摘要
为实现山羊痘病毒(GTPV)和口疮病毒(ORFV)的同步快速检测,基于GTPV的ITR基因及ORFV的059基因,分别设计用于普通双重PCR的引物2对、用于二联实时荧光定量PCR的特异性引物及探针2套;探针分别用5′JOE-Eclipse3′及5′FAM-TAMRA3′荧光染料标记物进行标记。结果显示,所建立的GTPV-ITR-L-ORFV-059双重普通PCR方法,可实现对GTPV与ORFV的特异性双重检测,检测敏感度可达10~4copies/μL DNA;所建立的ITR-059二联探针实时荧光定量PCR方法可同时特异性检测ORFV和GTPV产生特异性荧光信号,二联探针的检测敏感度可分别达10^(2.87)和10^(2.52) copies/μL DNA。本研究初步建立了可同步特异性检测GTPV和ORFV的双重普通PCR及二联探针实时荧光定量PCR检测方法。
To detect the goatpox virus（GTPV） and orf virus（0RFV） simultaneously and quickly,two pairs of primers for routine PCR assay,and two pairs of primers and relative probes for real-time PCR assay were designed based on the inverted terminal repeat（ITR） segment of GTPV and the 059 gene of OR- FV,respectively. The probes were labeled with different fluorescent materials 5＇JOE-Eclipse3＇ and 5＇FAM-TAMRA3＇,respectively. The results showed that the developed GTPV-ITR-L-ORFV-059 duplex routine PCR assay was specific for GTPV and ORFV with the detection sensitivity of 104copies/μL DNA. The established duplex ITR-059 probe real-time PCR assay was also proved specific for ORFV and GTPV,and the fluorescent signal could be detected with a detection limit of 102＇87copies/μL DNAand 10Z52copies/μL DNA for GTPV and 0RFV,respectively. Finally,in the study the duplex routine PCR and the duplex real-time PCR assays for simultaneous detection of GTPV and 0RFV were established preliminarily.
出处
《中国兽医科学》
CAS
CSCD
北大核心
2017年第5期569-576,共8页
Chinese Veterinary Science
基金
云南省重点新产品开发计划项目(2014BB014)
云南省现代农业奶牛产业技术体系建设项目[云财农(2009)171号]
