Establishment And Preliminary Application Of A Detection Method For Porcine Pseudorabies Virus Based On RAA-CRISPR/Cas13a

Porcine pseudorabies is an acute infectious disease caused by pseudorabies virus（PRV）,which belongs to herpesviridae,a herpesvirinae,swine herpesvirus type I.It can infect pigs of different ages,and the mortality of newborn piglets can reach 100%,which can cause reproductive failure of sows,causing huge economic losses to the pig industry at home and abroad.Because PRV has no characteristic anatomical changes and often shows invisible process after infection,it can only be diagnosed by rapid diagnosis and molecular biology methods.At present,although the detection methods for PRV have their own advantages,most of them still need large-scale experimental equipment and professional operators to complete,the process is cumbersome and time-consuming,therefore,it is of great significance to establish a simple,fast and sensitive detection method with strong specificity for the prevention and control of PRV.CRISPR/Cas system is the main gene editing tool,in which CRISPR/Cas13a protein not only cleaves target RNA but also has accessory cleavage activity,once activated,it can non-specifically cleave single-stranded RNA,and has stronger cleavage specificity and recognition ability compared with traditional RNA interference technology.By utilizing the characteristic,ss RNA reporter molecules are added into a reaction system,so that visual reading of a result can be realized;and in addition,by combining with a flow measurement test strip and utilizing the characteristic of rapidness and convenience,the detection with high sensitivity and high specificity can be realized without the operation of professionals.Recombinase-mediated isothermal amplification（RAA）is a new technique for in vitro nucleic acid amplification,which uses recombinase,single-stranded binding protein and DNA recombinase to complete the amplification of target in a short time at constant temperature（37℃）.Because it is fast,low price and can be operated without thermal cycle instrument,it is widely promoted in grass-roots and field detection.At present,the combination of RAA and CRISPR/Cas13a detection system has been applied to the detection of ASFV、CSFV、PED and other viruses,but there is no method to detect PRV.In this study,based on the above two detection techniques,RAA and CRISPR/Cas13a cleavage reaction were combined to establish a detection method without large-scale equipment and professionals.The specific results are as follows:1.The LbaCas13a protein is successfully expressed and purified,and the cleavage activity of the LbaCas13a protein is verified;after the LbaCas13a plasmid is transferred into Escherichia coli BL21（DE3）,the required protein is successfully induced for 20 h under the conditions that the IPTG concentration is 0.5 m M,the temperature is 16℃,and the rotating speed is 150 r/min;LbaCas13a protein was purified by Ni-NTA column,and its cleavage activity was proved.2.The cr RNA for PRV and LbaCas13a was designed successfully.The conserved sequences of the same gene of different strains were compared and the Protospacer sequence was found according to the PFS sequence characteristics of Cas13a and the cr RNA was designed.The designed cr RNA was proved to be useful for detection.3.The primers for RAA and PCR were designed and screened successfully,and the T7promoter sequence was added to the 5’end of the primers.The results of isothermal amplification and fluorescence color experiment showed that the designed primers could amplify the target sequence and be used for detection.Four pairs of primers were designed according to the RAA design principle,and the best pair was selected for RAA detection according to the comparison results.4.The detection method of PRV based on CRISPR/Cas13a was successfully established and optimized.The results showed that one tube reaction was the best,and the optimal concentration system of Cas13a and cr RNA was optimized.The results showed that when the concentration of Cas13a was 400 n M and the concentration of cr RNA was 400 n M,the fluorescence monitoring system could get reliable results in 1 h reaction,and the test strip monitoring system could get reliable results in 20 min reaction.5.The specificity,sensitivity and reproducibility of the method were tested.The positive samples of PRV,PCV2,PCV3,PRRSV,CSFV and ASFV are detected by the detection method,and only the detection result of PRV is positive,so that the detection method has good specificity;The PCR products of 10¹⁰-10¹copies/μL were detected,and the results showed that the lower limit of PRV detection was 10 copies/μL.After multiple batches of positive samples with different copy numbers were detected,the intra-batch coefficient of variation was less than 5%,and the inter-batch coefficient of variation was less than 10%,which indicated that the detection method had good repeatability.In summary,this study established a method for rapid detection of PRV under constant temperature conditions,the results can be directly interpreted in the fluorescence and test strip,the method can be completed without large-scale equipment,with simple operation,low requirements,short time,which has become a powerful tool in the prevention and quarantine of pig industry.It provides a new technique for the diagnosis and epidemiological investigation of PRV.