小反刍兽疫病毒及山羊痘病毒N-ITR基因二联实时荧光定量RT-PCR检测方法的建立  

Development of the Duplex Real-time RT-PCR Assay with TaqMan Probe of N-ITR Genes for Detection of Peste des Petits Ruminants Virus and Goat Pox Virus

作　　者：赵文华[1] 杨仕标[1] 高华峰[1] 王金萍[1] 

机构地区：[1]云南省畜牧兽医科学院、云南省热带亚热带动物病毒病重点实验室,云南昆明650224

出　　处：《中国畜牧兽医》2014年第3期65-71,共7页China Animal Husbandry & Veterinary Medicine

基　　金：云南省应用基础研究面上项目(2009ZC182M);公益性行业(农业)科研专项(200903037)

摘　　要：为实现山羊2种疫病病原体——小反刍兽疫病毒(PPRV)和山羊痘病毒(GTPV)的同步快速检测,基于PPRV的N基因及GTPV的ITR基因,分别设计合成2套特异性引物及探针;探针分别用5′FAM-TAMRA3′及5′JOE-Eclipse3′标记物进行标记以实现同步检测。试验结果显示,所建立的N-ITR二联探针实时荧光定量RT-PCR方法可同步检测PPRV和GTPV,产生特异性荧光信号,而对禽痘病毒(FPV)、犬瘟热病毒(CDV)等相关病毒无荧光信号检出。以PPRV-N和GTPVITR的pMD18-T载体质粒为标准品,构建了二联标准曲线,N-ITR探针对PPRV和GTPV的检测敏感度可分别达102和103拷贝/μL。本研究初步建立了可同步快速特异性检测PPRV和GTPV的二联实时荧光定量RT-PCR方法。Peste des goats＇ diseases--peste viruses simultaneously petlts ruminants des petits ru and quickly, mlna two virus （PPRV） and goat pox virus （GTPV） are the causative agents of two kinds of nts and goat pox which can cause disaster economic losses. In order to detect the two sets of primers and relative probes were designed based on the nucleoprotein （N） geneof PPRV and the inverted terminal repeat （ITR） segment of GTPV, respectively. In order to work together in the same reac- tion, the probes were labeled with different fluorescent materials 5rFAM-TAMRA3′and 5′JOE Eclipse3′ ,respectively. Results showed that the duplex Real time RT-PCR assay was identified to be specific for PPRV and GTPV only and specific fluorescent signal could be detected, but the related viruses including fowl pox virus（FPV）and canine distemper virus （CDV） had no spe- cific fluorescent signal. Positive recombinant plasmids （PPRV pMD18 T N and GTPV pMD18-T-ITR） were built and used for positive quantitative templates to establish duplex standard curves. The developed assay based on the probe N-ITR was found to be highly specific and sensitive with a detection limit of 102 copies/μL cDNA and 103 copies/μL DNA for PPRV and GTPV, respectively. Finally, the duplex Real time RT-PCR assay for simultaneous detection of PPRV and GTPV was established pre liminarily in the study.

关 键 词：小反刍兽疫病毒 山羊痘病毒 二联实时荧光定量RT—PCR 探针 

分 类 号：Q78[生物学—分子生物学]