Study On Detection Of African Swine Fever Virus Using CRISPR-Cas12a System

In recent years,African swine fever?ASF?has raged around the world,posing a huge threat to the aquaculture industry.In the absence of commercial vaccines,rapid and reliable laboratory diagnosis is essential for disease prevention and control.The traditional African swine fever detection method has the disadvantages of complicated steps and the need for professional and technical personnel,which is difficult to meet the needs of efficient detection of African swine fever.At present,the emerging gene editing tool CRISPR-Cas12a has been applied to nucleic acid detection,which is faster and easier to implement than qPCR and ELISA.Based on the activity of Cas12a after cutting target DNA,it can cut ssDNA,combined with the isothermal amplification system,add one end One end of the modified fluorophore modified the quenching group's ssDNA reporting system,which can detect trace amounts of DNA.In this study,an improved Cas12a DNA detection method was established and applied to the detection of ASFV DNA.?1?CRISPR-Cas12a protein expression purification and detection of ASFV DNA method.This experiment was based on the recombinant expression vector Cas12a-pet-28a,Induced expression and purification of Cas12a protein,and further verified the target protein by western blot.Based on the ASFV conserved region p54,3 sites were selected as ASFV detection sites,Cas12a detected 1ASFV,2ASFV and 3ASFV sites,and the experiment proved that Cas12a protein It can specifically recognize these three target DNA sites and cleave the ssDNA reporting system tzCas12a-j,which produces a fluorescence difference with the control group,that is,the Cas12a protein can effectively detect these three sites,and successfully established the Cas12a detection ASFV DNA method.?2?CRISPR-Cas12a detection DNA method optimization.Beca.u.se the fluorescence signal of the1ASFV experimental group?322010±39442 a.u.?was significantly higher than the2ASFV group?206115±29239 a.u.?and the 3ASFV group?159120±12190 a.u.?.Therefore,1ASFV was selected in this experiment The temperature,pH,ssDNA reporter system and reaction buffer components of the detection system were optimized at the site,and it was found that the reporting system tzCas12a-j,reaction temperature37°C,pH 8.0,Mg²⁺5 mM and contained 1mM DTT,5%glycerol and 50?g/mL Heparin.The system is the best condition for Cas12a detection of ASFV DNA.?3?Evaluation of the specificity and sensitivity of CRISPR-Cas12a for ASFV detection.The detection results of different concentration gradient target DNA based on 1ASFV site show that the detection limit can reach 10^-18M.1ASFV fully matched crRNA detects porcine pseudorabies virus?PRV?and porcine circovirus type 2?PCV2?.Studies have shown that tzCas12a-j cannot be cut open and almost does not emit fluorescence.It proves that the test has good specificity,and has with no cross reaction with PRV and PCV2.In summary,this study successfully established a detection method based on Cas12a for detecting ASFV DNA with high sensitivity and high specificity.This method provides a new idea for the rapid detection of African swine fever and promotes Cas12a to rapid Detection of pathogenic microorganisms.