猪伪狂犬病病毒SYBR-GreenⅠ实时荧光定量PCR检测方法的建立

Development of a SYBR-GreenⅠreal-time fluorescent quantitative PCR method for detection of pseudorabies virus

根据GenBank中猪伪狂犬病病毒(PRV)gE基因的序列(EF552427),设计并合成1对引物。通过常规PCR扩增猪伪狂犬病病毒gE基因,将鉴定正确的gE基因片段克隆入pTG19-T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作为模板建立SYBR-GreenⅠ荧光定量PCR标准曲线和溶解曲线,并做灵敏性试验、特异性试验和重复性试验。结果表明,标准曲线循环阈值与模板浓度呈良好的线性关系,溶解曲线特异,相关系数为0.9999,最低检测的拷贝数为35.4拷贝/25μL,比常规PCR高10倍,特异性和重复性较好并能对样品进行定量检测等优点。本研究成功的建立了检测猪伪狂犬病病毒的SYBR-GreenⅠ荧光定量PCR方法,为该病的早期诊断及定量分析猪伪狂犬病病毒感染程度奠定了基础。

According to genome sequences of gE of PRV published in GenBank,apair of primers was designed.The gE gene was amplified with traditional PCR.The PCR product was cloned into pTG19-T vector and sequenced.The positive recombinant plasmid was used as quantitative template to generate standard curve and melt curve.Sensitivity,reproducibility and specificity of the method were determined.The results demonstrated that standard curve established by recombinant plasmid was shown a fine linear relationship between threshold cycle and template concen- tration,melt curve was specificity and the correlation coefficient was 0.999 9.The detection limit of real-time PCR for PRV was 35.4 copy per 25μL,and the quantitative PCR had better reproducibility and specificity than traditional PCR.A SYBR GreenΙfluorescent quantitative PCR for detecting gE gene of PRV was developed for the basis of the early and rapid detection and quantitatively evaluating the infect degree of PRV.