PRRS流行毒株与减毒活疫苗TJM-F92株qPCR区分方法的建立与应用

Identification of PRRS Virus Strain and Gene-deleted Attenuated Vaccine Virus Strain TJM-F92 with qPCR

为快速准确区分PRRS流行毒株与减毒活疫苗TJM-F92株以及对流行毒株进行定量检测,按GenBank中发表的美洲型PRRSV SX-1分离株、弱毒疫苗株NSP2基因缺失部位的不同设计了一对特异性引物,通过RT-PCR和体外转录的方法,构建了体外转录RNA作为标准品,并对反应条件和反应体系进行优化,旨在建立一种敏感性高、特异性强、重复性和稳定性良好的qPCR鉴别方法。结果表明,该方法最低能检测出1.0×101拷贝/μL的模板,敏感性比常规PCR高100倍;应用该方法对218份临床样本进行鉴别与定量,qPCR检出率较常规RT-PCR高12.9个百分点。研究表明,qPCR鉴别方法的建立实现了对PRRS流行毒株与疫苗毒株的快速区分以及对流行毒株的定量检测,为PRRS的快速诊断提供了依据。

In order to develop a method based on SYBR Green I for identification of wild virus strain and gene-deleted attenuated vaccine virus strain TJM-F92 of porcine reproductive and respiratory syndrome virus（PRRSV）quickly and accurately,a pair of specific primers were designed according to the difference of the Nsp2 Gene deletion site between American type PRRSV SX1 isolates and the attenuated vaccine strain TJM-F92 which published by Genbank. Based on one-step RT-PCR and in vitro transcription for identification of constructed RNA as a standard sample, the reaction conditions and reaction system were optimized. The establishment was developed and verified for specificity, sensitiveness, sensitivity and reproducibility. Meantime,218 clinical samples were identified and quantified ,the results showed that quantitative fluorescent quantitative RT-PCR detection rate was significantly higher than that of conventional RT-PCR. This method might be used for clinical identification of wild PRRSV infection and PPRSV vaccination in pigs inoculated by vaccine prepared with TJM-F92 strain,which was helpful to evaluate the immune effect of vaccine and early diagnosis of wild PRRSV infection.