The invention relates to a multiplex real-time RT-PCR assay with a
heterologous internal control system (i.e., EGFP-RNA) for the simple and
fast diagnosis of classical swine fever virus (CSFV). Primers and
FAM-labeled TaqMan probes, specific for CSFV were selected by analyzing
the consensus sequence of the 5'-non translated region of various CSFV
strains. For determining the analytical sensitivity an in vitro
transcript (T7-PC3Alf) of the 5' NTR was constructed and tested.
Furthermore, a primer-probe system for the detection of the internal
control sequence was established, and a multiplex assay using CSF-System
1 and the IC real-time PCR could be performed as a one-tube assay without
loss of sensitivity or specificity.