Establishment And Application Of Rapid Detection Method For H1N1 Influenza A Virus Based On Ring Aptamer And Rolling Ring Extension

BackgroundAvian influenza is an avian viral disease caused by influenza viruses of different subtypes in the Orthomyxoviridae A influenza virus genus.According to the pathogenicity of the avian influenza virus to the infected birds,it is divided into highly pathogenic avian influenza and low pathogenic avian influenza.Low pathogenic avian influenza can be transformed into highly pathogenic avian influenza in a certain period of time.Traditional detection methods have cumbersome steps and long cycles.Molecular biology detection methods are sensitive and fast,and because of the need for expensive equipment and professional operators,it is difficult to popularize in primarylevel inspection work.Therefore,it is of great practical significance to find a quick and easy method to detect influenza virus.Compared with conventional detection,the rolling circle amplification(RCA)method can not only directly amplify DNA and RNA,but also realize the signal amplification of target nucleic acid with high sensitivity,so it has great advantages in nucleic acid detection.Application value and potential.ObjectiveIn this project,influenza ring aptamers are used to establish a double aptamer sandwich structure to detect influenza viruses.The visualization and rapid diagnosis of influenza viruses can be achieved through a color indicator,which provides new ideas for rapid influenza virus detection.Method(1)With influenza virus H1N1 as the target,two single-stranded DNA aptamers were obtained through literature review,and the secondary structure of single-stranded and circularized aptamers was fitted using Mfold software.According to the structure of the specific binding site after circularization,It is determined that the single-stranded aptamer is directly locked into a loop.(2)Verify the affinity and specificity of single-stranded aptamers and loop aptamers by dot blot,nano-gold colorimetric and ELISA characterization methods.(3)Construct a double aptamer sandwich structure to detect H1N1 influenza virus system,and use rolling circle amplification technology to detect influenza virus.(4)Choose different color reagents to optimize the reaction system and reaction conditions,realize the visual verification of the amplified products,and establish a visual detection method for influenza virus.Result(1)With influenza virus H1N1 as the target,two single-stranded DNA aptamers were obtained through literature review,and the secondary structure of single-stranded and circularized aptamers was fitted using Mfold software.According to the structure of the specific binding site after circularization,It is determined that the single-stranded aptamer is directly locked into a loop.(2)By ELISA method,the dissociation constant of the aptamer before and after the cyclization was calculated,and it was found that the dissociation constant of the single-stranded aptamer changed after the cyclization.The dissociation constant of the single-stranded aptamer Ap1 is 69.51 n M,the dissociation constant of the cyclized CAPT1 is 36.2 n M;the dissociation constant of the single-stranded aptamer Ap2 is13.53 n M,and the dissociation constant of the cyclized CAPT2 is 24.38 n M.(3)The color development verification and comparison of several reagents such as hydroxy phenol blue,calcein,neutral red and SYBR Green I were carried out,and the conditions were optimized to find the most suitable reaction indicator for specific detection of H1N1 influenza virus.Finally,it was determined that SYBR Green I had the best color development effect.(4)Combining the ring aptamer template and isothermal amplification technology,the RCA amplification is carried out based on the test protocol of magnetic bead aptamer,and the rapid detection of H1N1 influenza virus is realized.Its specific and rapid detection of H1N1 influenza virus,the shortest detection time is 2 hours,and the minimum limit of visual detection is 50 ng/μL.ConclusionThis study has successfully established a double aptamer sandwich method to detect influenza virus.This method has strong specificity,simple operation,convenient time-saving and specific results visualization.It provides technical support for the detection of influenza virus at the grassroots level and has broad development prospects.