DEVELOPMENT AND APPLICATION OF TAQMAN REAL-TIME RT-PCR FOR CLASSICAL SWINE FEVER VIRUS

In order to develop a TaqMan Real-time RT-PCR(qRT-PCR) assay for detection of C-strain and field strains of classical swine fever virus(CSFV),a pair of primers,a probe for qRT-PCR and 3 primers for preparing standard RNA were designed based on highly conserved 5'UTR sequence of pestivirus genome available in NCBI GenBank.Standard RNA that harbored the detection sequence for the TaqMan Real-time RT-PCR was also prepared using RT-PCR and in vitro transcription.Then,a TaqMan real-time fluorescence quantitative RT-PCR assay for CSFV was developed and optimized regarding reaction conditions and concentrations of primers and probe.The coefficients of variation were less than 2% for test trials performed for repeatability and reproducibility.The sensitivity of the assay was 1×102 copies/μL and test time was less than 60 min.The standard curve was established with slope,intercept and R2 of-3.97,47.41,and 0.999779,respectively.The assay was applied to quantitatively detect 3 clinical samples,2 spleen vaccines and 5 cell culture vaccines obtained from 6 commercial manufacturers.The data showed a significant difference in the number of CSFV copies per dose among 7 vaccines produced by f 6 commercial manufacturers.However,there was not a significant difference among RNA extracts from 3 clinical samples.The results showed that the TaqMan Real-time RT-PCR assay was suitable for early detection of CSFV in clinical samples and also for quality control for commercial vaccines.