Study On The Real-Time Fluorescent Quantitative PCR For The Detection Of CSFV

To create a real-time fluorescent quantitative PCR that can quickly detect classical swine fever virus with high sensitivity and strong specificity, we designed a suit of specific primer and probe according to gene sequence of classical swine fever virus the Gene bank had announced. The RNA of classical swine fever virus were extracted by extraction kits for the RNA of virus and reverse transcription to synthesis cDNA and amplificated by PCR, The PCR product were ligated with PMD19-T vector, to creat the pMD19-T-CSFV and transformed into DH5a cells, extracted plasmid, the recombinant plasmid obtained were certified by PCR and sequenced. use the plasmid as template to optimize the density of primer and probe and the annealing temperature so as to create the reaction system and parameter of the real-time fluorescent quantitation PCR, specificity and sensitivity of the real-time fluorescent quantitation PCR were tested with the classical swine fever virus and used to detected clinical samples.The results showed that the method we had created could distinguish the swine fever virus, bovine diarrhea virus, orf virus, and sheep pox virus, and the relatively results of clinical detection. The detection method of variation coefficient was below0.01, indicating a good stability and repeatability.The sensitivity could be as high asl02copies of order of magnitude and showed stability.