| According to the published VIR gene sequence of CEV (strain NZ2) in GenBank, one pair of primer was designed and synthesized. Then the VIR gene of OV/nm-05isolated in Inner Mongolia was amplified by PCR and cloned into pEASY-T1vector. Before sequenced, the recombinant plasmid obtained was dentified by PCR and restriction endonucleases analysis. The acquired sequence which contained the552bp complete VIR gene was779bp. The homology of nucleotide sequences analysis showed that OV/nm-05shared highest identity (98.0%) with1710/03and lowest identity (94.2%) with513/04. The results demonstrated that there was a little variation among different strains.The annealing temperature was optimized with the primers mentioned earlier. To determine that the PCR method was specific, the DNA of OV/nm-05, calf testis cell and goatpox virus (strain GVX/nm) were all amplified with by PCR. Only OV/nm-05was amplified approximately779bp and others were negative. After the template of OV/nm-05was10times gradient dilution, the template was amplified. The minimum detection level of DNA was38pg. The result demonstrated that the PCR method was high sensitivity. |
