猪瘟病毒野毒株和兔化弱毒疫苗株复合实时荧光定量RT-PCR鉴别方法的建立

Development of a multiplex TaqMan real-time RT-PCR for quantitative differentiation of wild-type viruses from C-strain of CSFV

根据GenBank中的猪瘟病毒强毒和弱毒株基因组序列设计了1对针对猪瘟病毒的通用引物和2条分别针对猪瘟病毒强毒和猪瘟兔化弱毒疫苗株的特异性TaqMan水解探针，建立了一种能区分猪瘟病毒强毒和兔化弱毒疫苗株的复合实时荧光定量RT—PCR检测方法。结果显示，该方法能将我国大陆流行的不同基因亚群的猪瘟病毒强毒株与猪瘟兔化弱毒疫苗株完全区分开来，而不与其他猪源病毒发生非特异反应，分别可检测到初始模板中41．8和81．5个拷贝的病毒RNA，与已建立的复合RT-套式PCR的敏感性相近，两种方法对152份样品检测的符合率达96．9％～100％。通过对8份猪瘟兔化细胞疫苗效价的检测，证实本方法与兔体反应热测定法有一定的相关性，可用于猪瘟病毒强毒株和兔化弱毒疫苗的定量和鉴别检测。

Based on the alignment of genomic sequences of 34 strains of CSFV available in GenBank,a pair of common primers for CSFV and 2 differently-labeled TaqMan probes were designed in the 5′- untranslated region. With the primers and probes,a multiplex real-time fluorescent quantitative RT-PCR was developed for the differentiation of wild-type viruses from C-strain vaccine CSFV. The 2 TaqMan probes could differentiate different subgroups of wild-type CSFV prevailing in China's Mainland from Cstrain vaccine CSFV,without specific hybridization to other pestiviruses or other viruses of porcine origin in the multiplex real-time RT-PCR. The sensitivity of the assay for wild-type viruses and C-strain vaccine CSFV was 41.8 and 81.5 copies of viral RNA, respectively. The agreement rates between the multiplex real-time RT-PCR and the previously-described multiplex RT-nPCR were 96.9%-100% in 152 samples. There was a good correlation between the titers of 8 batches of C-strain vaccines titrated in rabbits and the RNA copies quantitated by the multiplex real-time RT-PCR. The multiplex real-time RT-PCR can be used for the quantitative differentiation of wild-type viruses from C-strain vaccine against CSFV.