Establishment And Application Of Four Detection Methods For African Swine Fever

African swine fever(ASF)is an acute,haemorrhagic and highly contagious disease caused by African swine fever virus(ASFV),which infects domestic pigs and various wild boars.ASF has been introduced into China since August 2018,causing massive pig deaths in China and a a significant decline,causing huge economic losses.Currently,no commercial vaccine or drug has been developed globally that can effectively prevent ASF.Accurate,simple and reliable laboratory testing methods are essential for the prevention and control of ASF.With the genetic recombination between different strains in the field,the use of illegal vaccines and the emergence of type I ASF,many weak strains are prevalent in the field.weak strains of ASFV can cause persistent infection in animals,and these strains often fail to cause typical clinical signs in infected pigs,are somewhat insidious,carry low virulence and are difficult to diagnose accurately by nucleic acid testing alone.In this study,we established a fluorescent PCR method for ASFV based on the ASFV B646 L gene,an ASFV antibody detection method based on magnetic particle chemiluminescence,and an ASFV blocking ELISA antibody(Ig G)and indirect ELISA antibody(Ig M)detection method.The method was also used for the systematic detection of ASFV nucleic acid,Ig G and Ig M antibodies after the attack of ASFV strong and weak strains of ASF,in order to provide technical and theoretical support for the precise prevention and control and comprehensive diagnosis of ASF,as follows:1.Establishment and application of a fluorescent PCR method for ASFV detection.Fluorescent PCR can detect ASFV at the early stage of ASFV infection,and is characterized by high specificity,sensitivity and accuracy,making it an important tool for early monitoring and diagnosis of ASF.In this study,a fluorescent PCR method was developed for the detection of ASFV by comparing the p72 gene of the current classical epidemic strains and designing primer probes targeting the conserved region of the p72 gene,using the Taq Man probe method.The optimal primer-probe ratio for this method was found to be 1:1:1,the annealing temperature was 57°C and the detection limit was800 copies/m L.In addition,a standard curve for the detection of ASFV was established by applying standards with a correlation coefficient of 0.9991(Y=-3.3652X+36.272)and an amplification efficiency of 98.21%.The established fluorescent PCR method was highly sensitive and comparable to the detection capacity of the WOAH recommended primer probes for the clinical diagnosis of ASF.2.The Chemiluminescent magnetic microparticle immunoassay(CMIA)is a new method for the detection of ASFV antibodies.CMIA is a new analytical method combining magnetic separation,immunoassay and chemiluminescence techniques.The technique has demonstrated an irreplaceable role in the field of bioanalysis and has been widely used in various fields of diagnosis.p30 protein is a protein that is expressed in large quantities in the early stages of ASFV infection and is an ideal candidate antigen for serological diagnosis.In this study,purified p30 protein was coupled to magnetic beads and a magnetic particle chemiluminescence antibody assay was developed by applying alkaline phosphatase-labelled porcine secondary antibody as the detection antibody.Based on ROC curve analysis,the critical value of CMIA was 104315(area under the curve,0.998;Youden index,0.974).The sensitivity was higher than that of the commercial blocking ELISA kit.Specificity tests found no cross-reactivity with other porcine disease virus positive sera.Repeatability tests found the coefficient of variation to be less than 10% for both intra-and inter-batch replicates.p30 magnetic beads can be stored for more than 15 months at 4°C.The Kappa value between the CMIA and INGENASA blocking ELISA kits was 0.946,with good agreement.The CMIA developed in this study has the advantages of sensitivity,strong specificity and high reproducibility and can be applied to the detection of ASFV antibodies in clinical samples.3.Establishment and application of an ASFV blocking ELISA antibody assay based on ASFV p30 monoclonal antibody.In this study,monoclonal antibodies specifically targeting the p30 protein were obtained by screening the ASFV p30 protein through prokaryotic expression and purification,and hybridoma cell fusion after immunization of the purified p30 protein by emulsification in BALB/c mice.The purified p30 protein was used as the encapsulated antigen and the HRP-labelled monoclonal antibody was used as the detection antibody.a series of reaction conditions were optimised to establish an ASFV blocking ELISA antibody detection method.the ROC curve results showed that the critical value of this method was 28.61%(area under the curve,0.9992;Youden index,0.970).Specificity tests found negative sera with the method for other common swine disease virus positive sera,indicating good specificity.Repeatability tests found that the coefficient of variation for both intra-and inter-batch replicates was less than 10%.The method was applied to 220 clinical samples tested simultaneously with the commercial kit and the Kappa value between the two assays was 0.98,showing good agreement.In conclusion,this study successfully expressed and purified the p30 protein,screened a monoclonal antibody specifically against the p30 protein,and established a specific,highly sensitive and reproducible blocking ELISA antibody method,which can be used for the detection of ASFV antibodies and provide technical support for the epidemiological investigation of ASF and the evaluation of antibodies against weakly virulent strains of vaccines.4.A method for the detection of African swine fever virus Ig M antibodies was established based on the p30 protein.In this study,a method for the detection of African swine fever virus Ig M antibody was developed by optimizing a series of reaction conditions using purified ASFV p30 protein as the encapsulated antigen and goat antipig Ig M-HRP secondary antibody as the detection antibody.p30 protein was optimally encapsulated at a concentration of 0.5 μg/m L and serum at a dilution ratio of 1:100,and the optimal blocking solution was 1% BSA.The optimal reaction time for both the serum and the secondary antibody was 30 min.The threshold value was OD450 nm =0.3887.specificity tests showed that all positive sera for other common swine diseases were negative,indicating good specificity.The sensitivity test found that when the positive sera were diluted 1:512,they were also detected as positive.Repeatability tests found that the coefficient of variation for both intra-and inter-batch replicates was less than 10%.This method can be used for the detection of early antibodies to ASFV and provides technical support for the early diagnosis of ASFV.5.Currently commercialized ASFV antibody diagnostic kits are mainly used for the detection of ASFV Ig G antibodies.For the current situation in China,combining q PCR assay,Ig M antibody assay and Ig G antibody assay is the most accurate and reliable means.In this study,based on the established ASFV fluorescence PCR assay,ASFV indirect ELISA antibody(Ig M)assay and ASFV blocking ELISA antibody(Ig G)assay,we systematically detected ASFV nucleic acid,Ig G and Ig M antibodies after attacking animals with strong and weak ASF strains,and elucidated the results of ASFV nucleic acid,Ig G and Ig M antibodies after attacking animals with strong and weak ASF strains.The results showed that the nucleic acid,Ig G and Ig M antibodies of the wild-type ASFV strains were not changed.The results showed that ASFV nucleic acid could be detected in the blood of sick pigs from the 5th day after infection with the wild-type strong strain of ASFV,followed by an increase in the viral load and animal death.After the wildtype ASFV strain has attacked the animals,the blood is detectable at d 5,then the amount of toxicity increases and then decreases,the Ig M antibody starts to respond at d 7,then the Ig M antibody increases and then decreases,the Ig G antibody starts to respond at d 9,and the Ig G antibody titer gradually increases and reaches a plateau.In conclusion,q PCR is recommended for wild-type strong strain infections,while fluorescent PCR,Ig M antibodies and Ig G antibodies are more accurate when weak strains are infected.This study systematically investigated the changes of serum Ig M,Ig G and nucleic acid after ASFV infection,which can accurately and timely diagnose ASF and lay a solid foundation for the comprehensive diagnosis and prevention and control of ASF.