Differentiation of Infectious bursal disease viruses isolated from Iranian poultry flocks using real-time RT-PCR and high resolution melt curve analysis

Infectious bursal disease (IBD) is a highly contagious disease of young birds. Differentiation between classical virulent and very virulent IBDV (vvIBDV) isolate is very important for the poultry industry to choose the right vaccination program. Molecular and serological tests are time consuming and have variable sensitivity. However, the melting curve analysis is relatively fast method with high precision. This study was designed to evaluate the efficacy of the melting curve analysis for differentiation of some Iranian IBDVs which their identity had been previously determined by RT-PCR/RFLP analysis. In this study, after RNA extraction and reverse transcription and Real Time RT- PCR of IBDVs, high melting resolution at temperatures ranging from 81 to 92°C were performed. The findings of this study showed that in the high resolution melting curve analysis, the viruses were classified from A to D. Three vaccine strains of D78, Gumbokal, Bursa CE; IBD L; Bursine 2; and all field viruses were placed in groups A, B, C, and D, respectively. High resolution melting curve analysis after normalization also showed all viruses of this study were placed in 4 HRM genotypic group. Three strains, D78, Gumbokal, Bursa CE, produced similar and non-differentiable curve but were different from other vaccine and field strains. Two other vaccine strains, IBD L and Bursine 2, were different from each other and other viruses. We concluded that the real-time RT-PCR HRM technique is cost-effective and reliable among the currently used methods and can be used for differentiation of IBDV isolates.