Establishment and application of an NASBA-ELISA for detection of classical swine fever virus.

In order to establish a nucleic acid sequence-based amplification(NASBA) method for the detection of classical swine fever virus(CSFV),specific NASBA primers and probe of CSFV E2 gene were designed using Primer 5.0 and Blastn.Based on solution hybridization and enzyme-linked technology,the NASBA-ELISA method for the detection of CSFV were developed successfully,and the optimized amplification condition of nucleic acid isolation is 42 ℃ and 1.5 h.The result of clinical detection indicated that specific fragments of 155 bp were obtained from the CSFV positive samples by NASBA,other than in TGEV,PRRSV,PCV2,PPV,PEDV,APP and PK-15 cell samples.The NASBA assay had a detection limit equivalent from 1 to 10 CSFV copies/μL.The sensibility of NASBA-ELISA was similar to that of the RT-PCR.A total of 125 clinical samples of pigs were analyzed for CSFV with a positive rate of 5.6%,which showed that this assay had a coincidence rate of 94.4% with NASBA-ELISA and RT-PCR.In conclusion,this NASBA-ELISA method was simple to perform and can be applied to the rapid identification and detection of CSFV.