Establishment Of DDPCR Method For African Swine Fever Virus And Development And Application Of Nucleic Acid Reference Materials

African swine fever(ASF)not only severely harms the pig industry,but also has a huge impact on my country's national economy.When no vaccine is available,laboratory testing is the most effective way to identify and eliminate African swine fever.P72 protein is the main structural protein of African swine fever virus(ASFV),encoded by the B646L gene,with a molecular weight of about 73.09 k Da.As an important component of the virus capsid,the P72 protein participates in the process of virus adsorption and assembly,and has good immunogenicity and antigenicity.In addition,because the B646L gene encoding P72 protein has high homology,it has become an internationally recognized target gene for ASFV nucleic acid detection.It is not only suitable for the study of nucleic acid detection methods,but also for the construction of standard quality control products for various experiments.Positive quality control or proficiency verification of laboratory ASFV nucleic acid testing.Based on the gene sequence of ASFV B646L Gene ID:22220311 registered in Gen Bank,this study used primer5.0 software to design primers and probes for droplet digital PCR(Droplet Digital PCR,DDPCR)detection.After the reaction conditions were optimized,The sensitivity,specificity,and reproducibility of this method are verified for absolute quantification.At the same time,a pair of specific primers used to amplify the full-length B646L gene were designed to amplify the ASFV nucleic acid.The amplified products of the B646L gene after sequencing were verified by double enzyme digestion,ligation,transformation and other steps to screen out positives containing the B646L gene.Clone the p Fast Bac HTB-p72 plasmid.The p Fast Bac HTB-p72 plasmid was directly transformed into Escherichia coli containing the shuttle rod Bacmid.The transformed Escherichia coli was screened in blue and white,and the white positive bacteria were selected for PCR amplification and sequencing identification,and then the correct Bacmid-p72 recombinant bacmid was packaged into virus-like particles through the insect baculovirus expression system,and prepared after passage ASFV nucleic acid standard.Apply ASFV nucleic acid droplet digital PCR detection method to evaluate the uniformity and stability of ASFV nucleic acid standard substances in CD cell culture medium and CD cell culture medium containing 10%fetal bovine serum and different storage temperatures.To determine the best storage substrate and storage conditions.Subsequently,samples for comparison between ASFV nucleic acid testing laboratories were prepared,and ASFV nucleic acid testing capabilities were verified in 150 slaughter companies across the province,and the results were counted.The results show that the established ASFV nucleic acid DDPCR detection method has good sensitivity,specificity and reproducibility,and can be used for absolute quantification of the national AFSV B646L gene plasmid standard material and the ASFV-positive nucleic acid retained by this laboratory.The minimum detection limit is 0.7 copies/?L.At the same time,the full-length gene of B646L was successfully amplified with a size of 1941bp.Sequencing confirmed that its gene sequence was completely consistent with the B646L gene sequence of African swine fever virus isolate Pig/HLJ/2018(MK333180)included in Gen Bank.The ASFV B646L gene homology in 13 different countries is 99.3-99.8%.After that,sequencing confirmed that the constructed virus bacmid Bacmid-p72 contained the complete ASFV B646L gene,and the reading frame was correct without frameshift mutations.The transfected Bacmid-p72 virus bacmid was successfully passaged in Sf9 insect cells.The prepared ASFV nucleic acid standard material was confirmed by DDPCR method that it was stable in freezing conditions and had good uniformity.The initial concentration was 6.0×10~3copies/?L.The analysis of the comparison results of 150 slaughter companies across the province showed that the nucleic acid standard material is suitable for the AFSV nucleic acid detection reagents used by various companies,and has achieved good accuracy and uniformity.In summary,this study has successfully established the ASFV nucleic acid DDPCR detection method and standard materials,which can be widely used in the quality control and proficiency verification of ASFV nucleic acid detection,providing technical support for the prevention and treatment of ASF.