猪瘟和猪伪狂犬野毒与其疫苗株多重PCR鉴别方法的建立

Establishment of a multiplex PCR method for the differentiation of wild-type and vaccine viruses of classical swine fever and pseudorabies virus

为建立一步法鉴别检测猪瘟病毒(CSFV)和猪伪狂犬病病毒(PRV)野毒株与其疫苗株感染的多重PCR方法(m PCR),本实验通过比对分析Gen Bank中登录的CSFV和PRV的相关基因序列分别设计可以区分CSFV与PRV及其疫苗株的5对特异性引物,建立了其多重PCR鉴别检测方法。结果表明,建立的多重PCR方法对CSFV和PRV扩增为阳性,而对PCV2、PRRSV、JEV和BVDV扩增结果均为阴性;最低核酸检测量分别为1.7×10~3拷贝/μL(CSFV野毒株)、9.9×10~3拷贝/μL(CSFV-C疫苗株)、1.3×10~3拷贝/μL Guizhou-DY)、6.9×10~3拷贝/μL(Bartha-K61)和5.5×10~3拷贝/μL(SA215)。采用该方法对134份可疑临床样品进行检测,结果表明CSFV和PRV疫苗株与野毒株在能繁母猪、育肥猪、保育猪和哺乳仔猪中的5重感染率分别为2.6%(1/38)、7.7%(2/26)、8.3%(3/36)和8.8%(3/34)。本研究建立的多重PCR方法为从病原学方面快速鉴别CSF和PR疫苗毒与野毒提供了新的技术支撑,为规模化猪场实现猪瘟与猪伪狂犬的净化提供一种新方法。

To distinguish wild classic swine fever virus （CSFV） from the CSFV vaccine C strain and pseudorabies virus （PRV） from the PRV gene deleted vaccine strains of Bartha-K61 and SA215, a multiplex PCR （mPCR） method was established with 5 pair primers for differential detection of the wild-type and vaccine viruses of PRV and CSFV. The results showed that the assay was specific for differential detection of the wild-type and vaccine viruses of PRV and CSFV, but no any amplification was found from PCV2, PRRSV, JEV and BVDV. In addition, the minimum detections of the mPCR were 1.7x10^3 copies/μL of wild CSFV, 9.9x 10^3 copies/μL of CSFV vaccine C strain, 1.3 x 10^3 copies/μL of wild PRV, 6.9 x 10^3 copies/μL of Bartha-K61 and 5.5 x 10^3 copies/μL of SA215, respectively. Moreover, a total of 134 clinical samples were detected by the mPCR, and the positive rateof the mixed infections in breeding sows, fattening pigs, nursery pigs and suckling piglets were 2.6% （1/38）, 7.7% （2/26）, 8.3% （3/36） and 8.8% （3/34）, respectively. These results indicated the mPCR could be applied for the programs for eradications of CSFV and PRV from pig farms.