Research On Aptamer Selection And Application Of H5N1 AIV

Avian influenza（AIV）is a kind of global epidemic diseases,and the H5N1 of avian influenza virus is the main epidemic strain spreading wildly.Since the outbreak of H5N1subtype avian influenza virus,it has not only brought great losses to poultry breeding industry,but also posed a great threat to human beings.The H5N1 of avian influenza virus has been detected in more than 60 countries as migratory birds move through the world.Prevention and treatment of the H5N1 of avian influenza virus is now a top priority.Traditional virus detection methods have shortcomings in detection time,detection sensitivity,detection procedures and so on,and are not suitable for field rapid detection.Aptamers refer to single-stranded DNA or RNA with high affinity and specificity for target molecules,which are screened in vitro by SELEX（Systematic Evolution of Ligands by Exponential enrichment）technique.Aptamers are regarded as substitutes for antibodies because of their advantages such as wide range of target substances,short screening cycle,high affinity,good specificity,and easy preservation and transportation.In recent years,Raman spectrum gradually enters the public view.Surface-to-enhanced Raman scattering（SERS）is a phenomenon where special Surface adsorbable molecules enhance Raman scattering signals,which serves as a method of new pathogen detection.Compared with the original ELISA,LAMP,RT-PCR,and other detection methods,it has the advantages of fast,no culture,automation and high sensitivity.This detection method has been widely used in the field of detection.The research content of this topic is as follows:（1）Screening and evaluation of specific aptamers of H5N1 subtype avian influenza virus.Two aptamers of H5N1 influenza virus were obtained by magnetic bead SELEX screening and high throughput nucleic acid sequencing.Aptamer 2198 and Aptamer 3498 were detected by surface plasmon resonance technique with affinity values of 1.85e^-9 and 1.26e^-9,respectively.The aptamer was used as the secondary antibody for the detection of H5N1 subtype avian influenza virus.The aptamer coupled to the magnetic bead can capture H5N1 subtype avian influenza virus and achieve enrichment of H5N1 subtype avian influenza virus.（2）Rapid detection of H5N1 subtype avian influenza virus based on aptamer surface enhanced Raman spectroscopy.The two selected aptamers with good specificity and sensitivity for H5N1 subtype avian influenza virus were coupled with magnetic beads to establish a capture system for H5N1 subtype avian influenza virus.By in-situ reduction of the capture system,silver nanoparticles were generated to enhance Raman signals.An in situ,rapid and nondestructive Raman spectroscopy technique was developed for the rapid detection of H5N1subtype avian influenza virus.In this study,magnetic bead SELEX screening technique was used to screen out two aptamers with high specificity and strong affinity for H5N1 subtype avian influenza virus,which laid the foundation for the establishment of H5N1 subtype avian influenza virus.The rapid detection of H5N1 subtype avian influenza virus by Raman spectroscopy was established by aptamer capture.The detection method has good specificity,sensitivity,and stability.The method showed that H5N1 subtype avian influenza virus had a specific Raman peak spectrum at1058 cm^-1,and the minimum detection limit was 10¹⁰ times the dilution of the original virus venom.This method can be used to quickly detect the presence of H5N1 subtype avian influenza virus in the samples to be tested within 10 minutes and can quickly detect the target pathogen in the samples of large volume and low concentration,which can meet the requirements of quick field detection of H5N1 subtype avian influenza virus and has certain development prospects.