Establishment And Application Of A Method For The Detection Of Natural Variants Of African Swine Fever Based On LF-RPA

African swine fever(ASF)is caused by African swine fever virus(ASFV)with high mortality infection.Due to acute,high fever and contact transmission characteristics,it lead to the serious impact and devastating blow to the pig industry.For immune escape characteristics and numerous genotypes,an effective vaccine for preventing and controlling the virus has not yet been developed.Therefore,early rapid diagnosis has become one of the most effective means to prevent and prevent the spread of the disease.Based on the above mention,this study was to develop the nucleic acid amplification method of recombinase polymerase amplification(RPA)to provide technical support for rapid clinical diagnosis of the disease.Firstly,we collected bloodsucking insect samples from field,genomic DNA was extracted,African swine fever CD2v gene was detected by using PCR method,and the amplified product was constructed the recombinant plasmid.Using the recombinant plasmid p MD19-CD2v as the template,real-time quantitative PCR was also established,it could be used as RPA control experiments.The results showed that the CD2v gene was successfully amplified from bloodsucking insects PCR method with the size of 201 bp;And successfully constructed the recombinant plasmid p MD19-CD2v,the positive plasmid was identified by PCR,meanwhile real-time quantitative PCR method for detecting ASFV was successfully constructed.The result indicated that it has good specificity and sensitivity,with the lowest copies of 2.51×10~1/μL in sensitivity.Next,establishment of RPA method for detecting ASFV.According to the ASFV gene sequence MW521382.1 from Gen Bank,three groups of RPA primers were designed for CD2v gene region,then they were applied to detect samples of African swine fever virus,Agarose gel RPA reaction system and LF RPA reaction system were explored respectively,and the reaction temperature and time conditions also were optimized.At the same time,the sensitivity and specificity tests were carried out.The results indicated that the established LF-RPA method has high specificity,it did not react with other viruses;in sensitivity,it could detect as low as 10 copies of DNA molecules;The reaction conditions were optimal under the constant temperature of 39℃and 15 minutes,and the detection efficiency was significantly higher than that of established real-time quantitative PCR method.Finally,the established RPA method was used to detect environmental samples from pig farms,and it was compared with the PCR method.The results showed that 12 positive samples could be determined from 50 samples,the detection rate was 24%(12/50),but PCR method only could detect10 samples,the detection rate was 20%(10/50).In conclusion,both the basic RPA detection method and the side flow chromatography strip RPA detection method has been established in this study,it could be used for the early and rapid diagnosis of African swine fever virus,it will provide technical support for the prevention of the disease in pig farms,it has certain practical application value to prevent it,this method is suitable for use and promotion at the grassroots level.