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

发布时间：2018-03-18 18:41

本文选题：猪瘟病毒　切入点：野毒　出处：《中国畜牧兽医》2016年09期 　论文类型：期刊论文

【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2.2pg(单一RT-PCR中的1对引物)和1.7pg(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8.2pg(CSF野毒)和6.7pg(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。
[Abstract]:In order to establish a rapid and simple method for the detection of classical swine fever virus (CSFV) from classical swine fever virus (CSFV), and to distinguish the field and vaccine infection of CSFV, and to lay a foundation for the purification of CSFV on a large scale, CSF wild virus in GenBank was obtained. The whole genome sequence alignment of vaccine virus and near-source virus showed that there was T-rich insertion sequence in CSF attenuated rabbit-attenuated vaccine strain 3ntr. According to this characteristic, the insertion sequence was found. Two pairs of single RT-PCR primers were designed at both ends of the study, and the specific conserved region was selected to design double RT-PCR primers. The conditions of PCR reaction were optimized for the identification of CSF wild virus from rabbit attenuated vaccine. A single and double RT-PCR differential diagnosis method for differentiating CSF wild virus from vaccine virus was established. The sensitivity and specificity analysis showed that, The minimum nucleic acids detected by single RT-PCR detection CSFV primers were 2.2 PG (one pair of primers in a single RT-PCR) and 1.7 PG (another pair of primers in a single RT-PCR; the lowest nucleic acid detection of each primer of double RT-PCR for CSFV was 8.2 PG CSF wild virus) and 6.7 pgCSF were detected respectively. The DNA / RNA of PRVV VV VV VV2 PCV2 was not detected by both methods. 146 suspicious clinical samples were detected by this method. The results showed that the double infection rates of CSF vaccine and wild virus in fertile sows, fattening pigs, care pigs and suckling piglets were 6. 3% and 7. 4% and 8. 6%, respectively. The single and double RT-PCR methods developed in this study were highly sensitive, specific and reproducible. This study has very important reference value for the purification of swine fever in scale pig farm.
【作者单位】：贵州大学动物科学学院;贵州省动物疫病预防控制中心;
【基金】：贵州省2014年农业攻关项目(黔科合NY字[2014]3055号) 贵州省科学技术基金项目(黔科合J字[2013]2110号) 贵州大学研究生创新基金(农研[2015]030号)
【分类号】：S852.651

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