Deveiopment Of A Rapid Detection Method For African Swine Fever Virus In Pork

African swine fever virus(ASFV)is the pathogen of African swine fever(ASF),an acutely,severely and highly fatal disease of pigs.ASFV fatality rate is up to 100%.In china,ASFV has posed a new serious threat to the pig industry and seriously impacted the international trade of pig industry.There is still no effective treatment at present,biosafety is the only effective prevention and control approach.Early diagnosis of ASFV is the key to prevent and control the spread of ASF.ASFV spread through pork products is more hidden,more risky and more difficult to control.Detection of ASFV in pork products is of great significance to to prevent contaminated pork materials from entering food processing.At present,the Taq Man probe-based real-time quantitative PCR and the universal probe library quantitative PCR are the most widely used ASFV laboratory diagnostic methods in EU reference laboratories and OIE,but remote pig farms,small-sized and medium-sized pig farms and rural farmers of the Pig Group cannot establish diagnostic laboratories or apply real-time quantitative PCR for nucleic acid detection of ASF virus in large-scale pig farms.Therefore,it is necessary to establish a sensitive,specific and rapid on-site detection method for ASFV in pork products,that rapid screening and pre-detection of pig meat samples of farm can provide clues for the prevention and control of African swine fever.1.Establishment of a recombinase aided amplification for African swine Fever virus.In this study,multiple pairs of primers and probes were designed for ASFV highly conserved gene B646L/p72.The sensitivity was screened by real-time quantitative PCR.Based on this,the recombinase aided amplification(RAA)of ASFV was established.The specificity and sensitivity of the method were evaluated by ordinary PCR combined with agarose gel electrophoresis and non-denatured polyacrylamide gel electrophoresis.The verification results are as follows:This method is specific to ASFV with no cross-reactivity to pseudorabies virus(PRV),porcine circovirus type 2(PCV2)and porcine reproductive and respiratory syndrome virus(PRRSV).The method also highly showed sensitivity with a detection limit of 100 copies/ul for ASFV plasmid templates containing B646L/p72 gene.2.Establishment of African swine Fever virus quantum dot microspheres-based test strip method.In this study,the FITC polyclonal antibody coupled QDMs(QDM-FITC Pc Ab)were prepared based on the condensation between amino and carboxyl groups as described previously!QDMs based test strip was established based on the established RAA system.The verification results are as follows:This method is specific to ASFV with no cross-reactivity to pseudorabies virus(PRV),porcine circovirus type 2(PCV2)and porcine reproductive and respiratory syndrome virus(PRRSV).This method was ultrahigh sensitive to ASFV plasmid templates with a detection of 1 copy/μl per reaction.The reproducibility of the established method was also evaluated by analyzing the intra-and inter-assay coefficients of variance(CV)with 1 copy/μl and 10~2 copies/μl of ASFV plasmid templates.The CV values for 1 copy/μl of the intra-and inter-assay were 8.621%and 8.259%and 10~2 copies/μl of the intra-and inter-assay were 7.062%and 5.832%.respectively.3.Evaluation of clinical sample detection efficiency.Finally,a total of 120 clinical samples included 100 ASFV-negative pork samples and 20 simulated ASFV-positive pork samples were detected using the QDMs based fluorescent test strip assay established here.The results are as follows:The minimum detection limit of the established ASFV QDMs test strip method for positive pork simulated clinical samples was 100 copies/g.The good stability of the QDMs based test strip assay was confirmed,for the CV values as 9.916%,8.681%and 5.729%of blank control,negative pork sample and positive pork sample,respectively.Compared to the real-time PCR of OIE,the ASFV detection conformance of the QDMs based test strip assay was identified as 99.17%for 120 clinical pork samples.In conclusion,this study established QDMs based test strip assay and RAA method for on-site detection of ASFV with high specific,sensitive,stable and real-time detection method,that the result can be observed by naked eye.This detection method can complete sample detection within 25 min.The technology pretests samples in meat,small and medium scale or remote area pig farm,which has a good application prospect for control of ASF virus in pork processing industry.