Development and clinical application of Taqman real-time RT-PCR assay for detection of classical swine fever virus

According to the conservative sequences located on the 5′ untranslated region(5′-UTR) of classical swine fever virus(CSFV),a pair of specific primers and Taqman probe were designed and synthesized respectively,and a Taqman real-time fluorescent quantitative reserve-transcribed polymerase chain reaction(real-time RT-PCR) for detecting the CSFV was established in this study.Test results showed that the method had a detection limit of 10 copies of target RNA per reaction,and there was a good linear relationship between Ct value and copy numbers in diluted samples.The variation between batches was less than 1%.The RNA of porcine reproductive and respiratory syndrome virus,bovine viral diarrhea virus were detected by the Taqman RT-PCR,and the results were all negative.The CSFV-positive rate was 71.9% in 192 samples collected from Jiangsu and Xinjiang areas.Real-time RT-PCR detection showed that the different organs of swine including hearts,lungs,livers,kidneys,brains,spleens,lymph nodes and ascites were CSFV-positive,indicating that the method were more sensitive and effective than traditional RT-PCR.