Development of a triplex TaqMan real-time RT-PCR assay for differential detection of wild-type and HCLV vaccine strains of classical swine fever virus and bovine viral diarrhea virus 1

In this study, genomic sequences of pestiviruses available in GenBank were aligned to design three primer pairs and TaqMan probes: two targeting the NS5A region of the viral genome of classical swine fever virus (CSFV) for the differentiation of wild-type CSFV and hog cholera lapinized vaccine (HCLV) vaccine, and one targeting the 5'-untranslated region of bovine viral diarrhea virus 1 (BVDV-1). With these primers and probes, a triplex TaqMan real-time RT-PCR assay was developed for differentiating wild-type CSFV, the HCLV strain, and BVDV-1. The detection limit of the assay was 4.5 TCID(50) for wild-type CSFV, 10 TCID(50) for HCLV-strain CSFV, and 3.2 TCID(50) for BVDV-1. The triplex real-time RT-PCR had at least 98% (248 samples) agreement with other RT-PCR methods. The assay provides a sensitive tool for simultaneous detection and differentiation of wild-type CSFV and HCLV from BVDV-1.