Development of fluorescent real-time RT-PCR for rapid detection of classical swine fever virus

A fluorescent real time RT-PCR for quick detection of classical swine fever virus(CSFV) was developed.Primers and FAM-labeled Taqman-probes specific for CSFV were selected.Optimal concentrations of transcriptase,Taq polymerase,Mg2+ and dNTPs;annealing temperature;and PCR cycles of the real-time RT-PCR were identified using the Thiveral strain(T strain) of CSFV. Specificity evaluation of the fluorescent RT-PCR showed that the assay detected T and the lapinized Chinese(C) strains of CSFV;no amplification signal was observed in any of the non-CSFV pestiviruses which included the following: bovine viral diarrhoea virus,porcine respiratory and reproductive syndrome virus(PRRSV),porcine parvovirus,swine transmissible gastroenteritis virus(TGEV),porcine respiratory coronavirus(PRCV),pseudorabies virus(PRV),and cell lines such as PK-15 cells and primary bovine testicular cells.Sequence of the PCR product of T strain was expected.The detection limit of the assay was 1 TCID50/mL.Of 75 visceral organ samples from suspected-CSFV infected pigs(e.g.,liver,spleen,heart and kidney) and 132 other swine products tested,the RT-PCR and the commercial antigen-capture ELISA assay showed a concordance of 95.7%(198/207).The RT-PCR assay could be performed within 2 h or less.The above results demonstrated that the RT-PCR could be used as a method for quick diagnosis and detection of CSFV.