The Development Of Rapid Diagnosis For IBDV By Using Molecular Techniques

Infectious Bursal Disease Virus has been a great concern for the poultry industry for a long time, particularly for the past decade when its "re-emergence" in variant or highly virulent forms. In this study, two sets of primers (Pta and Pts, IBDa and IBDs), flanking the hyper-variable region of VP2 gene, were designed to run a reverse transcription polymerase chain reaction (primary RT-PCR) and Nested-PCR. Both of these assays can amplify all of 12 reference strains which including pathotypes cIBDV, vvIBDV and vIBDV, but not the 5 negative reference pathogens of chicken. 34 field samples, which were collected from different places of Guangxi during 1993 to 2003, were detected by using developed primary RT-PCR and Nested-PCR. 11 samples were positively detected by the primary RT-PCR, while 23 samples were positively detected by the Nested-PCR, significantly increasing the sensitivity of the technique.Virul isolation had been done for the 23 positive samples by using CAM or CEF. We only got 11 strains of IBDV. It indicated that developed primary RT-PCR and Nested-PCR are rapid diagnostic method for IBDV.For the products of primary RT-PCR and Nested-PCR are all encompassing the hyper-variable region of VP2 gene, so we can take a restriction enzyme analysis (REA) directly. Two useful restriction endonucleases (Sspl and SacI) were choosed to type the different pathgenic IBDV strains. The result is SacI only cleaved cIBDV (4vaccine strains: BJ836, B87, D78, BDC and 6 standard cIBDV strains: Hel, He2, He3, He4, SD3/98, ZJ1/98) and vIBDV (American variant-E) RT-PCR products, whereas products obtained with vvIBDV strains (YL1,YL2,YL5,YLZ) were only cleaved with Sspl. But the vvIDBV strain GX8/99 that had been identified by GUI only have SacI side, and no Sspl, the reason need farther research.According to the specific different sequence between cIBDV and vvIBDV in vVP2 gene. We also designed two sets of primers (cIBDa+cIBDs and vvIBDa and vvIBDs). The primers designed for cIBDV can amplify all the strains. But the primers designed for vvIBDV can only amplify vvIBDV strains. So we can typing strains between vvIBDV and cIBDV by using vvIBDV-specific primers by onereaction .Typing the pathotype of field IBDV in Guangxi, 23 field sample' fragments were amplified by the primary RT-PCR or Nested-PCR.They were cleaved with Sspl and Sacl. And wIBDV specific RT-PCR also uesed to detected these samples In the years from 1993 to 2000, there are 8 strains belong to wIBDV and 13 belong cIBDV. Two strains can't typing for they can be cleaved with both of enzymes Sspl and SacI..