Establishment Of A Semi-Nested RT-PCR Assay And Analysis Of The Genome Of Canine Astrovirus

Canine astroviruses are non-enveloped single-stranded positive sense RNA viruses with a·ve to six point star-like appearance. Generally, astrovirus infections are associated with enteric diseases, with mild to severe signs such as diarrhea, vomiting and abdominal pain. So, it is important to develop a method for canine astrovirus detection. About the semi-nested RT-PCR detection method of canine astrovirus, primers were designed, targeting a relatively conserved region at the 5′-end of ORF2 based on the published genomic sequence of canine astrovirus and this generated a 480-bp amplicon. The best reaction conditions of the method is as follows: the optimal concentration of primers was 0.24umol·L^-1, the optimal concentration of dNTP was 0.4mM, the optimal concentration of Mg2+ was 1.5mmol·L^-1, the optimal concentration of Taq enzyme is 0.05U/ul and the optimal annealing temperature is 56℃. Stool specimens were collected from 183 dogs with diarrhea and 138 healthy controls in Shanghai. Canine astrovirus were detected by using this method. 22 (12.02%) specimens from the 183 dogs with diarrhea and none (0%) from the 138 healthy controls were positive for astrovirus. Phylogenetic analysis indicated that the new isolates and the Italy strain were divided into two different clusters and the new isolates may represent a new strain of canine astrovirus. The result showed that the semi-nested RT-PCR assay had the advantage such as high sensibility, strong specificity and this method can be used for the epidemiological investigation and diagnosis of canine astrovirus.Using a combination of LA PCRTM and Genome Walking, 4 pairs of primers are devised based on those sequences available in GenBank. 5011 nucleotides of the genomic RNA of canine astrovirus were obtained, including the entire ORF2 gene (2475bp), the entire ORF1b gene (1536bp) and partial ORF1a gene (1198bp). Then a relatively conserved region at the 5′-end of ORF2 gene (P1 segment) was amplified by PCR, and inserted into the prokaryotic expression vector pET-30a. After identified by restrict enzyme and sequencing, the constructed recombinant expression plasmid was transformed into the receptive cells of E.coli BL21 (DE3) and induced by IPTG with a finial concentration 1mmol·L^-1, and the recombinant protein was expressed in the form of inclusion bodies. After the bacterium cell walls were disrupted by ultrasonication, the inclusion bodies of recombinant proteins were dissolved, and then were purified by Ni²⁺ affinity columns following with renaturation. The purified protein may make senses in the coming application in a serological test.