山羊痘和小反刍兽疫病毒双重实时荧光定量RT-PCR检测方法的建立

Development of the Duplex Real-time RT-PCR Assay for Simultaneous Detection of Goat Pox Virus and Peste des Petits Ruminants Virus

为实现对山羊痘病毒(GTPV)和小反刍兽疫病毒(PPRV)的同步快速检测,基于GTPV的ITR基因及PPRV的M基因,分别设计合成两套特异性引物及探针;探针分别用5′-FAM-TAMRA-3′及5′-JOE-Eclipse-3′标记物进行标记以实现同步检测。结果显示,所建立的ITR-M二联探针荧光定量RT-PCR方法可同时特异性检测GTPV和PPRV产生特异性荧光信号,而对禽痘病毒、犬瘟热病毒等相关病毒无荧光信号可检出。以GTPV-ITR和PPRV-M的pMD-18T载体质粒为标准品,构建了双重标准曲线,ITR-M探针的检测敏感度可分别达103拷贝/μL及101.2拷贝/μL cDNA。本研究初步建立了可同步快速检测GTPV和PPRV的二联实时荧光定量RT-PCR法。

Goat pox virus（GTPV）and peste des petits ruminants virus（PPRV） are the causative agents of the two kinds of goats＇ diseases-goat pox and peste des petits ruminants which can cause disaster economic losses. In order to detect the two viruses simultaneously and quickly, two sets of primers and relative probes were designed based on the ITR（inverted terminal repeat）segment of GTPV and the M gene of PPRV respectively. The probes were labeled with different fluorescent materials 5＇FAM-TAMRA3＇and 5＇ JOE-Eclipse3＇respectively in order to work together in the same reaction. Results showed that the duplex real-time RT-PCR assay was identified to be specific for GTPV and PPRV only and specific fluorescent sig- nal could be detected, but not for the related viruses including fowl pox virus（FPV）and canine distemper virus （ CDV ） without specific fluorescent signal. Positive recombinant plasmids （GTPV pMD-18T-ITR and PPRV pMD-18T-M） were built and used for positive quantitative templates to establish duplex standard curves. The developed assay based on the probe ITR-M was found to be highly specific and sensitive with a detection limit of 10acopies/μL and 101＇2copies/μL for GTPV and PPRV respectively. Finally, in the study the duplex real-time RT-PCR assay for simultaneous detection of GTPV and PPRV was established preliminarily.