Study On Low-cost Centrifugal Droplet Generation Method And Its Digital Loop-Mediated Isothermal Amplification Application

The droplet digital nucleic acid detection technology plays a huge role in the fields of biological detection,chemical sensing,medical diagnosis,etc.The technology distributes the target nucleic acid into tens of millions of droplets for amplification reaction,and each droplet contains at most one target.molecular.The droplet containing the target emits a fluorescent signal,and the quantity conforms to the Poisson distribution.Through statistics,the absolute quantitative detection of the target nucleic acid is realized.Micro-droplets have become the first choice for commercial digital nucleic acid detection instruments due to their high throughput and high heat transfer efficiency.However,the existing droplet generation methods mostly rely on microfluidic chips and sophisticated syringe pump devices.The complicated preparation process makes the micro-droplet technology have certain research barriers.Based on this background,this article explores a low-cost microtube-based centrifugal droplet generation method,combined with loop-mediated isothermal amplification technology to detect Staphylococcus aureus nucleic acid and coronavirus nucleic acid.The main content and research results of this paper are as follows:(1)A centrifugal droplet generation method and device based on microtubes was proposed and built.The device was manufactured by relying on conventional consumables and microtubes.By characterizing the physical and chemical properties of the droplets,the preparation conditions were optimized and the preparation was successful.A water-in-oil droplet with uniform size,good dispersion,good thermal stability,and high flux is produced.The generation method and device avoid complex processing techniques(such as photolithography,etching)and precise syringe pump requirements,and provide a solid foundation for subsequent digital detection of staphylococcus aureus nucleic acid and coronavirus nucleic acid.(2)A digital nucleic acid detection system based on centrifugal micro-droplets was constructed,and its detection performance was evaluated using Staphylococcus aureus nucleic acid plasmids.The lowest detection limit can reach the order of fM,within the concentration range of 0.1 fM?0.1 pM has a good detection effect.(3)Based on the construction of a digital nucleic acid detection system based on centrifugal micro-droplets to detect COVID-19 nucleic acid standards,the method in this article reduces the detection limit by an order of magnitude compared with the traditional fluorescence quantitative amplification method,which is an early stage Disease detection provides a program.