Establishment Of A Blocking ELISA Antibody Detection Method And A CRISPR/Cas13a Strip Detection Method For African Swine Fever Virus

African swine fever(ASF)is a highly contagious animal disease caused by the African swine fever virus(ASFV),which can infect almost all ag es and breeds of domestic pigs.ASF is classified as a reportable disease by the World Organization for Animal Health(WOAH),and a class I animal epidemic disease in China.ASFV is a large double stranded linear DNA virus with a complex structure,and is also the only member of the currently known African swine fever virus genus.African swine fever was first reported in China in August 2018,which immediately brought heavy economic losses to China’s pig industry.So far,there is still no vaccine or antiviral drug that can effectively prevent or treat ASF.Therefore,culling affected pigs,infected pigs,and pig herds that have been in contact with them,and conducting harmless disposal,are the most effective measures to control the situation of African swine fever outbreaks.Accurate and rapid diagnosis is essential for implementing this measure.Therefore,developing diagnostic methods for ASFV is crucial for curbing its spread.In this study,the P30 protein of ASFV was used as the target antigen to screen the monoclonal antibody with blocking activity,which then was used toestablish the blocking ELISA antibody detection method.At the same time,B646L gene of ASFV was selected as the target,based on the CRISPR/Cas13a system,combined with recombinase-aided amplification(RAA)and a lateral flow strip technology,an antigen detection method for ASFV was established.The main research contents are as follows:1.Screening of monoclonal antibodies against African swine fever virus P30 proteinIn order to improve the expression level of P30 protein in insect cells,the gene sequence encoding ASFV P30 protein was optimized and synthesized,and pFast Bac1-P30 donor plasmid was constructed.Subsequently,the correctly constructed pFast Bac1-P30 plasmid was transformed into DH10Bac competent cells to obtain recombinant bacmid rBacmid-P30.The recombinant bacmid was transfected into Sf9 cells to obtain P1 generation recombinant baculovirus.Western Blot and indirect immunofluorescence(IFA)experiments confirmed that P30 protein can be efficiently expressed in Sf9 cells.On this basis,after amplifying the recombinant baculovirus in suspension Sf9 cells,P30 protein was purified by nickel column affinity chromatography.Then,purified P30 protein was imm unized into 6-week-old SPF-grade BALB/c mice to prepare and screen monoclonal antibodies.After identification,two monoclonal antibodies against ASFV P30protein were successfully screened out,named 4A1 and 5D8 respectively.Their heavy chains were both IgG1 type and light chains were Kappa chains,and their antigenic epitopes were both located at C-terminal 134 aa-194 aa region.2.Establishment of a blocking ELISA antibody detection method for African swine fever virus.After measuring the blocking rates of the two monoclonal antibodies mentioned above,we found that monoclonal antibody 4A1 had a blocking rate of56.7%,while monoclonal antibody 5D8 had a blocking rate of 50.3%.Therefore,we used monoclonal antibody 4A1 to establish a blocking ELISA method.After coating purified P30 protein on enzyme-linked plates and optimizing the reaction conditions,we successfully established a blocking ELISA method.When the protein coating amount was 12.5 ng/well and the enzyme-labeled monoclonal antibody dilution was 1:1600,the blocking rate of this method was maximal,and S/N values less than 58.67%were determined as positive,while S/N values greater than 65.98%were determined as negative.The intra-and inter-assay coefficients of variation of this method were both within 5%,indicating a good reproducibility.After testing enzyme-linked plates stored for different periods,we preliminarily determined that their shelf life was eight months.When compared this method with the ASFV detection kit produced b y Wuhan Keqian Biological Co.,Ltd.The positive coincidence rate of the two methods was 90.73%,the negative coincidence rate was 100%,and the total coincidence rate was 91.48%by testing 223 clinical serum samples.3.Establishment of CRISPR/Cas13a dete ction method for African swine fever virusReferring to the sequences of African swine fever virus genotypes Ⅰ,Ⅱ,Ⅲ,Ⅳ,Ⅴand Ⅹ,primers were designed in the conserved regions of B646L gene in each genotype,and a total of 5 pairs of RAA primers and corresponding crRNA were designed.Firstly,5 pairs of RAA primers were tested.After measuring the size and concentration of amplified fragments,results showed that all primers could be selected for subsequent experiments.Then different primer and probe combinations were tested,the results showed that all primer and probe combinations could be used in the detection reaction.In the sensitivity test of different primer probe combinations,it was found that the combination of primer3+crRNA 3 showed the highest sensitivity,and its detection limit could reach 10~2copy/μL.By optimizing the reaction conditions,the detection limit of this method could reach 10~1 copy/μL,which was close to qPCR method in sensitivity.In addition,the results of specificity test for primer 3+crRNA 3 combination also showed that this method had no cross-reaction with other eight pig pathogens commonly seen in clinic,including classical swine fever virus(CSFV),porcine circovirus type 2(PCV2),encephalomyocarditis virus(EMCV),porcine reproductive and respiratory syndrome virus(PRRSV),Japanese encephalitis virus(JEV),porcine epidemic diarrhea virus(PEDV),porcine circovirus type 3(PCV3)and pseudorabies virus(PRV),indicating that it had strong specificity.Finally,83clinical samples from different tissues were tested by this method.The results showed that this method was completely consistent with qPCR method in detection results,indicating that the coincidence rate between these two methods reached100%.At the same time,retesting by this method also showed that both results were completely consistent,proving that this method had good repeatability.