猪链球菌2型粘附相关因子荧光定量PCR检测方法的建立  

Development of real-time PCR assays for detection of adhesion related-factors of Streptococcus suis type 2

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] 郭容利[1] 

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：《江苏农业学报》2014年第3期560-566,共7页Jiangsu Journal of Agricultural Sciences

基　　金：国家自然科学基金项目(31072155);江苏省自然科学基金重点项目(BK2010068);江苏省农业科技自主创新基金项目[CX(11)2060];公益性行业(农业)科研专项(201303041)

摘　　要：为了建立定量检测猪链球菌2型(SS2)粘附相关因子mRNA转录水平的荧光定量PCR方法,根据已报导的SS2粘附相关因子甘油醛-3-磷酸脱氢酶(GAPDH)、分选酶A(SrtA)、6-磷酸葡萄糖酸脱氢酶(6-PGD)及其管家基因aroA,用GenBank登录的核苷酸序列,设计特异性引物,提取SS2总RNA,经RT-PCR扩增和克隆了各粘附相关因子的核苷酸片段,以构建含有各自引物扩增序列的重组质粒,建立检测各粘附相关因子SYBR GreenⅠ荧光定量PCR方法。该方法起始模板数的对数与荧光信号达到所设定荧光阈值所经历的循环数(Ct)之间线性关系好,相关系数均达到0.995以上;扩增产物形成单一的特异性熔解峰;初始模板的检出下限达到了1μl 1.0×102拷贝数;组内变异系数小于2%,说明该方法特异性强、敏感性高、重复性好。To establish a method for quantitative detection of mRNA transcriptional level of adhesion related-factors of Streptococcus suis type 2（SS2）by real-time PCR, the nucleotide fragments of the adhesion related-factors gene, such as GAPDH, 6-PGD, SrtA, and housekeeping gene aroA of SS2 were amplified,cloned,and sequenced. The recombinant plas-mids containing the target gene were constructed. Real-time PCR assays based on SYBR GreenⅠfor detection of GAPDH, 6-PGD, SrtA and aroA were established. A line-ar relationship between template copy number and circula-tion number was detected by the method, with the correla-tion coefficients being over 0. 995. There was a single spe-cific melting peak for each gene. The detection limit of in-itial templates was 1. 0＆#215;102 copies. A coefficient of varia-tion for intra-assay was less than 2%. These data suggest＆amp;nbsp;the method extablished is highly specific and sensitive.

关 键 词：猪链球菌2型 粘附相关因子 STREPTOCOCCUS SUIS 2 

分 类 号：S852.61[农业科学—基础兽医学]