Establishment And Preliminary Evaluation Of Human Adenovirus 55 RPA Combined With Capillary Electrophoresis For Detection And Antigen Determination

Human adenovirus（HAdV）,a type of unencased linear double-stranded DNA viruses with a diameter of about 90nm,was the main pathogen of human acute respiratory tract infection.There are many HAdV types,and recombination may occur between different types.HAdV55 is a new virus recombination of HAdV11 and HAdV14 discovered in recent years.HAdV55 is mainly transmitted by droplets and is susceptible to infants,students,military recruits and immunocompromised patients.At present,there is no specific treatment for adenovirus infection in clinic,and symptomatic treatment is usually used.In order to reduce the harm caused by the HAdV55 epidemic,early detection of virus infection has been the focus of prevention and control.Up to now,studies on the rapid detection of HAdV55 are scarce.Recombinase Polymerase Amplification（RPA）technology is an application of recombinase polymerase amplification（RPA）technology,which can replace traditional PCR and complete the application of single-molecule nucleic acid amplification within15 minutes at normal temperature.At the same time,Capillary electrophoresis（CE）has the advantage of high sensitivity and qualitative analysis of nucleic acid products.At present,RPA combined with capillary electrophoresis has been well used in the detection of many pathogens.Adenovirus antigen detection is characterized by the specific recognition of antigens in antibodies and samples and the amplification of binding signals.It has the characteristics of simple and rapid detection of antigens,which is the direction of the development of diagnostic reagents.At present,there are few rapid detection methods for HAdV55,and no commercial detection reagent has been applied in the market.Objectives:1.In this study,HAdV55 was selected as the research object,and a RPA-capillary electrophoresis method was established to screen specific RPA primers for Hexon coding genes,and the method was optimized and evaluated.2.Specific monoclonal antibody（m Ab）of HAdV55Hexon was prepared,and a method for detection of human adenovirus 55 based on double-antibody sandwich ELISA was established.Methods:1.The HAdV55-Hexon plasmid was constructed as the amplification template,and the RPA-specific primers were designed for Hexon gene sequences in the conserved region of HAdV55.The primers were screened and optimized,and the RPA amplification and combined capillary electrophoresis detection system were established to evaluate the specificity,sensitivity and reliability.Rpa-capillary electrophoresis was used to detect 18clinical samples.2.The prokaryotic and eukaryotic expression plasmids of HAdV55,3,4,7,16 and21Hexon were constructed;the purified protein of Hexon was obtained by prokaryotic expression plasmid p ET28a-Hexon55;the eukaryotic expression plasmid p CAGGS-Hexon55 was used to immunize BALB/c mice by cupping,and the anti-HAdV55Hexon m Ab was prepared by hybridoma technique,and the antibody titer and antibody subclass were determined.The specificity of the antibody was identified by Westernblot and immunofluorescence assay（IFA）by p CAGGS-Hexon transfection into293murt T and BHK cells,and the cross reactivity of Westernblot and IFA was further analyzed by p CAGGS-Hexon3,4,7,16,21 and 55 transfected cells.The six antibodies were labeled with HRP and verified by pairwise pairing.the similarity of the epitopes recognized by the 6 antibodies was confirmed by competitive ELISA.Furthermore,using Hexon protein as the detection target,the antibody was screened by chessboard method,and a better antibody was obtained,and a double antibody ELISA sandwich method was established.Finally,the sensitivity and specificity of the established system were evaluated,and the method was preliminarily applied to the detection of cultured HAdV55.Results:1.The plasmid containing the full-length HAdV55 Hexon gene was constructed,and a pair of RPA primers with the highest amplification efficiency were obtained.The detection limit of RPA-capillary electrophoresis system of HAdV55 was2.9×103copies/m L,and the whole process was completed in 30 minutes.The standard extracts of coronavirus（HCo V-229E,HCo V-OC43,HCo V-HKU1）,influenza A B virus（Flu A-B）,parainfluenza virus（PIV）and respiratory syncytial virus（RSV）were all negative when tested by this system.In addition,common human adenovirus types（HAdV3,HAdV4,HAdV7,HAdV16,HAdV21）were also negative,which suggested that HAdV55 had strong specificity.The RPA-capillary electrophoresis assay can effectively detect human adenovirus type 55 in pharyngeal swabs of clinical patients.2.The expression plasmids of PET28a-Hexon55 and p CAGGS-Hexon55,3,4,7,16,21 were constructed successfully;IPTG induced PET28a-Hexon transformed BL21bacteria,Hexon protein mainly expressed in the form of inclusion body,after denaturation and renaturation ultrafiltration to obtain purified Hexon protein;Six hybridoma cell lines secreting HAdV55 Hexon m Ab were obtained.Antibody subclass analysis showed that 2 were Ig G2a subtype and 4 were Ig G2b.Two highly valent specific Hexon55 antibody strains were obtained,which had no cross-reactivity with Hexon3,4,7,16 and 21.The six strains were labeled by HRP and verified by pair-to-pair pairing.Six antibodies were obtained by competitive ELISA to identify two different epitopes.That is,2D3,1E2,1A12,2B2,and 3E7 identify one epitope,and 2H8 identify one epitope.A double antibody sandwich ELISA was used to pair each other,and finally a double antibody sandwich ELISA system with 2H8 as capturing antibody and 2D3 as detecting antibody was selected.When the sensitivity and specificity of the system were evaluated,the system did not bind to other viral proteins,and the lowest detection concentration of Hexon protein was 0.1ng/m L,indicating that the sensitivity and specificity of the method were good.Further using cell culture HAdV55 as the detection object,the double antibody sandwich method established in this study can detect 10^-3 PFU/ml at the lowest.Conclusion:In conclusion,this study established a detection method for HAdV55 type based on RPA combined with capillary electrophoresis.The detection limit was 2.9×10³copies/m L and the whole time was about 30min.A double-antibody sandwich ELISA was established using Hexon-specific monoclonal antibodies to identify HAdV55.Both of these methods provide experimental basis for the early diagnosis of HAdV55 infection in clinic.