Development Of CRISPR/Cas-based Nuleic Acid Detection Technique For Classical Swine Fever Virus And African Swine Fever Virus

Based on collateral RNase/DNase activities of Cas effectors,CRISPR/Cas diagnostic techniques have been rapidly developed in recent years.With labeled ssRNA/ssDNA reporter,CRISPR/Cas techniques can transform target RNA/DNA into fluorescent or visual readout at attomolar level with single-base mismatch specificity.CRISPR/Cas techniques have been widely used in infectious disease diagnosis since the first report of CRISPR/Cas 13a in 2017.In this study,CRISPR/Cas 13a and CRISPR/Cas 12a were used for classical swine fever virus and African swine fever virus detetion.A point of care testing with isothermal amplification and lateral flow dipstick was also established for African swine fever virus diagnosis.Combined with fluorophore-quencher-labeled RNA substrate as a readout for the presence of target RNA,CRISPR/Cas13a is a promising next-generation diagnostic strategy that can detect nucleic acid target,which had been widely used in resistance gene detection,cancer related gene mutation identification,human genetic information recognition and infectious disease diagnosis.As an OIE reportable disease,classical swine fever(CSF)is under eradication in China since 2017.Discrimination of CSFV infected and vaccinated animals is urgent for eradication of CSF.Cas13a is a single-component RNA-guided RNA-targeting CRISPR effector that can non-specically cleave ssRNA with its collateral RNase activity.In this study,CRISPR/Cas 13a based detection method was established for discrimination between virulent strains(Shimen and genotype 2.1 strain)and vaccinated HCLV with a detection limit of 3×102 copies/?L.In addition,with the use of heat and chemical reduction,HUDSON treatment was also introduced to achieve robust pretreatment of tested samples without extracting viral nucleic acids.HUDSON-RT-RAA-CRISPR/Cas13a assay can detect Shimen and HCLV cell culture samples with a detection limit of 3.45×102 copies/?L and 1.77×102 copies/?L,respectively.A total of 50 pig clinical samples were also assayed with HUDSON-RT-RAA-CRISPR/Cas13a,nPCR and antigen ELISA in parallel.HUDSON-RT-RAA-CRISPR/Cas13a showed 100.0%nPCR and 92%coincident rate with antigen ELISA,respectively.Compared Cas13a,Cas12a is a RNA-guided DNase that non-specically cleaves ssDNA with its collateral DNase activity.Thus,as a more stable,simpler and cheaper technique,CRISPR-Cas12a gradually replaces CRISPR/Cas13a for infectious disease diagnosis.African swine fever(ASF)is a highly contagious and usually deadly porcine infectious disease,bringing huge economic losses worldwide,especially since the first outbreak in China in 2018.In this study,AsCas12a was expressed in E.coli and purified by Ni affinity chromatography.AsCasl2a collateral DNase assay was firstly optimized,followed by ASFV candidate gRNA screening.Among 55 gRNAs designed according to the full length of ASFV B646L gene,gp72-P01,gp72-P02,gp72-N31 and gp72-N34 were determined as gRNAs for ASFV CRISPR/Cas12a diagonsis with a detection limit of 6×108 copies/?L Combined with RAA amplification,the sensitivity of RAA-CRISPR/Cas12a was greatly increased with a detection limit of 6×102 copies/?L.To avoid the use of fluorescence detection device for CRISPR/Cas12a diagnosis,we prepared a lateral flow dipstick for visual fluorescent readout.The detection time decreased from 1 h to 30 min when lateral flow dipstick was used,maintaining the same detection sensitivity.Finally,RAA-CRISPR/Cas12a showed 100%coincident rate with OIE-PCR when detecting 15 clinical blood samples and 15 serum samples.To make ASFV diagnosis can be performed in areas far from city with limited resouces and poor settings,a sensitive,specific,rapid and simple molecular point of care testing that combined RAA with lateral flow assay(LFA)was established.The workflow included treatment of blood samples with simple dillution and boiling for 5 min,isothermal amplification with RAA at 37? for 10 min,and visual readout with lateral flow dipstick at room temperature for 10-15 min.Without the need to extract viral DNA in blood samples,the intact workflow from sampling to final diagnostic decision can be completed with minimal equipment requirement in 30 min.The detection limit of RAA-LFA was 10 copies/?L,which was 10 fold more sensitive than OIE-PCR and quantitative PCR.Evaluation of clinical blood samples of RAA-LFA showed 100%coincident rate with OIE-PCR,in testing both extracted DNAs and treated bloods.We also found that some components in blood samples greatly inhibited PCR performance,but had little effect on RAA,indicating RAA is a better choice than PCR when blood is used as detecting sample.