Isolation,Identification And Detection Method Of Avian Pox Virus From Turtledove

Avian pox is a highly contagious infectious disease caused by Avian pox virus（APV）.In recent years,the incidence of domestic fowlpox has been gradually increasing,and cases of infection have also appeared in wild birds.Rapid detection is the key to the prevention and control of the disease,but traditional detection methods have a long cycle and complicated operations.Therefore,the establishment of a rapid and sensitive detection method will contribute to the clinical diagnosis and prevention and control of the disease.1.Isolation and identification of avian pox virusIn this study,the pathological autopsy of turtledoves suspected of infection in the campus was carried out.It was found that the turtledoves had severe acne scabs and hyperplastic nodules on the coat,legs and eyes,and no obvious lesions in the internal organs.The tissue suspension was prepared from the pox spots at the lesions,and the artificial air chamber method was used for passage on the chorioallantoic membrane（CAM）of chick embryos.After three consecutive generations,pox spots appeared.It was preliminarily confirmed that a pox virus was isolated and named as TDPV-HB,ELD50was 10^-3.5/0.2ml,and obvious lesions were seen after infection of DF-1 cells.The virus P4b gene was amplified and the genetic evolution analysis was carried out.The results showed that the P4b gene sequence of the TDPV-HB strain was homologous to the corresponding sequence of the known APV and had a similarity of more than 90%.Further research remains to be done.2.Establishment of fluorescent quantitative PCR assay for APVThe APV DNA Polymerase gene was selected as the detection target and the SYBR Green I real-time quantitative PCR（q PCR）detection method was established.With reference to the APV standard strain in Gene Bank（Gen Bank:AF198100.1）,primers were designed,standard plasmids were constructed,q PCR reaction conditions were optimized,and the performance of the method was determined.As a result,the q PCR reaction conditions were optimized and it was determined that when the primer concentration was0.4μmol/L and the annealing temperature was 60.3°C,the amplification curve was typical,the melting curve had a single peak,and the specificity was good.It has no non-specific reaction with other common pathogens such as avian adenovirus,avian parvovirus,and Escherichia coli.The minimum detection amount is 10² copies/μL,which is 10 times more sensitive than conventional PCR,and the coefficient of variation within and between batches is less than 1%,with good repeatability and stability.The q PCR method established in this study and conventional PCR were used to detect laboratory mock samples and clinical samples at the same time,the detection rate of q PCR was 100%,and the detection rate of PCR was 83.3%.It shows that the sensitivity of q PCR method is better than that of conventional PCR.The above results show that the APV q PCR detection method established in this study has high sensitivity and good specificity.Can be used for APV diagnosis and quantitative analysis.In this study,a fowlpox virus TDPV-HB was successfully isolated,and the genetic evolution of the isolate was analyzed.And successfully established a rapid and sensitive APV SYBR Green I fluorescence quantitative PCR detection method,which provided technical support for early clinical diagnosis and quantitative analysis.