Establishment And Application Of A Method To Identify The Infectivity Of African Swine Fever Virus

African swine fever（ASF）is an acute and severe infectious disease of pigs caused by African swine fever virus（ASFV）.A virulent virus can cause a high mortality rate of more than 90%in pigs.ASFV has spread from Africa to Europe and,in recent years,it has spread to China and other high-yield pig producing countries in Southeast Asia,threatening global pork production and food security.Due to the serious harm of the disease and the complexity of pathogenic mechanism,and there is no effective vaccine to prevent and control it,it is listed as a notifiable animal disease by the Office international des epizooties（OIE）,and it is listed as a Class I animal disease in China.In order to carry out ASFV epidemic prevention and control more accurately,we established a method to identify whether ASFV has intact capsid structure in this study,used this method to detect the inactivation rate of ASFV by various commonly used veterinary disinfectants,and simulated the application of this method in the detection of meat samples and environmental samples.1.PMA-qPCR method establishmentIn this study,based on ASFV P72 gene synthesis primers and TaqMan probes,the synthetic plasmid containing P72 gene amplification fragments was used as a template,and the established method showed a good linear relationship in the plasmid concentration range of 1.25×10⁴ copies/μL～1.25×10¹⁰ copies/μL.A method was established to detect whether ASFV had intact capsids by using propidium bromide bromide（PMA）,verify the feasibility of the method,and optimize the experimental conditions of the method,such as PMA concentration,dark reaction time,exposure reaction time and other conditions.The results showed that after PMA was added,the virus,which was disrupted in the sample,would not be amplified in qPCR,proving that the method was effective.The optimal working concentration of PMA is 350mg/L.5 min is the optimal dark reaction time.10 min is the optimal exposure response time.2.To detect the inactivation rate of ASFV by disinfectantsUsing the established PMA-qPCR method,the disinfectants commonly used in farms such as phenols,aldehydes,alcohols,acids,alkalis,halides,chlorine peroxide and quaternary ammonium salts were collected,and the inactivation rate of ASFV by various disinfectants was detected by referring to the disinfectant instructions,and the test results showed that benzalkonium bromide,povidone-iodine,hydrogen peroxide,sodium dichloroisocyanurate,75%ethanol,potassium bisulfate,and 25%glutaraldehyde could reach more than 99%inactivation rate of ASFV The inactivation rate of potassium bisulfate and 25%glutaraldehyde on ASFV can reach more than99.9%.Optimization of treatment conditions such as treatment time,storage time,temperature,etc.The results showed that potassium bisulfate had the highest inactivation rate of ASFV when it was reacted for 5 minutes.After potassium bisulfate was stored for 360 days,the inactivation rate of ASFV could still reach more than99.9%.When the temperature is 0°C,10°C,20°C,30°C,40°C,the inactivation rate of potassium bisulfate on ASFV can reach 99.99%.3.Detection of ASFV capsid integrity in pork productsSimulate positive viral fluid,simulated inactivated virus fluid,and a mixture of the two.These three liquids are added to the collected pork products and then tested for ASFV.The results showed that the larger the proportion of simulated positive viral fluid,the lower the Ct value of qPCR,and a method for detecting the integrity of ASFV capsid in pork by PMA-qPCR was successfully established.The integrity of ASFV virus capsids in pork samples was detected by PMA-qPCR and the results were all negative.4.ASFV capsid integrity testing in farm environmental samplesCollect environmental samples from all parts of the farm,such as the off-site ground,fence,gate,first,second and third level isolation points,personnel ingredients,on-site living area facilities,on-site roads,pig farm door handles,pig feeding troughs,pig positioning pens,etc.The PMA-qPCR method was used to detect the integrity of the viral capsid of ASFV in the samples,and the results were all negative.