Here we report the construction of sheep PrP gene standard plasmid DNA and curve using real-time RT-PCR.Total RNA was extracted from each sample and the fragments of target gene were amplified by RT-PCR.The plasmid wa...Here we report the construction of sheep PrP gene standard plasmid DNA and curve using real-time RT-PCR.Total RNA was extracted from each sample and the fragments of target gene were amplified by RT-PCR.The plasmid was constructed for calibrating unknown samples.In this study,the constructed plasmid containing only the target gene was used to construct a calibration curve.The absolute standard curve method was shown to be of high linearity,sensitivity and reproducibility.The purpose of this study is to investigate the quantification of PrP mRNA expression for knowing the scrapie pathogenesis and providing powerful tool for further studies on prion diseases pathogenesis.展开更多
为建立猪传染性胃肠炎病毒(TGEV)SYBR Green Ⅰ荧光定量RT-PCR检测方法,根据GenBank收录的TGEV-Miller毒株的S基因序列,设计合成1对特异性引物,用RT-PCR方法从疫苗株中扩增TGEV S基因的部分保守片段,并克隆到pGEM-T Easy载体上,得到重...为建立猪传染性胃肠炎病毒(TGEV)SYBR Green Ⅰ荧光定量RT-PCR检测方法,根据GenBank收录的TGEV-Miller毒株的S基因序列,设计合成1对特异性引物,用RT-PCR方法从疫苗株中扩增TGEV S基因的部分保守片段,并克隆到pGEM-T Easy载体上,得到重组质粒作为荧光定量RT-PCR检测的标准模板,进行SYBR GreenⅠ荧光定量RT-PCR扩增,并制作标准曲线,建立TGEV的荧光定量RT-PCR检测方法。结果表明:该方法检测灵敏度可达30拷贝/μL,与猪圆环病毒、猪乙型脑炎病毒、猪伪狂犬病毒、猪瘟病毒和猪流感病毒和猪细小病毒不发生交叉反应,具有重复性好、特异性强、灵敏度高等优点,可用于临床TGEV感染的早期诊断以及分子流行病学调查。展开更多
文摘Here we report the construction of sheep PrP gene standard plasmid DNA and curve using real-time RT-PCR.Total RNA was extracted from each sample and the fragments of target gene were amplified by RT-PCR.The plasmid was constructed for calibrating unknown samples.In this study,the constructed plasmid containing only the target gene was used to construct a calibration curve.The absolute standard curve method was shown to be of high linearity,sensitivity and reproducibility.The purpose of this study is to investigate the quantification of PrP mRNA expression for knowing the scrapie pathogenesis and providing powerful tool for further studies on prion diseases pathogenesis.
