Development Of Recombinase Polymerase Amplification Method For African Swine Fever Virus

African Swine Fever(ASF)is an acute,feverish,and highly infectious disease caused by African Swine Fever Virus(ASFV)infection,which has caused serious economic losses to the pig industry.As ASFV anti-infection mechanism is extremely complex and genotype,there is no effective vaccine for control.Thus,preventing the outbreak of the disease mainly based on early rapid diagnosis and prevention.Considering the factors above,in this study,we established ASFV Recombinase Polymerase Amplification(RPA)basic detection method and Recombinase Polymerase Amplification(RPA)probe detection method,and from the analysis of sensitivity,specificity,repeatability,detection time,detection temperature and other aspects,confirmed that the RPA-based nucleic acid amplification method has a strong sensitivity,high specificity,rapid response,constant temperature characteristics.According to the ASFV gene sequence FR682468 in GenBank,three groups of RPA primers were designed for the sequence of group p72 gene area,to To establish a recombinant enzyme polymerase chain polymerization(RPA)basal detection method,and optimize the reaction conditions in order to examine sensitivity,specificity,and repeatability of the RPA primers.The results showed that the optimum reaction conditions were 39? for 20 min.Sensitivity test can detect a minimum of 5.5×101 copies/?L,the same as the data of classical PCR method,and there was no crossreaction among CSFV(Classical swine fever virus),PCV-2(Porcine Circovirus Virus),PEDV(Porcine Epidemic Diarrhea Virus),TGEV(Transmissible)Gastroenteritis of Swine Virus).The experimental result demonstrated that the established Recombinase Polymerase Amplification(RPA)basal test method can detect ASFV rapidly,sensitively and specifically.In order to establish Recombinase Polymerase Amplification(RPA)probe detection method,three sets of primers and probes were designed for ASFV B646L(p72)gene,and the reaction conditions were screened and optimized,the sensitivity,specificity,and repeatability were tested.The results showed that the real-time fluorescent RPA method at 39? and 20 min can be detected within 10 copies of the DNA molecule,and had no cross reaction among swine fever virus,porcine circovirus type 2,porcine epidemic diarrhea virus and porcine transmissible gastroenteritis virus.The fluorescence intensity of the samples was detected at 5.5 × 106-5.5 ×100 copies/?L in 7 dilution samples at all time points,and the coefficient of variation ranged from 0.38%-28.30%.The lateral flow dipstick(LFD)RPA can detect a minimum of 5.5 ×101 copies/?L of the plasmid.At the current stage,the isothermal and rapid amplification methods can be used for the qualitative detection of ASFV,which can provide technical support for the early diagnosis of ASFV infection in China,and it has a great significance for the establishment of the corresponding control plan after the outbreak of the epidemic.