Development Of A Triplex TaqMan Real-time RT-PCR Assay For Quantitative And Differential Detection Of Wild-type And HCLV Vaccine Strains Of Classical Swine Fever Virus And Bovine Viral Diarrhea Virus 1

Classical swine fever (CSF) is a highly contagious, economically important infectious disease caused by classical swine fever virus (CSFV). It is one of World Organization for Animal Health (OIE) listed diseases and one of Class A diseases in China. The genome of CSFV is a single-stranded, positive-sense RNA of 12.3 kb in length, containing a large open reading frame (ORF) and the highly conserved 5′-untranslated region (5′-UTR) and 3′-UTR on both sides.Both bovine viral diarrhea virus (BVDV) and CSFV are members of the Pestivirus within the family Flaviviridae. In accordance with the sequence difference of the 5′-UTR, BVDV can be divided into two types, namely, BVDV-1 and BVDV-2. Since BVDV can infect not only cattle but pigs and results in clinical signs and pathological changes similar to those of CSF. Besides, BVDV may be misdiagnosed because of serological cross-reaction between pestiviruses. In addition, Hog cholera lapinized virus (HCLV) has been used widely in China, so it is difficult but necessary to distinguish CSFV field strains from vaccine strain. Therefore, a rapid, efficient, sensitive and specific detection method is in urgent need for precise differential diagnosis of CSFV, HCLV and BVDV.This study was aimed to establish a sensitive, specific and reproducible triplex real-time RT-PCR that can differentiate CSFV, HCLV and BVDV-1. Primers and TaqMan hydrolysis probes were designed in the NS5B gene for CSFV and HCLV and in the 5′-UTR region for BVDV. A triplex real-time RT-PCR assay was established for the simultaneous differentiation among CSFV, HCLV and BVDV-1. The results showed that the assay can discriminate HCLV, BVDV and wild-type CSFV of several subgroups (1.1, 1.2, 2.2 and 2.3 subgroups) completely in mainland China without non-specific reaction for other swine viruses. Within the 107-101 copies /μL range, the assay has a good linear relationship among CSFV, HCLV and BVDV testing standard template. The detection limit of the assay was 4.5 TCID50 for wild-type CSFV, 7.8 copies of viral RNA for HCLV strain, and 3.2 TCID50 for BVDV-1. The triplex real-time RT-PCR had at least 97% (248 samples) agreement with other established methods (the multiplex real-time RT-PCR and the multiplex RT-nested PCR for differential detection of wild-type and HCLV and the RT-PCR for BVDV-1). Thus, the method can detect and differentiate wild-type CSFV, HCLV, and BVDV-1 simultaneously and quantatively.