An Integrated Nucleic Acid Detection Method For Purification And Lysis Of Rice False Smut Spores Based On The Microfluidic Chip

Rice false smut(RFS)is one of the main fungal diseases in rice.The occurrence of rice smut not only affects the yield and quality of rice but also produces various toxins,posing a threat to human and animal health.The occurrence and prevalence of rice false smut are closely related to the bacterial source of rice false smut in the air,so real-time detection of rice false smut spores is particularly important.At present,common detection methods include liquid chromatography,flow cytometry,the microscope image recognition method,the biosensor method,the molecular biology detection method,etc.,but the accuracy and speed of these methods cannot be guaranteed at the same time.And there are several disadvantages to the detection of rice false smut using airborne spores as the medium:(1)There are many types of microorganisms in the air,including bacteria,fungal spores,PM2.5,aerosols,etc.It is difficult to directly separate the spores of rice false smut.(2)The pretreatment of the collected spore samples takes a lot of time,and the experimental operation is complicated.(3)The detection device is bulky and difficult to use to meet the needs of real-time detection of rice false smut spores.Therefore,this subject designed a microfluidic chip-based nucleic acid detection method that integrates the separation and enrichment,lysis,and nucleic acid amplification of rice false smut spores.The main research content of the thesis is as follows:1.Analyze the flow rule of airborne spores in the microfluidic chip and analyze the influence of secondary flow on spore purification.Analyze the effects of temperature and time on the efficiency of spore lysis based on the principle of spore lysis.Analyze the impact of different primers on the amplification effect based on the principles of loop-mediated isothermal amplification(LAMP)and SYBR Green Ⅰ detection.The purification structure design method of the chip was compared and analyzed,and the fabrication scheme of the microfluidic chip was designed.2.The purification structure of the microfluidic chip was designed,and the COMSOL Multiphysics simulation software was used to analyze and select the best purification structure of the chip.The microfluidic chip purification structure was used to purify the spores,which reduced the impurity interference in subsequent experiments on the detection of rice false smut spores.3.Design a plan for the spore lysis experiment based on the principle of spore lysis and conduct data analysis on the results.Test the nucleic acid release effect of the microfluidic chip,and finally couple the purification area of the chip with the nucleic acid release module.Using the benzyl chloride method to lyse spores and simplifying the method of nucleic acid purification,the lysis rate of spores could reach 80% within50 minutes,meeting the needs of nucleic acid amplification.4.A recombinant plasmid template for the target gene was prepared,and LAMP primers were designed and screened for sensitivity and specificity testing.Based on the principles of LAMP amplification and SYBR Green Ⅰ fluorescence,the reaction system and conditions for LAMP-SYBR Green Ⅰ were set.Verify the effect of LAMP-SYBR Green Ⅰ visual detection in the chip and study the integrated detection methods of purification,nucleic acid release,and amplification in the microfluidic chip.The sensitivity of the screened primers is 100 copies/μL.The specificity of the primers is good.Compared with reagent kit detection,the accuracy of on-chip detection reached 96.67%.The integrated nucleic acid test method of purification and lysis of rice false smut spores based on a microfluidic chip can realize real-time detection of rice false smut.