Research And Application Of Key Technologies For On-site Nucleic Acid Detection

Cancer and infectous disease still pose great threat to health,and the rapid and accurate detection of related biomarkers is the prerequisite for effective response.Protein and nucleic acid biomarkers are the main target molecules for in vitro diagnosis.However,the immunoassay based protein detection methods are generally not sensitive.Therefore,the detection technology of nucleic acid has become the mainstream of rapid detection.The PCR and its derivative techniques in nucleic acid detection have limited the application scene because of strict temperature control,and sophisiticated instruments.The new technology represented by loop-mediated isothermal amplification(LAMP)has the advantages of simple,rapid,high specificity and high sensitivity.However,the primer design of LAMP and other isothermal amplification methods are complex and subject to strict patent protection,which restricts the application.Microfluidic chip can integrate steps of nucleic acid detection by take the merits of precision fluidics control and large-scale integration,which will greatly reduce the manual operation steps,shorten the detection time.And the inherent miniaturization of microfluidic chip will also make the field portable multi-target nucleic acid analysis possible.The work was centered on key technologies of on-site nucleic acids detection by multiplex microfluidic chip,invention of novel isothermal amplication and rapid sample pretreatment.The main contents and results were as followed:(1)Foundation of visually and fluorescent multiplex microfluidic detection chip based on LAMP method for on-site nucleic acid detetion.The microfluidic chip for 6 targets visually detection at once for malira prevention was developed.And the microfluidic chip for 10 targets fluorescently detection detection at once of Middle East Respiratory Syndrome Coronavirus was developed.At last,the freeze-drying technology for room temperature storage reagents of LAMP was explored.(2)The Competitive annealing mediated isothermal amplification of nucleic acids(CAMP)was invented.CAMP takes the advantage of hybrid primers design and DNA polymerase with strand displacement activity.Through the cycle of primer annealing,extension,competitive annealing and extension,the purpose of nucleic acid amplification at isothermal conditions is achieved.In comparasion of LAMP,the principle of primer design is simpler and the required nucleic acid fragment is shorter in CAMP.Moreover,the outer primers and loop primers were introduced in order to improve the reaction rate of CAMP.At last,the reaction system of CAMP was optimized in compotents,primer design and fluorencent agents for the establishment of the novel nucleic acid isothermal amplification method.(3)The established CAMP method was sucssessflully applied for the detection of pathogens such as HIN1 Influenza A virus and dog virus.And the microfluidic based CAMP method coupled with rapid nucleic adids extraction was developed for the on-site detection of Koi herpesvirus.(4)In addition,another isothermal nucleic acid amplification method called helix loop mediated isothermal amplification of nucleic acids(HAMP)was aslo verified,would be of great promise for further application.