Establishment And Application Evaluation Of Dual Fluorescence Quantitative PCR Detection Method For ASFV And CSF

African Swine Fever(ASF)and Classical Swine Fever(CSF)are animal diseases that must be reported by the World Organization for Animal Health(OIE).In our country has classified it as a Class I animal disease.At present,the CSF epidemic has been well controlled in China,but ASF is still in an unstable state,and there is no targeted vaccine available.ASF and CSF usually show similar characteristics,clinical symptoms and pathological changes,so it is difficult to distinguish between clinical manifestations and pathological necropsy,and laboratory testing techniques are required to confirm the diagnosis.To this end,this study intends to establish a dual fluorescent PCR detection method for African swine fever virus(ASFV)and Classical swine fever virus(CSFV),so as to provide a scientific and accurate detection method for the rapid clinical diagnosis of ASF and CSF.Download the ASFV and CSFV genome sequences from NCBI,and design 2 specific primers and Taq Man probes for the conserved target sequences of ASFV B646L and CSFV 5’-UTR through sequence alignment.The reaction conditions of the method were optimized,and the specificity,sensitivity and reproducibility were verified.The results showed that the correlation coefficients R~2of the standard curves of ASFV and CSFV were 1 and 0.999,respectively,and the linear relationship was good;they did not react with other common pig pathogens,and the specificity was good;the lower detection limits of ASFV and CSFV were 2.4 copies/μL and 1.3copies/μL,respectively.high sensitivity;inter-assay and intra-assay coefficient of variation is less than 2%,good stability.The above results show that this experiment successfully established a dual fluorescence quantitative PCR method with high sensitivity and specificity for ASFV and CSFV.In order to verify the application effect of the dual fluorescence quantitative PCR detection method established in this experiment,a total of 225 clinical samples were collected from 10large-scale farms in Harbin,Daqing and Qiqihar City,Heilongjiang Province,double fluorescence quantitative PCR and common PCR were used to amplify the treated samples.E2gene was sequenced in clinical samples that were positive by ordinary PCR method.Phylogenetic tree was constructed and its homology was analyzed according to the sequencing results.The results showed that all ASFV samples were negative in the double fluorescence quantitative PCR method,10 cases were positive for CSFV,the positive rate was 4.44%,and 6cases were detected by ordinary PCR amplification,and the positive rate was 2.66%.2 of the 6positive samples were sequenced successfully and named HLJ-11 and HLJ-22 respectively.Phylogenetic tree and homology analysis showed that HLJ-11 and HLJ-22 were both 1.1genotypes,HLJ-11 was closely related to Alfort/187(X87939),a moderately virulent strain from France,with the highest homology of 99.8%.The HLJ-22 strain was most closely related to HCLV(AF531433.1)and GXXJC1(EF014338.1),and the highest homology was 99.5%.In conclusion,this study successfully established a dual fluorescence quantitative PCR method for ASFV and CSFV,and the sensitivity was better than that of ordinary PCR methods.The clinical application effect is good,and it provides technical support for the clinical detection and epidemiological investigation of ASF and CSF.