| Goose circovirus was a new member of Circoviridae recognized recently. Circoviruses invade lymphoid tissue and lead to immuno-suppression, growth retardation and an increased probability of secondary infections.In 2005, we detected GoCV in a dead goose infected with avian influenza virus from Yongkang, and constructed the pMDT-GoCV1-yk01 plasmid with 1813bp GoCV nearly full length genome. A pair of primers with BamH I restriction site was designed to amplify the complete genome of GoCV. The amplified DNA was cloned into pGEMT-easy vector, and a positive recombinant plasmid (the pGEMT-GoCV-Bam) was selected. The 1.8 kb DNA fragment of full length GoCV genome was cut from the pGEMT-GoCV-Bam by BamH I, self-ligated and then ligated to an adapter. The 3.6 kb DNA with two GoCV genome copies was amplied with the adapter primer and was then cloned into pGEM-T Easy. An infectious GoCV DNA clone with tandem two GoCV genome copies was identified. The infectious DNA clone with Lipfectamine was injected into negative embryos though allantoic cavity and embryo, and into goose though enterocoelia and vein, respectively. PCR detection result showed that there were positive bands amplified from a BF and 6 sera samples.Conserved regions were identified by an alignment of 24 published sequences of GoCV. Three pair of primers was designed with Primer Premier 5.0 and the best one was selected using conventional PCR. The pMDT-GoCV-yk01 plasmid was served as a standard template to establish the real-time PCR method and to construct the standard curve. The analysis of melting curve was also carried out to elucidate the specificity of amplification. It was concluded that the established real-time PCR assay had a wide detection range (Ct:12～32) and low detection limit (as low as 1.46×102 copies of virus DNA). The established assay could also detect GoCV in tissue and serum from transfected geese. They were 1.57×106 copies/mg in the BF and within 4.96x 104-5.92×105 copies/μL in sera. |
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