Development And Evaluation Of A Rapid And Low-Cost Detection System For Apple Stem Groove Virus Based On Crispr-Cas12a Technology

Apple(Malus domesica)is one of the largest fruits in the world,mainly planted in the temperate region of the world,and has a high commercial value.However,the apple industry is facing a serious apple virus disease problem.Recessive viruses such as apple chlorotic leaf spot virus(ACLSV),Apple stem pitting virus(ASPV),Apple stem grooving virus(ASGV)are common in China,and are harmful.Apple latent virus usually does not show obvious symptoms and there is no effective chemical or biological agent.Once infected,it will carry the virus for life and suffer long-term damage.Therefore,strengthening the virus quarantine of seedlings and trees in the park is the only way and fundamental guarantee to prevent and control virus diseases.CRISPR-Cas technology is a newly emerging genome editing technology.CRISPR-Cas technology is a new genome editing technology.Recent studies have found that the Cas12a protein can stimulate trans-cleavage activity after binding to the target,cleaving any single-stranded DNA in the system.Gold nanoparticles(AuNPs)detection is a simple and cheap method,The aggregation of AuNPs will cause color changes.In this study,CRISPR technology was combined with DNA AuNPs probe.Relying on cas12a to identify the virus and prepare cheap DNA-aunp probes to realize the visual detection of ASGV.The results as follows:1.Establish ASGV fluorescence detection system based on CRISPR-Cas12a technology.Explore the conservative region of ASGV and select the target sequence,design RPA primers and cRNA.Optimize the reaction system Determine the optimal reaction buffer and the optimal reaction concentration ratio of Cas12a/crRNA complex.The results show,Cas12a can bind to specific sites of ASGV and activate trans-cleavage activity,Produce a strong fluorescence difference with the control group.The best buffer for the reaction is commercial NEBuffer 2.1,When the concentration of Cas12a/cRNA was 50 nM and 62.5 nM,the cleavage activity of Cas12a was significantly enhanced,and the fluorescence signal reached the peak within 30 min.2.Establish ASGV visual detection system based on CRISPR-Cas12a technology.Rapid extraction of RNA from plant leaves by(PEG)solution,Preparation of DNA-AuNPs probes for colorimetric detection.Explore the effect of Linker-ssDNA concentration on the speed of DNA-AuNPs cross-linking.Establish the optimal reaction conditions for the ASGV visual detection system.The results show,Saturated sodium chloride can accelerate the crosslinking speed of DNAl/2-AuNPs.When the concentration of Linker-ssDNA is higher than 40 nM,DNA1/2-AuNPs can be observed to change from red to colorless in 1 min.Therefore,this study established a method for rapid detection of ASGV based on CRISPR-Casl2a technology and DNA-AuNPs probes-Achieve rapid extraction of RNA from apple leaves,RT-RPA directly amplifies the crude extract of apple leaves.Add the amplified product to the Cas12a/crRNA and 100 nM Linker-ssDNA reaction system,Mix with an equal volume of DNA-AuNPs after 30 min,and detect the virus by observing the color change after centrifugation.3.Research the specificity and sensitivity of ASGV detection.13 apple leaf samples were randomly selected for CRISPR-Cas12a/AuNPs detection and RT-PCR detection.Compare the detection results of the two methods to evaluate the specificity of CRISPR-Casl2a/AuNPs for ASGV detection.Under the optimal reaction conditions,serially dilute the ASGV virus standard plasmid by 10-fold,perform CRISPR-Cas12a/AuNPs detection and RT-PCR amplification,Determine the detection sensitivity of CRISPR-Cas12a.Detect 51 field plant samples,Investigate the applicability of this method in international detection.The final result shows that the detection method has good specificity,The sensitivity of this method is 2.5×103 copies/reaction,which is 100 times higher than that of RT-PCR In the field decection,the decection results of 51 samples were consistent with the RT-PCR decection results.In summary,we have established a method for detecting ASGV based on CRISPR-Cas12a technology and DNA-AuNPs probes.The method is a constant temperature reaction condition of 37 ℃.The sensitivity was greatly improved by RT-RPA.The DNA-AuNPs probe of this method is versatile,does not require expensive fluorescent probes,does not require large-scale detection equipment and the process is simple.Low-speed centrifugation can detect multiple samples within 1 hour.Its sensitivity is 2.5×103 copies/reaction.It has good specificity and high sensitivity,low cost and high efficiency.In actual production,it can detect a large number of field samples and identify the virus-free apple seedlings.