Establishment Of A Magnetic Ionic Liquid-based Nucleic Acid Extraction And Detection Method Of African Swine Fever Virus

DNA detection and analysis is an important technique widely used in agronomy,medicine,genetics and other fields,and the purity and quality of DNA extraction has a critical impact on the reliability of the analytical results.In biological samples,the specific extraction of target DNA with high purity from complex mixtures is complex and challenging.African swine fever（ASF）,a highly contagious disease of domestic and wild boars caused by African swine fever virus（ASFV）,has been spreading rapidly in 31provinces across the country since 2018 and is severely affecting pig performance and hindering economic development.Currently,no effective commercial African swine fever virus vaccine and potent drugs have emerged globally;therefore,early prevention has become an important tool to control African swine fever disease.In this context,a magnetic ionic liquid（MIL）-based nucleic acid purification and extraction method for ASFV and a real-time fluorescent quantitative PCR assay for ASFV were developed in this study.The specific study is divided into two parts as follows:1.In this study,six pairs of fluorescent quantitative PCR primers and Taq Man probes were designed based on the conserved sequence of ASFV VP72 gene,and the fluorescent quantitative PCR primers and corresponding probes with better amplification effect were screened based on the results of conventional PCR amplification,and a fluorescent quantitative PCR assay for ASFV was established after the optimization of the reaction system.The specificity test results showed that the nucleic acids of a total of four viruses,namely porcine group A rotavirus,porcine reproductive and respiratory syndrome disease virus,porcine epidemic diarrhea virus and porcine pseudorabies virus,were not amplified,but the nucleic acids of African swine fever virus were effectively amplified,which verified the better specificity of the method.The results showed that the method was specific for ASFV,as it did not amplify the DNA of four other porcine viruses（PRo V,PRRSV,PEDV,PRV）.However,it did amplify the DNA of ASFV,verifying the good specificity of the method.The method was also found to be sensitive,with a minimum detection limit of 10 copies/reaction.A standard curve was constructed based on the amplification results,and the correlation coefficient R²was 0.999,indicating good linearity.Finally,the reproducibility of the method was tested through three concentration gradients,and the results showed that the within-batch coefficient of variation was0.095%,0.131%,and 0.120%,and the between-batch coefficient of variation was 0.390%,0.401%,and 0.422%,all less than 0.5%,indicating good reproducibility.2.MIL-probe-based ASFV nucleic acid extraction method:This study established a MIL-probe-based ASFV nucleic acid extraction method.Single-factor experiments were performed to deter mine the optimal conditions for probe binding,DNA capture,and DNA desorption,and the optimal volume ratio,temperature,time,p H value,and ion strength were explored.The results showed that under the optimal conditions for probe modification and MIL synthesis,5μL of MIL-probe bound to the maximum amount of DNA（52,660 ng）.DNA was captured at p H 7 and a Na Cl concentration of 0.1 mol/L,and3 min of denaturation at 95°C and 5 min of capture at 58°C were optimal for DNA desorption.The maximum capture rate was 87.2%.In the DNA desorption experiment,the MIL-probe was eluted in dd H₂O at 70°C for 15 min,and the DNA desorption rate reached 90.51%.Based on the results of condition optimization,an optimal extraction protocol for target nucleic acids was established.Additionally,the reusability of the MIL-probe was tested by capturing and desorption four times,and the extraction efficiency of DNA was still 90.20±2.2%,verifying the good reusability of the MIL-probe in nucleic acid purification.Finally,a comparison of the optimized extraction method with a commercial extraction kit was performed,and the results showed that the minimum extraction concentration achieved using the optimized method was 10 times higher than that achieved using the commercial extraction kit,and the efficiency of DNA extraction at low concentrations was better with the optimized method than with the commercial extraction kit.In this study,an extraction method for ASFV nucleic acid purification based on oligonucleotide probes modified with magnetic ionic liquid and a fluorescent quantitative PCR assay for ASFV were successfully constructed.Among them,the extraction method can specifically purify and extract ASFV DNA with good reproducibility and reusability,and the extraction effect is better than that of commercial extraction kits,and combined with the established ASFV fluorescence quantitative PCR detection method,the early nucleic acid extraction and diagnosis of ASFV can be completed.