Differentiation of Italian field and South African vaccine strains of bluetongue virus serotype 2 using real-time PCR.

The current outbreaks of bluetongue (BT) disease in sheep in the central parts of the Mediterranean basin are being combated by extensive vaccination to control further spread of the virus and to suppress its long-term maintenance in the field. To be able to monitor the success of this campaign, and to be able to identify new foci of the disease, it is necessary to harness diagnostic methods, both rapid and sensitive, for differentiating reliably field from vaccine strains of bluetongue virus (BTV). A new method is described for their differentiation using fluorescence resonance energy transfer (FRET) probes with real-time PCR. The method is based on the principle that the melting temperature of a DNA duplex gives information about the sequence, and allows even double-base alterations in the amplicon to be identified. The RT-PCR, the generation of melting curves, and fluorescence detection were all performed using the LightCycler system (Roche Diagnostics, Mannheim, Germany).