Abstract:

For the distinguishment of wild-type and vaccine viruses of classical swine fever (CSF),a rapid and simple method was established,which laid the foundation for the purification of classical swine fever virus (CSFV) in large-scale farms.It was found that T-rich insertion sequence independently that 3'-NTR in Lapinized vaccine strain according to the genome sequence comparative analysis of CSF wild virus,vaccine virus and near origin virus published in GenBank,on the basis of this,two pairs of specific primers were designed in the upper and lower ends of the insertion sequence and duplex RT-PCR primers were designed.The anneal temperature of polymerese chain reaction (PCR) was optimized,then the two single and duplex RT-PCR method for the differentiation of wild-type and vaccine viruses of CSF were established.The sensitivity and specificity results showed that the minimum amounts of nucleic acid by the single RT-PCR were 2.2 pg (one pair of primers in single RT-PCR)and 1.7 pg (another pair of primers in single RT-PCR),respectively,and that of the duplex RT-PCR were 8.2 pg (wild-type virus of CSF) and 6.7 pg (vaccine virus of CSF), respectively, and no amplification of PRV,PRRSV,JEV,BVDV,PCV2 DNA/RNA were detected by these methods.Then the methods were used to detect 146 suspicious clinical samples,and the results showed that the positive rates of the mixed infection by wild-type and vaccine viruses of CSF in breeding sows,fattening pigs,nursery pigs and suckling piglets were 6.3%,7.4%,8.3%,8.6%, respectively.The results indicated that the single and duplex RT-PCR methods had high specificity,sensitivity,and good repeatability,and it had an important reference value for the purification of CSF in large-scale farms.

Key words: classsical swine fever virus; wild-type; vaccine viruses; single RT-PCR; duplex RT-PCR; differential diagnosis; purification and application