文献类型：期刊文献

中文题名：分子信标实时荧光定量PCR构绵羊PrP基因质粒和标准曲线

英文题名：Construction of Sheep PrP gene Standard plasmid DNA and curve using molecular beacon Real-time RT-PCR

作者：王川[1];吴润[1];李发弟[2];刁小龙[1];赵春林[1];王芳[3]

第一作者：王川

机构：[1]甘肃农业大学动物医学学院;[2]甘肃农业大学动物科学技术学院;[3]甘肃中医学院

第一机构：甘肃农业大学动物医学学院

年份：2011

卷号：27

期号：8

起止页码：708

中文期刊名：中国人兽共患病学报

外文期刊名：Chinese Journal of Zoonoses

收录：CSTPCD;;北大核心:【北大核心2008】；CSCD:【CSCD2011_2012】；

基金：教育部高等学校博士学科点专项科研基金项目(20060733006);甘肃省农业生物技术研究与应用开发项目(GNSW-2007-04);家畜疫病病原生物学国家重点实验室基金项目(2008-2009)

语种：中文

中文关键词：PrP;RT-PCR;实时荧光定量PCR;标准曲线;质粒;绵羊

外文关键词：PrP ;RT PCR ; real-time quantitative RT-PCR ; standard curve ; plasmid ; sheep

摘要：目的为能够用实时荧光定量PCR研究消化系统PrP mRNA的表达水平,构建目的基因PrP的标准品质粒和标准曲线。方法根据GenBank中绵羊基因编码区保守序列,设计特异性引物和分子信标探针;提取总RNA,经反转录、RT-PCR后,提取质粒,PCR-SSCP及测序鉴定,获得ARQ质粒;将质粒梯度稀释,进行荧光定量PCR,获得标准曲线及回归方程。结果结果显示,本实验设计的分子信标具有特异性强、灵敏度高和结果准确的特性;标准曲线相关系数r2=0.999,表明线性关系好,成功构建了目的基因的标准品质粒和标准曲线。
The aim of the present study was to prepare the plasmid and standard curve of real-time PCR for the quantita- tion of mRNA expression level of the digestive tract in sheep. Specific primer and molecular beacon were designed based on the conserved coding region from GenBank. Total RNA was extracted from each sample and the fragments of target gene were amplified by RT PCR. Plasmid DNA was purified and identified by PCR amplification and sequencing after recycling and purifica tion. The ARQ pattern plasmid was selected by PCR-SSCP, and was sequenced and analyzed. The positive plasmids were detected the realtime quantitative PCR after gradient dilution. The standard curve were generated automatically. The results showed that molecular beacons have high specificity and provide high sensitivity and accurate expression profiles. The correla- tion coefficients of the standard were 0. 999, suggesting the strong linear relationship between the groups. Therefore, the construction of the standard plasmid and standard curve were established.