豚鼠细胞因子信号抑制蛋白1基因SYBRGreenⅠ荧光定量PCR检测方法的建立  

Development of SYBR GreenⅠ real-time RT-PCR to detect SOCS1 in guinea pigs

作　　者：许微微[1,2] 孙丽娟[2] 张坤[2] 王铁成[2] 赵永坤[2] 黄耕[2] 冯娜[2] 杨松涛[2] 高玉伟[2] 夏咸柱[2] 

机构地区：[1]吉林农业大学动物科技学院,吉林长春130122 [2]吉林省人兽共患病预防与控制重点实验室,军事医学科学院军事兽医研究所,吉林长春130122

出　　处：《中国病原生物学杂志》2013年第8期701-704,共4页Journal of Pathogen Biology

基　　金：国家科技支撑计划项目(No.2010BAD04B02)

摘　　要：目的建立豚鼠细胞因子信号抑制蛋白(SOCS1)SYBR GreenⅠ荧光定量PCR方法。方法根据GenBank上公布的豚鼠全基因序列,设计SOCS1全长引物,并在其保守区设计Real-time PCR扩增引物。对SOCS1全长PCR扩增,构建pEASY-Blunt-SOCS1标准质粒。利用10倍梯度稀释标准质粒作为模版,建立SYBR GreenⅠ荧光定量PCR检测方法。结果成功建立了标准曲线,相关系数为0.996,扩增效率为103.7%,无非特异性产物和引物二聚体产生,初步应用结果显著。结论本实验建立的豚鼠细胞因子信号抑制蛋白SYBR GreenⅠ荧光定量PCR方法可用于细胞因子抑制信号蛋白变化的快速定量检测,能够为禽流感H5N1的研究提供一定的技术支持。Objective In order to study the expression of SOCS in guinea pigs infected with the avian influenza virus （AIV）, a rapid assay using SYBR Green I real-time RT-PCR was developed to detect SOCS1 in guinea pigs Methods The sequence of the SOCS1 gene of guinea pigs was obtained from GenBank and analyzed using the tool MegAlign in DNAStar software. A full-length primer was designed along with a primer for PCR amplification of its conserved region. The standard plasmid was diluted 10-fold. Results Results indicated that the correlation coefficient for the standard curve was 0. 996 and the efficiency of amplification was 103.70//oo. The standard plasmid was constructed to verify the ac- curacy of real-time PCR. Non-specific production and primer dimerization were not noted. In addition, the technique yiel- ded significant results in its initial use. Conclusion The SYBR Green I RT-PCR technique developed in this study can rapidly detect SOCS and it can facilitate study of the AIV.

关 键 词：荧光定量PCR SOCS1 豚鼠 禽流感 

分 类 号：Q78[生物学—分子生物学] S855.3[农业科学—临床兽医学]