A Method for Detecting Adhesive Related-Factors of Streptococcus suis Serotype 2 by Real-time PCR  

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(英文)

作　　者：汪伟[1] 何孔旺[1] 倪艳秀[1] 周俊明[1] 张雪寒[1] 俞正玉[1] 吕立新[1] 茅爱华[1] 温立斌[1] 王小敏[1] 李彬[1] 郭容莉 

机构地区：[1]江苏省农业科学院兽医研究所,农业部兽用生物制品工程技术重点实验室,国家兽用生物制品工程技术研究中心,江苏南京210014

出　　处：《Agricultural Science & Technology》2013年第10期1378-1382,共5页农业科学与技术（英文版）

基　　金：Supported by National Natural Science Foundation of China(31072155);Natural Science Foundation of Jiangsu Province(BK2010068);Fund for Independent Innovation of Agricultural Science in Jiangsu Province[CX(11)2060];Special Fund for Agroscientific Research in the Public Interest(201303041)~~

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. []Vlethod] 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 （FF） of the standard curves were over 0.995. Also, these as- says were highly specific a^d 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. Finally, it was highly repeatable and had a coeffi- cient of variation less than 2% for intra-assay. [Conclusion] This study will 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。

关 键 词：Streptococcus suis serotype 2 Adhesive related-factors （adhesins） Real- time PCR 

分 类 号：R341[医药卫生—基础医学]