Preliminary Study On The Detection Method Of African Swine Fever Virus Based On PfAgo Protein

African swine fever(ASF)is a highly contagious viral disease of livestock and wild boars,the main clinical symptoms are fever,bleeding and skin cyanosis,and the mortality rate of acutely infected pigs is as high as 100%.The ASF outbreak first broke out in Kenya in the 1920 s and has since become endemic around the world,posing a huge threat to the development of China’s aquaculture industry.Because there is currently no effective commercial vaccine or therapeutic,rapid,highly sensitive,and highly specific ASFV detection in the field is critical for early management.In recent years,a new gene-editing tool,a restriction enzyme based on the pfAgo protein,has been reported for nucleic acid detection,showing that the method is minimally detectable in single copies,easier to manipulate and more economical than traditional methods,and that pfAgo protein can recognize any region of the target DNA,with great advantages in vitro diagnostics.In this study,the recombinant plasmid of the synthesized pET28a-pfAgo was first converted to E.coli BL21(DE3)/p Lys S competent cells,followed by induced expression and purification of pfAgo protein,and the pfAgo protein was validated using Western Blot.Using 20% TBE-PAGE assay to detect whether the pfAgo protein has the desired activity.Download genome sequences of multiple ASFV p72 strains on the NCBI,and perform sequence comparison by Meglign to obtain a conservative sequence.ERA primers,g DNA primers,and fluorescent quantitative primers were designed for conservative sequences and screened,and after the fluorescence method and dipstick method reaction system based on pfAgo protein were initially established,they were optimized in many aspects,and the minimum detection limit and specificity were explored using the optimized conditions.Finally,clinical samples were tested,and the two methods established were compared with the results of the standardized detection of African swine fever kits.The results showed that the high-purity pfAgo protein was successfully prepared in this study and had cleavage activity.Primer screening yielded gp72-6-1 as the appropriate g DNA primer,F1 and R2 as the best ERA primer pairs,the template was better visualized after isothermal amplification,and the sensitivity test showed that the fluorescence method could detect as low as 4copies/μL,the dipstick method could detect as low as 80copies/μL.Specificity tests have shown that the two methods based on pfAgo protein are highly specific,do not cross-react with other important porcine viruses.Both methods have the potential to be applied to clinical samples compared to standardized kits.In summary,this study successfully established two assays based on pfAgo protein for rapid detection of ASFV-p72 gene,with high sensitivity and high specificity.This method provides new technical support for the detection of ASFV,and extends the pfAgo protein to the field of rapid detection of animal pathogenic microorganisms.