Development And Application Of CRISPR-Cas13a Technology For H9N2 Subtype Avian Influenza Virus

H9N2 subtype avian influenza is an acute and highly contagious disease caused by the subtype H9N2 of avian influenza A virus.When poultry are infected with the disease,it will not only cause respiratory symptoms,but also damage the reproductive system and lead to a decline in egg production.And it is difficult to return to normal levels after recovery.The virus can cause severe immunosuppression and cause a large number of deaths in infected poultry after secondary infection with other pathogens.In recent years,H9N2 subtype avian influenza has been prevalent in Asia,Europe,North America and other regions,which has brought serious harm to the breeding industry.At present,it is found that the epidemic mainly relies on daily monitoring,usually using emergency immunization and isolation treatment to control the spread of the disease.Therefore,it is of great significance to establish a convenient,sensitive and specific detection method for the monitoring and control of H9N2 subtype avian influenza.In order to establish a universal detection method,this study isolated a H9N2subtype avian influenza virus from a chicken farm in Xintai,Shandong Province,and analyzed the genetic evolution of HA gene and M gene respectively.The results showed that the HA gene of the isolate QS strain had 78.3%～93.5%nucleotide homology with the reference strains of each branch.The isolated strain belongs to the h9.4.2.5 branch,but it is not in the same branch as the ordinary vaccine strain and has a distant genetic relationship,indicating significant genetic variation in the HA gene of the isolated strain.The nucleotide homology of M gene with each branch reference strain was 82.7%～99.8%,Its sequence is more conservative.It is in the same branch with the vaccine strain WJ57 and has a close genetic relationship.Therefore,the conserved gene M gene of H9N2 subtype AIV was selected to establish a detection method.The CRISPR/Cas system provides a new idea for rapid detection of pathogens on the spot.The CRISPR-Cas13 a system can activate the incidental cleavage activity after cutting the target RNA,and can non-specifically cut nearby ss RNA.According to this characteristic,the addition of RNA reporter groups can achieve visual detection of pathogens.In clinical applications,DNA is pre-amplified by recombinase polymerase isothermal amplification(RPA),and then transcribed into RNA by T7 in vitro.The CRISPR-Cas13a-cr RNA complex can activate and cleave RNA reporter groups,so that target RNA in clinical samples can be detected faster and more sensitively.In this study,RPA was combined with CRISPR-Cas13a system for H9N2 subtype AIV,and two detection methods were successfully established.The detection results can be interpreted by monitoring fluorescence signal,blue light irradiation and test strip.One is to establish a fluorescence detection method based on RPA-CRISPR-Cas13a and optimize the reaction system:Using the M gene of H9N2 subtype AIV isolate(QS strain)and the RPA design principle,four pairs of RPA primers were designed for isothermal amplification and electrophoresis verification,and a pair of RPA primers with the highest amplification efficiency was successfully screened.Three cr RNAs were designed,and the best cr RNA was cr RNA1.The optimal reaction concentration of cr RNA was 2.56 ng/μL,and the optimal reaction concentration of Cas13a protein was 3.937 ng/μL.At the same time,the one-step detection and two-step detection were compared,and the results showed that the one-step detection reaction was faster and more sensitive;the sensitivity results of fluorescence detection and blue light irradiation showed that the detection limit of plasmid was 10copies/μL.The specificity results of fluorescence detection and blue light irradiation showed that there was no cross reaction when detecting other common infectious pathogens of poultry,indicating good specificity.A total of 48 clinical samples suspected to be infected with H9N2subtype avian influenza were detected by the established detection method.The results were consistent with those of PCR,and the positive and negative coincidence rate was 100%.The other is a test strip detection method based on RPA-CRISPR-Cas13a:the sensitivity of the test strip showed that the detection limit of the test strip was 10~2copies/μL;the results of specific detection showed that when detecting other avian infectious pathogens,only the test band appeared for H9N2 subtype AIV,indicating good specificity.Using this method to detect 48 clinical samples,The positive coincidence rate was about 94%.In summary,the use of CRISPR-Cas13a detection technology to detect H9N2 subtype AIV is not only fast,sensitive and highly specific,but also does not require special instruments when using blue light irradiation and test strip detection.The naked eye can directly interpret the results,which is suitable for the field screening of H9N2 subtype AIV.The establishment and application of this detection method provides a new idea for the rapid diagnosis of H9N2 subtype AIV,which is of great significance for the prevention and control of H9N2 subtype avian influenza.