Screening And Application Of Nucleic Acid Aptamers Of Enterovirus EV71 And CA16

BackgroundHand,foot and mouth disease(HFMD)is an acute and highly contagious viral rash that usually occurs in symptomatic children under 5 years of age.The clinical symptoms of HFMD are usually mild and include fever,loss of appetite,rash,and blisters,which do not require specific treatment.However,there are some rare neurological or cardiac complications,such as meningitis and acute flaccid paralysis,that can be fatal.HFMD is generally considered to be a self-limiting disease.However,over the past 20 years,repeated population outbreaks in Asia and the Pacific have resulted in severe disease,debilitating complications and even death.More than 20 enterovirus genotypes can cause hand,foot and mouth disease,among which the main pathogens are human enterovirus 71(enterovirus 71,EV71)and coxsackievirus A16(coxsackievirus A16,CA16),both of which belong to A type enterovirus.However,the traditional virus detection method has a long cycle and complicated steps.Among them,the molecular biology detection method is sensitive and fast,but due to the need for professional operators and expensive instruments,it is difficult to popularize this method in the basic inspection work.Therefore,it is very necessary to propose a more rapid,simple and accurate in vitro detection method for the pathogen of HFMD.The emergence of aptamers provides new potential feasibility for ultrasensitive and rapid detection of viruses.ObjectiveThe purpose of this study is to screen aptamers for two main pathogens EV71 and CA16 in hand-foot-mouth disease by magnetic bead-SELEX technique,and then design an ultra-sensitive and rapid detection method for hand-foot-mouth disease pathogen with low cost,rapid detection,simple composition and operation.MethodTwo viruses,EV71 and CA16,were selected as targets,and the nucleic acid aptamers were screened by magnetic beads-SELEX method.During the screening process,methods such as gel electrophoresis,q PCR,and micro-nucleic acid assay were used to monitor the progress of aptamer screening.The enriched nucleic acid sequence was verified by monoclonal and sequenced,followed by homology analysis and secondary structure analysis,using dot blot hybridization,gold nanometer colorimetry,ELISA and other characterization methods to verify the binding force and specificity of the aptamer.The aptamer with high binding force and high specificity was selected for locked ring formation to characterize the specificity of the ring aptamer.Finally,a virus detection method including double aptamer sandwich and gold nano-gold visualization was established.ConclusionAfter 12 rounds of aptamer screening,6 EV71 and 3 CA16 candidate aptamers were screened respectively.Three EV71 aptamers and two CA16 aptamers were screened by dot hybridization.The affinity of the aptamers was verified by ELISA,and the equilibrium dissociation constants(Kd values)of the five aptamers were measured to be 9.64±1.25 n M,6.45±0.22 n M,17.53±2.92 n M,21.40±5.27 n M,and14.52±3.83 n M.The specificity of the aptamer was verified by the nano-gold colorimetric method,and the specificity and affinity of the nucleic acid aptamer were compared.One aptamer with high affinity to both EV71 and CA16 and one aptamer with high specificity and high affinity only to CA16 were selected.The two aptamers were locked and looped to verify the binding ability of the loop aptamers to the virus.A double aptamer sandwich method for indirect detection of viruses was established.After rolling circle amplification,gold nanoparticles were used to visualize the detection method.The shortest detection time of this method was2 hours,and the lowest detection limit was 1 fmol/L.