Study On The Rapid Collection Device Of Respiratory Tract Samples And Hybridization Chain Reaction-based Assay For The Detection Of Influenza A Virus

As a common pathogen of respiratory infectious diseases,influenza A virus can live wide range of hosts and spread rapidly by droplets to cause acute upper respiratory tract infection,which has been a serious threat to human life and health.Therefore,establishment a rapid detection technology for the early screening and diagnosis of influenza A patients is of great significance,which could provide technical support to respond the outbreak of such infectious diseases timely and efficiently.Objective:This study focuses on the frequent outbreak of respiratory infectious diseases to endangers public health,based on the technique of exhale breath condensing,want to develope a portable pipe-typed exhale breath condensate collecting device to meet the rapid collection of the upper respiratory tract infection pathogens.Meanwhile,we hope to establishe a simple,accurate and sensitive method for detecting influenza A virus by taking the advantages of nanomaterials and hybridization chain reaction technology for the detection of respiratory infectious diseases to solve the problem of complex sampling pretreatment,large sample background interference,complex sampling operation,large sample background interference,redundant operation steps,and dependence on large-scale instruments which influence the implementation of point of care test.Methods:Through investigating the related reports,documents,patents and commercial products of the exhale breath condensate（EBC）collecting device,a tubular exhale breath condensate collection device was designed for collecting pathogens of respiratory infectious diseases based on the principle of aerosol phase transition and the movement of fluid in confined space.To optimize the design and constructing of the EBC device,based on evaluating the working efficiency by measuring the cooling rate of the instrument,the cooling rate of the collecting tube and the collection amount of the condensateIn this paper,a generic influenza A detection primer based on influenza A M1 protein was designed and verified for hybridization chain reaction（HCR）and validate the result of the reaction by agarose gel electrophoresis experiment.Then fixed the concentration of H1,H2 modified by FAM group,the concentration of Itarget to optimize the quenching effect of concentration and time.of graphene oxide（GO）.Then fixed the GO quenching effect condition to optimize the HCR reaction time and determine the final reaction conditions.Finally,based on the optimized reaction detection conditions,the detection method was evaluated from four aspects:single base mismatch recognition ability,quantitative detection ability,the detection ability towards influenza A virus and clinical sample.Results:We developed a rapid exhale breath condensate collection device for respiratory aerosol,with the main performance:（i）cooling speed:the instrument refrigeration module can bring down from 28℃to-4℃in 10 minutes;（ii）good refrigeration effect:the collection tube is cooled from room temperature to 2℃within 1 minutes with stable temperature;（iii）the sample quantity can reach about 200μL in 1 minutes,sufficient amount for the subsequent detection.We established the detection method of influenza A virus based on HCR reaction and GO fluorescence quenching.The optimized the HCR reaction conditions were:50nM of H1and H2,2.5 h of the reaction time,after the HCR reaction the reaction product was quenched with the 20μg/mLof GO.quenching time is 30min;After quenching,the fluorescence intensity was detected with the excitation wavelength of 480nm and emission wavelength of520nm.The result shows that the established HCR detection method is specific to influenza A virus（H1,H5,H7 subtype）versus other groups（FluB,Adv）（P<0.01）.For the samples containing I_target,the fluorescence intensity was shown to be significantly higher than that in the I_mismatch groups（P<0.05）shows that this method has the ability of single base mismatch recognition.The positive detection range is 5-100n M.The fluorescence intensity in the range of 10-40nM shows a good linear relationship with the concentration of the target nucleic acid sequence（y=4.7576x-10.401 R2=0.9858）.The test results of 15 nasopharyngeal swabs showed that the established HCR detection method was consistent with that of real-time PCR.Conclusion:In this study,we developed a tube exhale breath condensate collection device for upper respiratory infectious disease samples,which has the characteristics of high cooling efficiency,controllable target temperature,small size and portable,easy to operate,etc.It can meet the need of rapid collection of upper respiratory tract infectious disease samples,and lay a foundation for the subsequent use of the combined use of biosensor detection equipment.In addition,based on the hybridization chain reaction（HCR）,a rapid detection method for influenza A virus was designed by using the quenching of fluorescent groups by graphene oxide.This HCR amplification detection technology has a good application prospects for point of care test,with the advantages of enzyme-free isothermal amplification,simple reaction system,simple operation steps and good sample detection ability...