Development and preliminary application of multiplex RT-PCR for classical swine fever virus and porcine reproductive and respiratory syndrome virus

Objective A multiplex RT-PCR method for detection of classical swine fever virus(CSFV)and porcine reproductive and respiratory syndrome virus(PRRSV)was developed,verified and preliminarily applied.Methods According to the genomic sequences of CSFV and PRRSV reported in GenBank,two specific pairs of primers were designed,based on which a multiplex RT-PCR method was developed and verified for sensitivity and specificity.Twenty suspected samples with CSFV,PRRSV or mixed infection were detected by the developed method,of which the results were compared with those by commercial RT-PCR kit.The PCR products of partial samples were sequenced,and analyzed for homologies of nucleotides to those of representative nine CSFV strains and ten PRRSV strains.Results Specific gene fragments at lengths of 664 and 286 bp respectively were amplified by RT-PCR using the mixed primers for CSFV and PRRSV.The viral RNA at a dilution of 1 ∶ 100 was detected by the developed method.The detection results of CSFV,PPRSV and mixture of the two viruses were positive,while those of porcine epidemic diarrhea virus(PEDV),transmissible gastro-enteritis virus(TGEV),porcine circovirus(PCV),porcine parvovirus(PPV) and porcine pseudorabies virus(PRV) were negative.Compared with that of commercial RT-PCR kit,the sensitivity of the developed method for 20 suspected samples was 100%.The homologies of nucleotides amplified CSFV to those of nine representative CSFV strains were 92.3% ~98.2%,while those of amplified PRRSV to ten representative PRRSV strains were 94.1% ~ 97.9%.Conclusion A multiplex RT-PCR method for detection of CSFV and PRRSV was successfully developed,which laid a foundation of early diagnosis and epidemiological investigation of classical swine fever and porcine respiratory and reproductive syndrome.