Screening And Application Of Respiratory Syncytial Virus Aptamers

BackgroundRespiratory syncytial virus(RSV)is one of the most common diseases infecting newborns,children,and the elderly.Its tremendous contagious potential and lethality harm human health all around the world.,yet existing RSV detection methods rely mainly on large instruments as well as professional technicians,resulting in high detection costs and long processing cycles.Therefore,creation of a low-cost,simple-to-use approach for rapid detection of RSV in everyday life has good prospects for application.ObjectiveAptamers have the advantages of more flexible spatial conformation,no immunogenicity and low synthesis cost,and have been widely used in rapid detection of viruses.In this study,aptamers specifically binding to RSV were screened and characterized by various methods.Finally,a visualization method for RSV detection based on aptamer-gold nanoparticle colorimetric method was established to provide a new method for simple and rapid diagnosis of RSV.At the same time,the study of cyclic aptamers was carried out to increase the application of cyclic aptamers in RSV detection,and the visual detection method of RCA amplification products was studied to provide new ideas for the detection of cyclic aptamers.MethodsIn this paper,magnetic beads-SELEX technology was used as an in vitro aptamer screening method for RSV,with RSV as the target and BSA as the anti-screening protein.The screening process was established,and the library concentration and binding effect were monitored.Then the last round of library sequencing analysis to obtain candidate sequences.Subsequently,the binding ability,specificity and affinity of the candidate aptamers were characterized by dot hybridization,gold nanoparticle colorimetry and ELISA,and the aptamer-gold nanoparticle colorimetric method was established to determine the minimum detection limit of RSV.Subsequently,the aptamers were cyclized,and the binding force and affinity of the cyclic aptamers were evaluated by QCM and gold nanoparticle colorimetry.Finally,the detection method of RCA amplification products based on ring was explored by protamine-gold nanoparticle colorimetric method.Results(1)After 14 rounds of screening,the sequencing of 9 RSV candidate aptamers was completed.The homology between sequences was low and the secondary structure was different.(2)The fitness specificity of R1,R3 and R7 for RSV recognition was verified by dot hybridization and gold nanoparticle characterization.The dissociation constants(Kd)of the three aptamers were 14.33 ± 4.340 n M,26.45 ± 6.970 n M and 3.047 ± 1.632 n M,respectively,measured by ELISA.The aptamer R7 with the best affinity was selected to detect RSV with the minimum detection limit of 0.1 ng/μL by gold nanoparticles colorimetric method.(3)The aptamer R1 was ringed,and the QCM method was used to determine that the cyclic aptamer Capt1 also had binding effect on RSV.The Kd value of Capt1 was 97.245 ± 9.872 p M determined by gold nanoparticle colorimetry,which proved that the cyclic aptamer had better affinity than the linear aptamer.(4)Using protamine-gold nanoparticle colorimetric method to detect RCA amplification products,RCA amplification results can be judged according to the difference of negative and positive colors,and finally determine whether there is a circular aptamer in the RCA reaction template.ConclusionsThis project initially established a new visual detection method for RSV,namely aptamer-based gold nanoparticle colorimetric method for RSV detection.This method has strong specificity,simple operation and visualization of experimental results.The whole detection process is only 1.5h,which provides a new detection method for the diagnosis of RSV.Meanwhile,the study of cyclic aptamers provides a new type of special probe for the design of RSV biosensors,which has potential application value.