Summary
As a consequence of the recent outbreaks of bluetongue (BT) disease amongst sheep in the Mediterranean Basin, and following the subsequent vaccination campaign to control further spread of the disease and its long-term maintenance, it has become most important to develop rapid and sensitive methods that can reliably differentiate between field and vaccine strains of the causative virus. The authors describe a new method to differentiate bluetongue virus serotype 2 (BTV-2) field and vaccine strains, using the VP2 gene sequence differences between the South African vaccine and the Italian field wild-type strains. The method is based on the principle that the melting temperature of a DNA duplex gives information on the sequence, which enables the identification of even single-base alterations in the amplicon. The real-time polymerase chain reaction the generation of melting curves and fluorescence detection were all performed using the light cycler system (Roche). Primers and probes were designed using VP2 gene sequences. After RT-PCR, the melting curves analysis, derived by the fluorescence resonance energy transfer (FRET) real-time PCR, was performed using the light cycler data analysis program (Roche). To assess the diagnostic value of the method, a BTV-2 vaccine strain (Onderstepoort Biological Products, South Africa) was first compared against a field strain of BTV-2 (isolated during an outbreak in 2000 in Sardinia). The ability of the method to reliably identify all the BTV-2 strains was tested using an array of eleven BTV-2 field strains isolated during outbreaks in various Italian regions between 2000 and 2002 and other serotypes (BTV-1, BTV-4, BTV-9 and BTV-16) that had been isolated during recent outbreaks of BT in the Mediterranean Basin. The method was clearly able to differentiate BTV-2 strains of vaccine virus from all wild-type strains of the same serotype tested. The resultant melting curves distinctly reveal the two strains to have differing peak values of 47.8°C F 0.6°C and 60.5°C F 0.6°C, respectively.

Keywords
Bluetongue, Culicoides, Energy transfer, Fluorescence resonance, Molecular differentiation, Polymerase chain reaction.