鉴别猪瘟野毒与疫苗毒的单一与二重 RT-PCR检测方法的建立及应用

摘要

为建立一种对猪瘟病毒(classical swine fever virus,CSFV)临床样本快速、简便的检测技术,以区分猪瘟(classical swine fever,CSF)野毒和疫苗毒感染,为规模化猪场 CSF 净化奠定基础。通过对 GenBank 中 CSF 野毒、疫苗毒及近源病毒的基因组全序列比对,发现 CSF 兔化弱毒疫苗株3′-NTR 独立存在富含 T 的插入序列,根据这一特点分别在该插入序列的上、下两端设计2对单一 RT-PCR 引物并选择特异保守区域设计了二重 RT-PCR 引物,优化筛选能够鉴别 CSF 野毒与兔化弱毒疫苗的 PCR 反应条件,建立了能鉴别 CSF 野毒和疫苗毒的单一与二重 RT-PCR 鉴别诊断方法。敏感性和特异性分析表明,单一 RT-PCR 检测 CSFV 各引物最低核酸检测量分别为2.2 pg(单一 RT-PCR 中的1对引物)和1.7 pg(单一 RT-PCR 中的另外1对引物)；二重 RT-PCR 检测 CSFV 各引物最低核酸检测量分别为8.2 pg(CSF 野毒)和6.7 pg(CSF 疫苗毒),两种方法均检测不到 PRV、PRRSV、JEV、BVDV、PCV2的 DNA/RNA。采用该方法对146份可疑临床样品进行检测,结果表明 CSF 疫苗毒与野毒在能繁母猪、育肥猪、保育猪和哺乳仔猪中的二重感染率分别为6.3%、7.4%、8.3%、8.6%。本研究建立的单一与二重RT-PCR 方法都具有敏感性强、特异性优、重复性好的特点,该研究对规模化猪场猪瘟的净化具有十分重要的参考价值。%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 classi-cal swine fever virus (CSFV)in large-scale farms.It was found that T-rich insertion sequence in-dependently that 3′-NTR in Lapinized vaccine strain according to the genome sequence compara-tive 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 inser-tion 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 dif-ferentiation of wild-type and vaccine viruses of CSF were established.The sensitivity and specifici-ty 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 ref-erence value for the purification of CSF in large-scale farms.