Establishment Of PCR And Fluorescence Quantitative PCR For Diagnosis Of African Swine Fever Virus

African swine fever(ASF)caused by African swine fever virus(ASFV)is a high pathogenic and contagious disease with severe economic loss in the swine industry worldwide.The main symptoms are severe haemorrhage of lymph nodes,skin and internal organs,high fever,oedema,and dyspnea.Because the spread of ASFV is across countries,it has been listed as an international quarantine object of the viruses,and Class I of animal epidemics in China.At present,there is still no effective vaccine and medicine to control the ASF.The necessary test of ASFV is an urgent need for some countries suffering from ASFV infection.Adequate monitor for ASFV infection is helpful for competent authorities to control of ASFV epidemic situation.Meanwhile,it can monitor the health status of swine in real-time.Conventional and fluorescent quantitative PCR methods of ASFV were established in this study,which provided references for clinical prevention.Primer Premier 5.0 was used to design three pairs of conventional PCR primers,named by 539-F/R,Premier1-F/R,Premier2-F/R referring to the gene sequence of ASFV VP72.Similarly,fluorescent quantitative PCR primers VP72-F/R were designed by Primer Express Software(v.3.0).And then,a standard positive plasmid was constructed by synthesising a complete sequence of ASFV VP72 gene and used to perform a specific experiment of established ASFV PCR methods.A comparative test about sensitivity and specificity between PCR method designed by us and recommended by OIE was carried out.As well as,effects of the quantitative fluorescent PCR method and the ASFV fluorescent PCR commercialisation kits were also compared.Through the specific detection,PCR and fluorescence quantitative PCR methods of ASFV were successfully established.Only the positive plasmid constructed by the method by this research has been effectively amplified.The six viruses CSFV,PEDV,RV,PRV,PCV2,and PPV were tested for the specificity of the established method,but no results were obtained amplify.The method constructed in this study has excellent specificity.In terms of sensitivity,the minimum detection limits of the two PCR methods designed in this experiment reached 10-10 and 10-9,respectively,while the minimum detection limit recommended by OIE for primers was 10-7.It is proved that the African swine fever virus PCR method in this study has higher sensitivity than the PCR recommended by OIE.The establishment of quantitative fluorescence PCR in this institute can detect positive sample plasmid.The minimum nucleic acid concentration range is 1.56×103 copies/?L,which is compared with the African swine fever virus fluorescent PCR commercialisation kit.The test results of this study are consistent with the results of the kit,indicating that the fluorescent quantitative PCR method established in this study is available.This study successfully established the PCR method of ASFV VP72 gene to improve the sensitivity of ASF diagnosis and increase the diversity of ASF diagnostic methods.In practical applications,it can be used for early detection of pig herds in farms significant advantage.It provides a new PCR detection method for the detection of African swine fever virus.It provides a particular reference value for the development of African swine fever prevention and control measures and purification strategies.