摘要
根据GenBank中猪伪狂犬病病毒(PRV)gE基因的序列(EF552427),利用Premier express设计并合成1对引物和相应的TaqMan探针,从猪伪狂犬病病毒(PRV)感染的细胞中提取DNA,进行PCR扩增。将鉴定正确的gE基因片段克隆入pGEM-T Easy载体中,转化大肠杆菌JM109,经PCR及测序鉴定后得到阳性重组质粒。以该阳性重组质粒为荧光定量PCR标准品模板建立标准曲线。对探针浓度、引物浓度、镁离子浓度和退火温度进行优化,建立了最佳的荧光定量PCR反应体系和扩增程序。经临床应用表明,该荧光定量PCR方法的建立为猪伪狂犬病病毒的早期诊断并定量分析猪伪狂犬病病毒感染程度奠定了基础。
A pair of primers and a TaqMan probe were designed according to gE gene nucleotide sequence （EF552427） of PRV in GenBank. The expected fragment was amplified from DNA of PRV-infected PK-15 cells,The PCR product was cloned into pGEM-T Easy vector and sequenced. The positive recombination plasmid was used as a positive quantitative template to establish a standard curve. By optimizing the probe＇s concentration, Mg^2＋ concentration,primers concentration and the annealing temperature, real-time fluorescent quantitative PCR was established. Clinical detection of PRV by real-time fluorescent quantitative PCR showed that real-time fluorescent quantitative PCR paved the way for the early and rapid detection of PRV and quantitative analysis for the infect degree of PRV.
出处
《中国兽医学报》
CAS
CSCD
北大核心
2009年第4期433-436,共4页
Chinese Journal of Veterinary Science
基金
河南省杰出人才创新基金资助项目(0621002100)
