Isolation,Identification And Mutation Analysis Of N-GAstV And Establishment Of Colloidal Gold Chromatography Detection Method

The N-GAstV is a newly emerged pathogenic virus in recent years,which can cause urate deposition in multiple organs of 5-20-day-old goslings.The high mortality had caused serious economic losses to the goose industry in my country.Therefore,it is great significance to strengthen the research on the pathogen of the N-GAstV.The rapid detection method to prevent and control the N-GAstV was established.It is of great significance to protect and promote the sustainable development of my country's goose industry.At present,methods such as RT-PCR,RT-q PCR and LAMP for the detection of N-GAstV have been established,but these methods all require the assistance of special instruments.The colloidal gold chromatographic detection method has the advantages of simplicity,rapidity,suitability for on-site detection,and no need for special experimental instruments.It is widely used in the diagnosis of epidemic diseases.In this study,7 N-GAstV strains were successfully isolated.The monoclonal and polyclonal antibodies against the N-GAstV were prepared.Based on this,a colloidal gold detection method for the N-GAstV was established.It can provide certain technical support for the prevention and control and rapid diagnosis of NGAstV.The contents of this study are as follows:1.Isolation and identification of the novel goose astrovirus pathogenIn order to understand the mutation of N-GAstV,this study collected 30 disease materials of geese of different days from 9 provinces in my country from 2019 to 2021.Necropsy showed that all the internal organs had lesions characteristic of urate deposition.The collected samples were tested by PCR,and N-GAstV was detected in all samples.And the positive samples were isolated by using chicken embryo hepatocytes(CEL).A total of 7novel goose astroviruses were isolated.The results of homology alignment and genetic evolution analysis showed that the nucleotide homology of the ORF2 gene of the 30 virus isolates was between 97.9% and 99.7%.There was little difference between the isolates.The nucleotide homology between the virus and the ORF2 gene of the N-GAstV SDPY strain was between 98.4% and 99.5%,and the amino acid homology was between 98.0% and 99.0%,They were closely related on the same branch of the evolutionary tree,indicating that NGAstV has not mutated significantly in recent years.The nucleotide homology between the virus isolates and the ORF2 gene of goose,chicken,duck and turkey-derived Ast V standard strains was low.Among them,the homology with GAstV FLX strain was between 42.6% and42.8%.The homology with DAst V YP2 strain was between 38.2% and 38.7%.The homology with TAst V MO/01 was between 38.2% and 38.4%.The homology with CAst V 4175 was between 37.0% and 38.4%,with the lowest homology and the farthest relationship.In addition,the isolates with Ast V from goose,chicken,turkey and duck were in different evolutionary tree branches.The genetic relationship was far away.In this study,the SDWF0601 isolate with the highest homology to the ORF2 genome sequence of the NGAstV SDPY strain and lower homology to other avian Ast V was selected as the experimental strain.And the viral nucleic acid was extracted to amplify the ORF2 target gene fragment.2.Preparation of monoclonal antibodies and polyclonal antibodiesIn this study,Primers were designed based on the published N-GAstV ORF2 genome sequence.The ORF2 protein was expressed prokaryotically.The protein was immunized with BALB/c mice aged 6 to 7 weeks that prepared spleen cells.After fusion with SP2/0,screening and subcloning,hybridoma cell that can stably secrete monoclonal antibodies.And obtained Mabs by inducing the production of ascites in mice by hybridoma cells.The type was Ig G2 a,and the antibody titer was 1:51200.IFA,WB and indirect ELISA identified that the monoclonal antibodies had good reactivity and specificity.The protein was mixed with Freund's adjuvant and incomplete Freund's adjuvant to immunize rabbits.After 4 times of immunization,blood was collected to prepare Pabs.And the titer of Pabs was 1:102400.IFA and WB identification showed that the polyclonal antibodies had good reactivity.3.Establishment of colloidal gold immunochromatographic detection methodIn this study,gold particles were prepared by reducing chloroauric acid with trisodium citrate.And the color of the prepared colloidal gold was clear and uniform.Optimized for each condition,the optimal binding concentration of colloidal gold and monoclonal antibodies were 6 ?g/m L.The optimal binding p H was p H 8.0.And the optimal immunocolloidal gold resuspension dilution was 0.01 mol/L Tris-Cl+ 1% sucrose+ 1% BSA.The prepared monoclonal antibodies were used as the gold-labeled antibody,and the polyclonal antibodies were used as the coating antibody The colloidal gold chromatography technique was established to detect N-GAstV.According to the optimization of each condition,the best coating conditions was PBS.The best coating concentration was 1.6 mg/ml.The sensitivity test showed that the test strip was 1.2 ?g/ml and the PCR was 0.12 ?g/ml of minimum amount of virus.The sensitivity was similar.The specificity test showed DAst V,GPV,H9-AIV,FAd V-4,DHAV-3 and TMUV were all negative,and no cross-reaction.The repeatability test showed that the virus can be detected using different batches of test strips.The clinical sample detection showed that 40 samples from different regions were collected.The test strip can detect 37 positive samples,and the coincidence rate with PCR method was92.5%.The stability test showed that the test strip can be stored stably for 3 months at room temperature of 25°C and 5 months at 4°C.Moreover,the method had a short reaction time,and results can be obtained within 10 min.Colloidal gold chromatography detection technology can be used as a simple,rapid,sensitive and specific method for the detection of novel goose astrovirus.