Establishment And Application Of Multiple RPA Method For Three Diseases Viruses ASFV,PDCoV,SVA In Pork And Its Products

African swine fever(ASF)is an acute,hot and highly infectious disease caused by African swine fever virus(ASFV)in domestic pigs and wild boars.Since its introduction into China in 2018,ASF has caused great damage to China's breeding industry.By April2019,the total number of pigs killed had reached 1.02 million.Both A-type senecivirus a(SVA)and pig delta coronavirus(PDCoV)have caused great damage to pig industry in foreign countries.Although they have not caused great impact on pig industry in China,they should be paid attention to.In April 2019,African swine fever prevention and control work of the State Council required pork product processing enterprises to test raw pig products and make records.Recombinase polymerase amplification(RPA)provides the possibility to realize this idea.This method can detect and react at a lower temperature(37?42 ?),and takes a shorter time(10?20 min).In this study,a rapid real-time fluorescent RPA detection method for ASFV,pdcov and SVA,and a multiple real-time fluorescent RPA method for simultaneous detection of the above three viruses were established.The specific research results are as follows:1.The fast real-time RPA fluorescence probe detection methods of ASFV,pdcov and SVA were established,which can detect the results in 20 minutes,with the sensitivity of94 copies/?L,77 copies/?L and 57 copies/?L respectively.The results of detection of suspected positive samples of ASFV were consistent with that of fluorescent PCR,and the detection rate was 100%.2.Food samples were simulated by adding common additives of edible salt,edible oil and nitrite to the positive samples of African classical swine fever.The results showed that common additives of edible salt,edible oil and nitrite in meat products would not affect the detection of African classical swine fever virus by real-time fluorescence RPA.The results showed that the common processing methods of food would not affect the detection of African swine fever virus by real-time fluorescent RPA.3.A multiple real-time RPA fluorescence probe detection method was established to detect ASFV,PDCOv and SVA simultaneously.The results were detected in 20 minutes with sensitivity of 940 copies / ?L,770 copies / ?L and 570 copies / ?L,respectively.The suspected positive samples of ASFV were detected and compared with the fluorescent PCR method.The detection rate was 66.67%.The real-time fluorescent RPA detection method established in this study has the characteristics of high sensitivity,strong specificity,fast speed and simple operation,which can meet the requirements of food processing enterprises,market supervision departments and other rapid detection.