Five microliters of extracted material was used per reverse transcription PCR (total volume 25 μL) by using the OneStep RT-PCR Kit (QIAGEN). The reaction mixtures contained 1 μmol/L of each primer and 0.2 μmol/L of probe. Design of the primers and probe was based on an alignment of all available SAFV sequences in GenBank (www.ncbi.nlm.nih.gov/genbank) in July 2010 by using ClustalW (www.clustal.org) and Primer3 (http:// frodo.wi.mit.edu/primer3) software. The primers and probe are selective for a highly conserved part of the 5′ untranslated region (Table) . The Strategene Mx3005P real-time thermocycler instrument (Agilent Technologies A/S, Horsholm, Denmark) was used for amplifi cation and detection with the following settings: 50°C for 20 min, 95°C for 15 min, followed by 45 cycles of 95°C for 15 s and 55°C for 1 min.