Genomic Characterization And Real-time PCR Assay For Detection Of Duck Astrovirus Type 3 Epidemic Strain

Duck viral hepatitis(Duck viral hepatitis,DVH)is a serious hazard ducklings acute,highly contagious disease.The infectious agents of this disease include Duck hepatitis A virus(DVAH)and Duck Astrovirus(DAstV).The existing research shows that there are four kinds of duck astrovirus(DAstV),namely duck astrovirus type 1(DAstV-1),DAstV-2,DAstV-3 and DAstV-4.Among them,the DAstV-3 caused a high infection rate and incidence rate in ducks,and prevailing in different areas in China.At present,there are few reports on the molecular characteristics of DAstV-3 and its rapid detection methods.The detection method for this virus is mainly the conventional RT-PCR method,which can only qualitatively diagnose DAstV-3,and can not accurately quantify the infection of the virus.In this study,we performed a whole genome sequence analysis of a strain of newly isolated DAstV-3 and established a SYBR Green I real-time PCR method for the virus detection.In this study,a strain of DAstV-3(named GX1806 strain)was isolated from clinical samples.The genome of GX1806 strain was amplified fragmentally by RT-PCR.The sequencing results were edited,and spliced into a whole viral genome of GX1806 strain.Genome sequence analysis showed that GX1806 strain had a total genome length of 7463nt and had typical characteristics of astrovirus genome,and had three open reading frames of ORF1a,ORF1b and ORF2.In the overlapping region of ORF1a and ORF1b,there was a ribosomal shift frame signal consisting of a7-base sliding sequence(AAAAAAC)and stem ring structure.ORF1b exists in the single strands are indispensable in the chain RNA virus replication of RNA dependent RNA polymerase(RDRP)gene.Sequence analysis showed that GX1806 strain was closely related to CPH strain in GenBank,and the whole genome sequence had 96.87%homology.To establish a Quantitative Real-time PCR(Q-PCR)method for the detection of DAstV-3,a 609 bp conserved region in the RDRP gene of DAstV-3 was amplified and cloned into the pMD18-T vector.The plasmid was subjected to 10-fold gradient dilution and used as a standard template for Q-PCR,and the amplification standard curve was prepared.When the content of RDRP gene was(1.15×10~2~1.15×10~8)copy/?L,the linear amplification was good,the amplification correlation coefficient was 0.997,and the amplification efficiency was 99.9%.The established DAstV-3 real-time fluorescence quantitative PCR assay was highly sensitive with a minimum detection limit of 1.15×10~2copy/?L;It is highly specific and has tested negative for common viruses(such as AIV,DRV,ATMUV,DHAV,DAstV-1,and DAstV-2).The repeatability results indicated that the intra-group repeatability coefficient was 0.14%~0.65%,and the inter-group repeatability coefficient was 0.53%~2.20%,indicating goodrepeatability.SYBR Green?real-time fluorescent quantitative PCR detection method of positive rate was 59.5%(22/37),conventional polymerase chain reaction(PCR)to the positive rate was 43.2%(16/37),two methods of coincidence rate was 100%.The above results show that the real-time fluorescence quantitative PCR detection method based on SYBR Green?established for the first time in this study can be used for early diagnosis of disease caused by DAstV-3 in clinic.