Detection Of H1N1 Influenza Virus Based On Magnetic Bead Aptamer And Isothermal Amplification Technology

BackgroundInfluenza is an acute respiratory infectious disease caused by influenza viruses.In recent decades,highly infectious influenza viruses can easily cause seasonal pandemics and pose a serious threat to global public health.H1N1 is a subtype of influenza virus.Since H1N1 can directly infect humans and cause human illness or death,strengthening surveillance and detection of H1N1 has far-reaching reality in reducing world economic losses and improving human health.significance.Existing detection methods such as traditional nucleic acid analysis usually require professionals to perform in laboratories with complex and expensive equipment.With the growing demand for H1N1 monitoring and inspection,low-cost,easy-to-operate,and fast inspection methods are receiving increasing attention.Therefore,it is particularly important to develop a method for detecting H1N1 influenza virus with simple operation,short response time,and constant temperature conditions.ObjectiveThis research group intends to establish a method for detecting H1N1 influenza virus based on magnetic bead aptamer and isothermal amplification technology,explore the amplification effect of two different signal amplification methods,and use visual colorimetric detection to detect the amplification result for H1N1 influenza.The simple and rapid diagnosis of the virus provides new ideas,and lays the theoretical foundation and practical basis for the early detection and control of influenza.At the same time,the research of circular aptamers has provided the possibility to enhance the applicability of aptamers by modifying them.Method(1)The aptamer affinity and specificity were verified by dot hybridization,gel blocking,and nano-gold colorimetric characterization methods.(2)Based on the preparation of magnetic bead aptamers,dual aptamers combined with dual primer rolling circle amplification and self-generated primer rolling circle amplification were used to detect the H1N1 influenza virus.(3)Optimize the reaction system and reaction conditions by selecting different chromogenic reagents,realize the visual detection of the amplified products,and establish a method for visual detection of the H1N1 influenza virus.(4)Existing aptamers were cyclized using the principle of lock probe looping,and their affinity was identified by point hybridization and 96-well plate method.Results(1)Taking aptamers targeting H1N1 influenza as the research object,and conducting literature experiments to obtain 3 aptamers against the complete virus.The aptamer was verified by different characterization methods,and an aptamer capable of specifically binding to the H1N1 influenza virus was clearly obtained.(2)Combining aptamer and isothermal amplification technologies,and designing two signal amplification methods,RCA and pg-RCA,based on the dual aptamer experimental scheme,to detect the H1N1 influenza virus,compare the two methods and optimize them,and finally Selected RCA method for amplification(3)Compare several chromogenic agents of hydroxynaphthol blue,calcein,neutral red,and SYBR GREEN I,and optimize the conditions to find the most suitable indicator for H1N1 influenza virus specific detection.The minimum detection limit is 10 ng / ?L,and realize the detection of actual samples.(4)On the basis of the existing linear aptamers,the aptamers were modified for circularization.By point hybridization and 96-well plate method,the affinity of uncirculated aptamers was compared,and it was confirmed that the circular aptamers also have specific binding.H1N1 influenza virus capabilities.ConclusionThis paper proposes a method for detecting H1N1 influenza virus based on magnetic bead aptamer and isothermal amplification technology.Different types of characterization methods are used to verify the aptamer.The feasibility of two signal amplification methods is explored.The amplified products are judged to establish a system for visual detection of the H1N1 influenza virus.Traditional detection equipment is not required.The detection method for the H1N1 influenza virus is added from the methodology.It is not affected by the environment and conditions and can be used for grass-roots detection.At the same time,research on ring-shaped aptamers shows that the modification of aptamers has an effect on aptamer functions,providing a theoretical basis for the establishment of new detection methods,and has broad application prospects.