The Establishment And Application Of Dot Blot Hybridization Assay For Detection Of CIAV And PBFDV

Multiple diverse circovirus are widely spread in livestock,such as porcine circovirus(PCV),chicken infectious anemia virus(CIAV)and psittacine beak and feather disease virus(PBFDV),which caused extensive infection in domestic animals and poultries.CIAV causes chicken infectious anemia(CIA),which is an immunosuppressive disease with characteristic by bone marrow fatty degeneration,systemic lymphoid tissue atrophy and aplastic anemia in chicks,also can infect other virus,bacteria and fungi.Psittacine beak and feather disease(PBFD)is the most common disease in parrots,it can cause the feather symmetry atrophy,shedding or abnormal growth,and abnormal deformation of beak and paw,eventually death caused by reduced immunity and is extremely serious to the parrots industry.At the moment,commercial vaccines against porcine circovirus disease have been widely used in commercialized swinery,.However,no effective vaccines are available to provide protection against CIA and PBFD,which provides convenience to use conventional molecular biology methods to detect.In this study,two dot blot hybridization assays for detection of CIAV and PBFDV were established,respectively,and the genome sequencing and analysis of CIAV and PBFDV which detected positive were performed by this method.In order to provide reference models for screening and detection of similar viruses among different species,and also provide reference data for the variation of pathogen molecules.1.Establishment of dot blot hybridization assays for detection of CIAV and PBFDVIn this study,digoxin labelled oligo-nucleotide probe targeting CIAV or PBFDV were used to hybridize DNA samples collected from broiler chickens or parrots suspicious of CIAV and PBFDV infection,respectively.At the same time,these DNA samples were screened by CIAV and PBFDV conventional PCR.The result showed that dot blot hybridization assays displayed high sensitivity and specificity,and was highly consistent with the results obtained by PCR methods.Thus,this study provides sensitive and specific methods for molecularepidemiology and clinical diagnosis of CIAV and PBFDV infection,and also for further understanding.2.Whole genome sequencing of CIAV and PBFDV samples and analysisThe dot blot hybridization assay established in this study was applied for screening broiler chickens and parrots samples with suspected CIAV and PBFDV infection.Primers for CIAV and PBFDV were designed and synthesized based on published sequences to amplify the positive samples of CIAV and PBFDV by dot blot hybridization assay,the homology and phylogenetic analysis were carried out as above.The results showed that the homology between the five CIAV strains sequence was 97.5%-99.2% at whole genome level,while the homology between the two PBFDV strains was 100%,which further demonstrated the highly homogeneity of circoviridae viruses infecting poultries and birds.Furthermore,whole genome sequencing would facilitate understanding the CIAV and PBFDV’s phylogeny and molecular epidemiology,and help to control these two diseases from spreading in China.