Phylogenetic Analysis Of Goose Astrovirus In Regions Of Anhui Province And Establishment Of Detection Methods Of Goose Astrovirus

Since 2015,a disease caused by goose astrovirus(GAst V)has emerged in China,with the typical symptoms of swollen joints and urate deposition in internal organs.The mortality rate can reach more than 30%,causing serious economic losses to the goose industry in China.At present there are no effective treatments and vaccines for the disease,and the prevention and control focus is on the early diagnosis and monitoring of the epidemic situation.Therefore,this study carried out research from the analysis of the genetic diversity of GAst V in some areas of the Anhui Province and the establishment of rapid detection methods,provide technical support for the epidemiological investigation and early diagnosis of GAst V.First,in order to better understand the prevalence of GAst V in Anhui province,32 clinical samples of liver and kidney of goslings with gout symptoms from some regions of Anhui province were selected in this study.RT-PCR was used to detect GAst V,and a total of 6 samples were found to be GAst V positive.We designed 14 pairs of specific primers and amplified the complete sequence of 5 strains of GAst V and named it AHAU1,AHAU2,AHAU3,AHAU4,AHAU5,submit it to Gen Bank,with nucleotide homology analysis and phylogenetic analysis.The results of nucleotide sequence similarity analysis showed that the similarity between the amplified 5 strains of GAst V and the reference GAst V strain was 97.4%-99.2%,and the similarity of nucleotide sequence with the SDPY strain was the highest,reaching 99%.A phylogenetic analysis showed that AHAU1 and AHAU4 are in the same branch as the reference strain SDPY(MH052598)and have a high relationship;AHAU2,AHAU3,and AHAU5 formed separate branches.Through the analysis of GAst V genetic situation,it provides data support for further understanding of the phylogenetic analysis of GAst V in Anhui Province.In order to diagnose GAst V quickly and accurately,this study designed primers based on GAst V's conserved gene ORF2 gene,and optimized the reaction conditions and reaction system,and finally established two detection methods of SYBR Green I based real-time RT-PCR and reverse?transcription loop?mediated isothermal amplification.SYBR Green real-time RT-PCR test results showed,the detection on fowl adenovirus serotype4(FAd V-4)?infectious laryngotracheitis virus(ILTV)?avian leukosis virus(ALV)?goose parvovirus(GPV)and infectious bronchitis virus(IBV)pathogens were negative except the GAst V.It indicated that the assay has good specificity.The lowest template detection ofthe assay was 37.5 copies/?L.The coefficients in the repeatability test were less than 1 %.Thirty-two clinical samples from Anhui province were tested using this assay.Using this method to detect 32 clinical samples,the positive detection rate was 25% higher than conventional RT-PCR,and the positive coincidence rate of the two methods was 42.85%,it shows that this method can be used to detect clinical samples.The conditions of LAMP amplifcation were optimized in terms of reaction time and temperature.The optimal conditions are 60 min in a 60°C water bath.No cross-reactivity was noted with FAd V-4?ILTV?ALV?GPV and IBV.The RT-LAMP sensitivity was comparable to that of nested RT-PCR.Thirty-two clinical samples of diseased goose livers and kidneys were tested the results showed that the positive detection rate of RT-LAMP was 9.33% higher than that of nested RT-PCR.The establishment of the two detection methods provides technical support for the rapid and efficient detection of GAst V in clinical samples.