猪链球菌2型粘附相关因子MRP、FBPS和 CPS2J荧光定量PCR检测方法的建立

摘要

[Objective] This study aimed to establish a method for quantitative detection of mRNA transcriptional level of SS2 adhesive related-factors of Streptococcus suis serotype 2 (SS2) by fluorescent quantitative PCR. [Method] The gene fragments en-coding SS2 adhesive related-factors MRP, FBPS and CPS2J and a housekeeping gene aroA were amplified by reverse transcription PCR from the total RNA of SS2, cloned, and sequenced. The recombinant plasmids containing the target genes were constructed, and used as templates in Real-time PCR. [Result] Dynamic curves, stan-dard curves and melting curves of the adhesive related-factors and aroA were ob-tained by the optimized Real-time PCR system. The standard curves showed a good linear relationship between template copy number and circulation number, and the correlation coefficients (R2) of the standard curves were over 0.995. Also, these as-says were highly specific and there was single specific melting peak for every gene. Moreover, the assays were highly sensitive and had a detection limit of 1.0 ×102 copies in 1 μl of initial templates. Final y, it was highly repeatable and had a coeffi-cient of variation less than 2% for intra-assay. [Conclusion] This study wil provide a way to reveal the adhesion mechanism of SS2 to different host cells at molecular level.%[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 RT-PCR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 Green Ⅰ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。