Research On Instant Detection Of Pathogen Nucleic Acid Based On Microfluidic Chip And Smartphone

It is of great significance to develop rapid and efficient pathogen detection technology in response to public health emergencies.Currently,nucleic acid detection is the mainstream method for pathogen detection,while traditional nucleic acid detection technology requires the use of large and complex instruments,making it unable to be applied to areas with limited infrastructure.With the development of microfluidic chip technology and smart phone technology,mobile sensing based on the integration of microfluidic chip and smartphone(MS~2)is applied to nucleic acid detection systems,which is an effective way to solve the problem.However,the current nucleic acid detection system based on MS~2 technology still has shortcomings,such as the lack of nucleic acid extraction function,single detection method and poor applicability,which limits their application in areas with limited infrastructure.Therefore,this paper designed a portable nucleic acid analysis system based on MS~2 technology.The volume of this system is 32.5×20×20 cm~3,and it integrates the microfluidic control module,temperature control module and fluorescence acquisition module.The microfluidic control module combined with a nucleic acid extraction chip can realize nucleic acid extraction by magnetic beads on the chip.The high precision temperature control module can realize isothermal temperature control and PCR temperature control.The fluorescence acquisition module uses six sets of filters to detect fluorescence in six different excitation bands.Smartphones control each module of the system through a self-made application(App),which can extract,amplify and detect nucleic acids on microfluidic chips.By combining different amplification chips,the system can realize qualitative detection of isothermal amplification detection,as well as quantitative detection of real-time fluorescence PCR(qPCR)detection and multiplex PCR(multiplex PCR)detection.In addition,smartphones can collect fluorescent images,analyze experimental data and share experimental results through the App.In order to verify the performance of the system,the human genome DNA was extracted by the system,which proved that the system has the capability of nucleic acid extraction similar to commercial instruments.qPCR was performed on the system using plasmid DNA samples from six influenza viruses to verify the nucleic acid detection capability of the six fluorescence channels on the system.The detection sensitivity of the FAM channel reached 10~1copies/?L through gradient detection experiments on the FAM channel.The applicability of the system was verified by isothermal amplification detection experiment and multiplex PCR experiment.Finally,the actual samples of African swine fever virus(ASFV)were successfully extracted and detected by qPCR using the system,which further proves that the system has the capability of nucleic acid analysis and can be applied to the real-time detection of pathogen nucleic acid.