CRISPR-Cas13a Based Diagnostic Technology For Avian Influenza A(H7N9)virus Infection

Influenza A?H7N9?is one of the few influenza A viruses that can infect humans.When the influenza A?H7N9?outbreak,the conventional detection method is difficult to function due to the needs for expensive and bulky testing instruments and a high standard of detection environment.Therefore,it is urgent to find a simple,less expensive and on-site detection method for avian influenza A H7N9.The CRISPR-Cas13a protein is a codon-optimized protein.It is expressed by prokaryotes and displaying RNase Activity.It is guided by single stranded RNA to degrade the target RNA non-specifically.Harnessing Cas13a,the paper aims to establish an underlying on-site H7N9 virus nucleic acid detection method.The main result are as follows:1.Protein expressed by E.coli was collected for SDS-PAGE.By staining with Coomassie blue,the protein bands were analyzed by gel imager.The results showed that the expression of LwCas13a protein in Rosetta 2?DE3?pLysS E.coli cultured in LB medium accounted for 8.27%of total protein.The factors affecting the expression of E.coli were univariately optimized,and the control conditions were used to change the induction conditions.Then the proteins expressed in E.coli were subjected to SDS-PAGE electrophoresis.After staining with Coomassie blue,the gel imager was used to analyze protein bands.The results showed that when the OD₆₀₀ value of the E.coli index was 1.0,the target protein in the induced expression accounted for the highest proportion of the total protein,reaching 5.17%.When the final concentration of the inducing agent IPTG was 0.1 mM,the target protein was induced to express.The ratio of total protein was 5.97%.When the induction temperature was set to25�C,the ratio of the target protein to the total protein was 7.43%.At 16 hours of induction,the ratio of the target protein to total protein was reached.The maximum is8.13%.When LwCas13a was purified by affinity chromatography,the purification effect was poor,and many heteroproteins existed.When LwCas13a was purified by enzymatic cleavage affinity chromatography,the purification effect was very good.The protein was subjected to SDS-PAGE electrophoresis,and stained using Coomassie blue.After staining,the purity was good using a gel imager.2.Designed two sets of negative controls to verify the activity and specificity of the Cas13a protein complex.The results showed that the fluorescence value of the experimental group was significantly higher than the negative control of the two groups,which proved that the Cas13a protein complex has RNase activity and has good specificity against the target RNA.The 1 IU Cas13a protein complex was defined indirectly by reference to RNase A,and the optimum amount of each component was measured using a dichotomy method.The results showed that the 1IU Cas13a protein complex consisted of 1.875 pmol of Cas13a,2.25 pmol of crRNA and 64.46 pmol of target RNA.The Cas13a protein and crRNA were diluted on the basis of 1 IU of Cas13a protein complex to determine the optimal amount to be used.The results showed that in the 20?L assay system,there should be 9.375 nM Cas13a and 12.5 nM crRNA,plus 1?L of RNase inhibitor,100 ng of human total RNA,a certain amount of RNA to be detected and 300 nM RNA probe to constitute detection.system.3.The concentration of HA and NA gene RNA extracted from the H7N9 strain was determined by real-time fluorescence absolute quantification.The results showed that the HA gene RNA concentration was 29.14 fM and the NA gene RNA concentration was 819.72 fM.The minimum detection limit and the minimum detection time of the detection system was evaluated using known sample concentrations with different time intervals.The results showed that 1 nM of NA gene RNA could be detected within 5 min.To improve detection sensitivity,RT-RPA and T7 in vitro transcription were introduced to form a new detection system.Three sets of 9 pairs of RPA primers were designed for HA and NA genes respectively.The SX-H3-F+SX-H1-R primer pair and SX-N2-F+SX-N3-R primer pair were determined by screening for HA and NA to find the best RPA primer for the NA gene.The T7 promoter sequence was added to the 5'end of the downstream primer and a new detection system was constructed according to the instructions of the RPA kit.The known concentration of H7N9 RNA sample was used to evaluate the minimum detection limit and the minimum detection time of the detection system.The results showed that 100 aM of NA gene RNA could be detected within 30 min.Influenza A virus RNA extracted from various subtypes was tested using a new assay system.The test results showed that only the H7N9 virus group showed a significant increase in fluorescence value,which proved that the new detection system has good specificity.Therefore,we induced the expression and purification of the active Cas13a protein,and based on the Cas13a protein,a detection system with good sensitivity and specificity against the H7N9 influenza A virus was constructed.It provides a theoretical basis for on-site detection of H7N9 influenza A virus.