猪源干扰素刺激基因PPBP荧光定量PCR检测方法的建立及初步应用

DEVELOPMENT AND PRELIMINARY APPLICATION OF SYBR GREEN Ⅰ REAL-TIME PCR FOR DETECTION AND QUANTITATION OF PORCINE PRO-PLATELET BASIC PROTEIN

根据猪源干扰素刺激基因PPBP(pro-platelet basic protein)基因全长序列,使用引物设计软件Primer Premier5.0设计合成特异性引物,经过PCR扩增,将基因扩增的片段连接到相应的载体上,成功构建重组质粒p3XFLAG-CMV-7.1-PPBP。重组质粒经过筛选、鉴定以及纯化后,经10倍系列稀释作为质控样品,用于实时荧光定量PCR中PPBP标准曲线的构建,并检测重复性、灵敏性和特异性。结果显示标准曲线线性关系R2>0.99;特异性试验中也能检测到PPBP扩增曲线;组间和组内变异系数均小于5%。使用建立的SYBR GreenⅠ荧光定量RT-PCR检测PRRSV感染组织和未感染组织中PPBP的表达情况,能够检测出组织中PPBP的含量存在差异。本研究初步建立了检测猪PPBP基因的SYBR GreenⅠ荧光定量RT-PCR的方法,为后续猪传染性疾病和猪PPBP之间相互关系的研究提供一个特异灵敏的检测和手段。

The study was to develop a real-time assay for detecting swine pro-platelet basic protein. According to PPBP gene sequence, specific primer was designed by primer design software Primer Premier5.0 The resulting fragments were cloned into the vector to construct recombinant plasmids of p3XFLAG-CMV-7.1-PPBE The recombinant plasmid was used as standard products to establish standard curve of porcine PPBP and detect repeatability, sensitivity and specificity. The results show a precise linear relationship with a correlation coefficient R2 〉 0.99. The amplification efficiency is 102%. Dissolve curve appears one peak. The variation coefficient is lessthan 0.5% within and between assays. The SYBR Green I real-time PCR assay was used to detect porcine PPBP from mock pigs and pigs infected with PRRSV. PPBP was found in this samples and was significant different between the infected and mock group.This SYBR Green I real-time PeR assay developed in this study has highly sensitivity, specificity, stability and repeatability, which could be a tool for detecting relationship between pig infectious diseases and porcine PPBR