| The LA-PCR method for IBDV genome was established in this paper. By this method, Cloning and sequence analysis of the genome of three isolates SD-LX,SD-RZ,HB-HD were carried out. The result of complete genome sequence and VP2 amino acid analysis showed that the three isolates have character of vvIBDV strain in the fields of phathogenicity, complete genome sequence and amino acid sequence, but SD-LX isolate has an amino acid on N279 position, which shows the character of attenuated strain. This result was not in accord with the previous study of Yamaguchi,Lim.So the virulence of four IBDV must be caused by different amino acid sites rather than two individual sites. Further verification should be done through Reverse Genetics.A high efficiency DNA polymeraseâ…¡-mediated Reverse Genetics System was established in this paper. The complete genome of genomes A and B in SD-RZ isolate had been cloned into the CMV enhancer of PVAX1 fused gene to construct infectious clone PVAX1-A and PVAX1-B.After purified with plasmid Midi Kit (Invitrogen), transfected by LipofectamineTM2000 to CEF, then tested by RT-PCR and AGP, the extricated IBDV were detected. This study will provide a solid basis for the further study on the function of IBDV and the knock out of VP5.A simple, quick RT-LAMP method was established to detect Infectious bursal disease viruses.the specific outer and inner primers were designed in the VP3 region conserved site of the genome of Infectious bursal disease viruses. MgSO4, Betaine, Bst DNA Polymerase, dNTP, AMV, two pairs of primers and other conditions were optimized, sensitivity and specificity were tested. In addition, as LAMP method was performed under isothermal conditions without special apparatus needed, which makes it more economical and practical than PCR. This new method might facilitate genetic analysis applied in clinical laboratory and field surveillance. Meantime, it also has some potentials for rapid test. |
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