Study On Microfluidic Device For Rapid Detection Of Nucleic Acid Based On Isothermal Amplification

For both animals and humans,various virulent infectious diseases are a serious threat to life and health,such as African swine fever and the outbreak of new crown pneumonia(COVID-19)at the end of 2019,which had a significant negative impact on agricultural biosecurity and public biosecurity respectively.In particular,COVID-19 has taken exceptionally large losses on many aspects of social,economic,and public health worldwide.Therefore,the research of various new disease detection techniques,methods and devices to achieve rapid,accurate and efficient detection of diseases is of great practical significance and value in the building of guardian defense line for the maintenance of human and even animal life and health.In addition to PCR-based nucleic acid detection techniques,isothermal amplification-based nucleic acid detection techniques are gaining increasing interest and playing an important role in life sciences,biomedicine,and the inspection and quarantine of animal and plant.Point-of-care testing(POCT)technology is a method of quickly detecting disease by a simple,convenient device in typical or atypical health care facilities,such as in the field,in the community,or even at home With the significant advantages of microfluidic chips in terms of functional integration,and the advantages of isothermal amplification technology in terms of simple reaction conditions and short detection time,the study of automated,integrated nucleic acid detection technology and portable devices based on the isothermal amplification principle is of great significance to further improve the level of rapid nucleic acid detection in POCT.This paper researches on two portable POCT nucleic acid amplification assays and devices.(1)Combining isothermal amplification technology and colloidal gold strip chromatography detection technology,this paper studied on a handheld isothermal amplification assay method and device,to realize the integrated operation of isothermal amplification and colloidal gold strip detection.In particular,the innovative,integrated and integrated design effectively avoids the risk of aerosol contamination during the process of detection.With simple thermostatic heating and strip color development,the highly sensitive nucleic acid amplification and visualization can be achieved in about 25 minutes by simple manual operation with minimal dependence on external equipment.(2)A nested isothermal amplification assay and device based on recombinant polymerase reaction(RPA)technology has been developed to further enhance the key performance of isothermal amplification assays.The isothermal amplification device is capable of performing a two-step nested isothermal amplification reaction,and achieving the detection of the amplified product by a colloidal gold strip.With different reaction chambers,and simplified fluidic control,which achieved a simple and convenient two-step nested isothermal amplification through coupled with a matching detection device within 30 minutes.Meanwhile combined with colloidal gold paper strip chromatography reaction,a rapid detection of amplified products was achieved,which laid a good foundation for further improving the performance of isothermal amplification detection.(3)For the detection of colloidal gold strips of isothermal amplification products,the algorithm for image detection and analysis was studied.By optimizing the image detection and processing strategy,the algorithm improved the recognition accuracy of the colloidal gold strip test line,which laid a good foundation for the realization of high-performance isothermal amplification detection.