鉴别小反刍兽疫病毒野毒和疫苗毒双重荧光RT-PCR检测方法的建立

Establishment of A Duplex Real-time RT-PCR Assay for Identification of Field and Vaccine Peste Des Petits Ruminant Virus

为快速准确检测小反刍兽疫病毒野毒和疫苗毒,建立一种特异性强、灵敏性高、经济实惠的实验室鉴别诊断方法,在同一反应体系内同时鉴别诊断小反刍兽疫野毒株和疫苗毒株用于应对该疫病诊断,使实验室检测工作更具有实效性。根据基因库中基因组序列设计2对特异性引物和探针,通过优化反应体系和扩增条件,建立能够同时检测并鉴别小反刍兽疫病毒(PPRV)野毒和疫苗毒的双重荧光RT-PCR检测方法。结果表明,建立的双重荧光RT-PCR可特异性检测小反刍兽疫野毒(FAM通道)和疫苗毒(TAMRA通道);敏感性实验表明,野毒(FAM通道)可检测到10^(-3)稀释度的RNA,疫苗毒(TAMRA通道)可检测到10^(-5)稀释度的RNA;用建立的二重荧光RT-PCR方法对20份样品进行检测,结果显示,该方法可以有效区分野毒和疫苗毒株,且检测结果与商品试剂盒检测结果一致,可用于临床检测。

In order to detect the field and vaccine virus of peste des petits ruminants(PPR)rapidly and accurately,this paper aims to establish a laboratory diagnosis method with strong specificity,high sensitivity and economic benifits,and simultaneously identify the field and vaccine PPRV in the same reaction system,so as to make the laboratory detection more effective.Two pairs of specific primers and probes were designed based on the reported genome sequences in the Genbank.By optimizing the reaction system and amplification conditions,a duplex real-time RT-PCR method was established for simultaneous detection and identification of field and vaccine PPRV.The results showed that the established duplex real-time RT-PCR could specifically detect field PPRV(FAM channel)and vaccine PPRV(TAMRA channel).Sensitivity experiments showed that 10^(-3) diluted RNA could be detected by field virus and 10^(-5) diluted RNA could be detected by vaccine virus.20 samples were detected by duplex real-time RT-PCR method.The results showed that the method could effectively distinguish field PPRV and vaccine PPRV,and the detection results were consistent with those of commercial kits,which could be used for clinical detection.