend.Finally,the droplet image imported into the wide channel was processed,the number and area of droplet were counted,and the coefficient of variation was calculated to be 6.19%,which ensured the stable generation of droplet with good homogeneity and was suitable for the subsequent test section.The final task is to identify the fluorescent signal of the nucleic acid amplified droplets in the detection section channel.The microfluidic chip designed for rapid detection enables it to hold hundreds of liquid droplets for simultaneous detection,thereby increasing the detection speed.Laser-induced fluorescence technology is used to induce fluorescence in droplets containing the nucleic acid amplification reaction system or alternative droplets containing fluorescent particles.The fluorescent droplets in the image are identified by machine learning methods,and the classifier is trained to distinguish between fluorescent droplets and the background area.After the fluorescent droplets are identified,the automatic particle counting analysis method is used to count.According to the Poisson distribution principle and the ratio of the fluorescent droplets to the total droplets,the initial concentration of the virus in the sample is obtained to achieve the purpose of quantitative detection.