高致病性猪繁殖与呼吸综合征病毒实时荧光定量PCR检测方法的建立

摘要

根据GenBank公布的24株高致病性猪繁殖与呼吸综合征病毒毒株和5株PRRSV经典毒株的保守区基因序列,使用PrimerExpress 3.0软件设计并合成实时荧光定量PCR（Real-timeFluorescent Quantitative PCR,Real-time FQ-PCR）用引物和探针,建立了Real-time FQ-PCR检测方法以鉴别检测高致病性猪繁殖与呼吸综合征病毒。用建立的检测方法对已定量的10倍倍比稀释的质粒pGET-258为标准品进行检测,并与常规PCR进行比较。结果显示,该Real-time FQ-PCR方法敏感度可达1.5个拷贝,比常规PCR敏感度高100倍,且批内和批间重复性检测结果的变异系数均小于2%。用该方法与常规PCR方法及病毒分离方法对18份临床样品进行对比检测,显示该方法灵敏度高、成本低,并且能够对样品中病毒进行定量,为高致病性猪繁殖与呼吸综合征的快速鉴别诊断提供了有效的技术手段。%Primers and probe were designed with PrimerExpress 3.0 and synthesized according to the conserved gene sequences of 24 highly pathogenic porcine reproductive and respiratory syndrome（HP-PRRSV） strains and 5 classical PRRSV strains available in GenBank.A real-time fluorescent quantitative PCR assay was established for detection,differentiation and quantitation of HP-PRRSV and classical PRRSV.A known concentration of 10-fold series of dilutions of pGET-258 plasmid DNA were detected by using the established real-time FQ-PCR assay,then compared to the detection result of routine PCR.The developed real-time FQ-PCR assay could detect 1.5 copies with plasmid DNA and its sensitivity was 100 times higher than that of the routine PCR.Three plasmid standards were examined using the real-time FQ-PCR repeatedly at three different time,the coefficient of variations were below 2%,and the results indicated that the real-time FQ-PCR assay was reproducible and could be used for the diagnosis and differentiation of HP-PRRSV infection.