Detection of classical swine fever virus by real-time RT-PCR

In this study,a real-time RT-PCR method was developed to detect and differentiate various genotypes of Classical swine fever virus(CSFV)from other pestiviruses using a set of degenerate primers and a TaqMan probe directed to the 5' NTR of classical swine fever virus genome.A serial cDNA standards were prepared by reverse transcription using RNA template prepared from blood stock of CSFV strain Shimen and used to optimize the real-time PCR.The method was successfully used to quantitate the virus load in tissue specimens,and was able to detect 10~(-0.82)TCID_(50) of the virus.Further detection of 108 clinical tissue specimens showed that 73 of them were CSFV positive by the real-time RT-PCR,which were subsequently confirmed by virus isolation after 1 to 3 passages on PK-15 cells.In comparison only 54 of them were detected positive in routine RT-PCR, indicating that real-time RT-PCR is more sensitive than routine RT-PCR,which has a potential application in clinical diagnosis.