Establishment Of Multiplex PCR Assay For Detection 5 Common Goose Viruses And Genetic Characteristic Of VP Gene Of Goose Plague Virus In Some Goose Farms In Hotan

Gosling plague virus(GPV),Newcastle disease virus(NDV),Goose Astrovirus(GAst V),Novel duck reovirus(ARV),Tembusu virus(TMUV)are pathogens that cause gosling plague,Newcastle disease,goose gout,goose hemorrhagic necrotizing hepatitis,and egg-laying decline syndrome.Those above five viruses can be transmitted through eggs,which risks carrying pathogens when introducing eggs.The death rate of infected geese is high,hindering the development of the geese industry and causing severe economic losses in Xinjiang.To help the rapid detection of the above five viruses in geese in Xinjiang,this study intends to establish a multiplex PCR assay for GPV,NDV,GAst V,ARV and TMUVSpecific primers were designed according to the conserved regions of GPV,TMUV,GAst V,ARV and NDV in Gen Bank and positive plasmids carrying viral gene as described above were constructed in order to established a single PCR assay for detecting 5 Kinds of common geese viruses.Based on the single PCR method,optimization of multiplex PCR conditions and analysis of its sensitivity,specificity and reproducibility were carried out and some clinical samples were tested by using multiplex PCR.The results showed that The multiplex PCR assay with annealing temperature of 57℃and primer concentrations of 0.8μm/L or 1.0μm/L has the better effectiveness and showed no cross-reactivity with avian influenza virus,avian pox virus and infectious bronchitis virus,and the detection limits of five viruses including GPV,TMUV,GAst V,ARV and NDV were 8.6×10 copies/μL,4.4×10~3 copies/μL,8.3×10~3 copies/μL,3.3×10~3 copies/μL and 8.8×10~3 copies/μL,respectively.The clinical samples were detected based on this detection.There were GPV 74.29%(26/35),NDV 20.00%(7/35)and GAst V 2.86%(1/35),the infections were 0%,mixed virus(5/35)in a goose farm in Hotan.The hepatic tissue samples were collected from a goose farm in Hotan and detected by the multiplex PCR method.After being treated,the single-infected samples were used to inoculate duck embryos for three serial blind passages.The specific primers of the GPV VP gene were designed and amplified by PCR,resulting in cloning vectors being constructing.Then the similarity and genetic relationship of VP1,VP2,VP3 gene were analyzed and advanced structure of viral VP protein was predicted.PCR results showed that the Goose Plague Virus was detected in the urinary bladder fluid of duck embryos,which was named as GPV/GOOSE/XJHT/2020,which was pathogenic to duck embryos.The results showed that the strain had a low affinity,with homology was 95%,and was indifferent branches compared with the vaccine strain(SYG61v)and low virulent strain(GPV-98E).However compared with the virulent strain,it had a high affinity,the homology was more than 99%and in the same branch.The secondary structure prediction of the VP gene showed that the protein had good immunogenicity.In this study,the single and multiplex PCR detection was established for GPV,TMUV,GAst V,ARV,and NDV virus.First,compared with the existing multiplex PCR detection,this method can detect more pathogens with high sensitivity,reasonable specificity and repeatability.Secondly,it successfully isolated a goose plague virus,sequenced its VP gene,and performed similarity analysis,genetic evolution analysis,and protein structure analysis.Multiplex PCR detection was established in this study to provide technical support for goose industrial disease detection,epidemiological investigation and prevention and control in Xinjiang.It provided essential data for the epidemic regularity of GPV and a theoretical basis for the construction of subunit vaccine of GPV in Xinjiang.