An Integrated Nucleic Acid Detection Method Based On Microfluidic Chip For Collection And Culture Of Rice False Smut Spores

Rice false smut spores,which are airborne fungal spores that infect rice ears and cause U.virens,not only cause severe yield loss and grain quality reduction,but also produce toxins that are harmful to humans and animals.Traditional nucleic acid detection methods require manual operations such as spore collection,DNA extraction,and partition amplification,which require high professional skills.Microfluidic chip has the characteristics of miniaturization,integration,etc.,which can realize the integrated detection of pathogenic bacteria.Yet there is a lack of portable methods that integrate the collection of airborne fungal spores and nucleic acid detection.Because rice false smut spores have thick cell walls and require liquid nitrogen grinding to extract DNA,breaking the walls on-chip is difficult.Therefore,the realization of spore wall breaking on-chip is a major difficulty and also a very meaningful study.Based on microfluidic chip,this study takes the chlamydospores of the rice false smut as the research object,provides a novel method of culturing spores on-chip,which realizes the integrated detection.The specific research contents include:（1）The microfluidic chip is designed according to the gas dynamics of airborne spores at the micro scale,the principle of double sheath flow focusing and inertial separation.COMSOL simulation was used to optimize the rate of the double sheath flow and verify the chip purification and collection effect.（2）The rice false smut spore culture on-chip experiment is studied and the results are analyzed,the optimal spore culture on-chip scheme is proposed.The temperature control module is built and its temperature stability is tested.At the same time,the module was used to test the effect of spore culture on-chip.（3）According to the micro mixing mechanism,the micromixer is designed,and the structure of the micromixer is optimized by COMSOL simulation.Based on the microvalve structure,the airborne spore collection chip and the micromixer are combined to design and fabricate an integrated microfluidic chip.（4）RPA primers are designed manually,and their specificity and sensitivity are tested.RT-PCR and RPA-LFD are used to determine the lower limit of detection time,and an integrated experimental method based on microfluidic chip is proposed.The specificity and sensitivity of the integrated experimental method are analyzed,and rice ear samples are randomly used to evaluate the detection effect of the method.When the flow rate of the sample inlet is 12.5 ml/min and the speeds of double sheath flow 1 and 2 are 20 ml/min and 55 ml/min,respectively,the spore concentration rate is as high as 98%,which is highly linear related to the number of spores collected by the spore trap（R²=0.94）.The fluctuation range of temperature control module is less than 0.2℃.The germination effect of spores is best when cultured on-chip under the condition of 2%sucrose and 28℃.The mixing efficiency reaches 0.95 when the square wave width P and the channel width W₂ are 1600μm and 600μm,respectively.The detection time limit of the integrated method is 12 hours,the detection sensitivity is 1×10² CFU/ml～1×10⁵ CFU/ml,and the detection effect of this method is highly consistent with that of the RT-PCR kit,its detection accuracy is 96.36%.Therefore,this method can be fully used for the integrated detection of rice false smut spores.