采用SYBR Green I实时荧光定量RT-PCR方法对猪瘟强毒与疫苗弱毒的鉴别研究

摘要

为建立一种能够区分猪瘟病毒(CSFV)强毒与弱毒疫苗C株的SYBR Green Ⅰ荧光定量RT-PCR结合熔解曲线分析方法,本研究对GenBank中登录的25株CSFV强毒株和兔化弱毒疫苗C株全基因组序列进行比较分析,设计一对共用下游引物以及分别针对CSFV强毒株与弱毒疫苗的特异性上游引物,其Tm值分别为84.5±0.5℃和88.5±0.5℃,熔解曲线分析显示为单特异峰.检测结果显示本实验建立的鉴别CSFV强毒感染与弱毒疫苗的荧光定量RT-PCR结合熔解曲线分析方法特异性强,对其他相关病毒无特异性扩增；敏感性高,最低检出量为5×RID50的细胞疫苗基因组拷贝；重复性好；并且扩增效率高、线性范围广、检测时间短,可对免疫猪群中CSFV强毒感染做出快速准确的鉴别检测,为有效防制猪瘟提供依据.%For establish a differential detection of wild-type and C-strain vaccine viruses of classical swine fever virus (CSFV), A SYBR Green I real-time RT-PCR was developed with a common reverse and 2 specific forward primers according to 25 CSFV reference strain and the C-strain vaccine virus strain in GenBank with Tm value of 84.5 ± 0.5 ℃ and 88.5 ±0.5 ℃, respectively. The test results showed that the method for detection of wild-type and C-strain vaccine viruses of CSFV was sensitivity, specificity and efficiency with the lowest detected limit of 5 × RID50 virus and had no amplification for other related porcine virus. This method was able to distinguish wild-type CSFV infection from vaccinated pigs and provided basis for effectively controlling classical swine fever.