论文部分内容阅读
[目的]建立定量检测SS2粘附因子mRNA转录水平的荧光定量PCR方法。[方法]根据已报导的SS2相关粘附因子(包括MRP、FBPS、CPS2J)及管家基因aroA,以GenBank登录的核苷酸序列,设计特异性引物,利用RTPCR扩增和克隆各粘附因子的核苷酸片段,构建含有各自引物扩增序列的重组质粒作为阳性模板,建立检测各粘附因子的SYBR GreenⅠ荧光定量PCR方法。[结果]利用优化的Real-time PCR体系建立了各粘附因子和aroA的扩增曲线、标准曲线和溶解曲线。标准曲线表明,起始模板数与Ct值之间线性关系好,相关系数R2均达0.995以上。此方法特异性好,扩增产物形成单一的特异性熔解峰;敏感性高,初始模板的检出下限达1.0×102拷贝数/μl;重复性好,组内变异系数均小于2%。[结论]该研究为在分子水平上探究不同SS2菌株对细胞粘附差异的机制提供了技术手段。
[Objective] To establish a quantitative real-time PCR method to detect mRNA transcription level of SS2 adhesion molecule. [Method] Based on the reported SS2-related adhesion factors (including MRP, FBPS, CPS2J) and the housekeeping gene aroA, specific primers were designed based on the nucleotide sequences registered in GenBank. RTPCR was used to amplify and clone the various adhesion molecules Nucleotide fragments were used to construct recombinant plasmids containing their respective primer amplification sequences as a positive template to establish a SYBR Green I fluorescence quantitative PCR method for detecting each adhesion factor. [Result] The amplification curve, standard curve and dissolution curve of each adhesion factor and aroA were established by optimized Real-time PCR system. The standard curve shows that the linear relationship between the initial template number and the Ct value is good, and the correlation coefficient R2 reaches above 0.995. The specificity of this method is good, and the amplification product forms a single specific melting peak. The sensitivity is high. The detection limit of the initial template reaches 1.0 × 102 copies / μl. The repeatability is good, and the coefficient of variation within the group is less than 2%. [Conclusion] The study provided a technical means to explore the mechanism of different SS2 strains on cell adhesion differences at the molecular level.