马Toll样受体表达水平SYBR Green Ⅰ荧光定量RT-PCR检测方法的建立  

Establishment of a Real-time RT-PCR Assay Based on SYBR Green Ⅰ for Detection of the Expression of Horse Toll-like Receptor Genes

作　　者：赵一萍[1] 白东义[1] 李蓓[1] 黄金龙[1] 张宇宏[1] 芒来[1] 

机构地区：[1]内蒙古农业大学动物科学学院,呼和浩特010018

出　　处：《畜牧兽医学报》2013年第2期220-227,共8页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：内蒙古自治区科技厅重大项目(20091404);内蒙古农业大学科技创新团队支持计划(NDTD2010-12)

摘　　要：旨在建立一种检测马Toll样受体(TLRs)mRNA表达水平的SYBR GreenⅠ荧光定量RT-PCR(Rverse Transcription-Polymerase Chain Reaction)方法。参照GenBank中马TLRs的基因序列保守区设计特异性引物,以马β肌动蛋白(β-actin)mRNA为内参,建立荧光定量RT-PCR方法。结果,扩增曲线在1×102～1×108copies.μL-1范围内有很好的线性关系,扩增相关系数在0.990以上,扩增效率在1.0左右。熔解曲线分析表明,产物为特异单峰,无引物二聚体,具有较高的特异性和灵敏度。重复性结果表明,该方法的组内、组间变异系数均小于3.50%,重复性较好。临床样品检测结果表明,TLR1、TLR2、TLR3、TLR4、TLR6、TLR7、TLR8和TLR9在骨髓中均有表达,其中TLR4mRNA表达水平最高,TLR9mRNA表达水平最低。本研究建立的检测方法能够成功用于临床样品的检测,为研究马匹TLRs在mRNA水平的定量分析提供技术平台。This study aimed to develop a real-time reverse-transcription polymerase chain reaction（RT-PCR） method based on SYBR Green Ι fluorescent for detection of horse Toll-like receptor mRNA.According to the gene sequence in conservative region of horse＇s Toll-like receptors available in GenBank,the specific primers were designed with β-actin as an internal control to construct Real-time RT-PCR assay.The standard curves showed good linear relationships with the correlation（r20.990） and efficiency about 1.0 in the range of 1×102 to 1×108 copies·μL-1.The melting curve analysis showed that the product was specific to a single peak,and no primer-dimers,with high specificity and sensitivity.The good reproducibility was obtained and the coefficient of variation were less than 3.5% for the intra-assay and inter-assay.The clinical sample test indicated that the TLR1,TLR2,TLR3,TLR4,TLR6,TLR7,TLR8 and TLR9 mRNA were transcripted in medulla,and TLR4 mRNA level was highest,but TLR9 mRNA level was lowest.This assay could be successfully used for the detection of clinical samples and provide a technical platform to research horse TLRs at the mRNA levels in the quantitative analysis.

关 键 词：马 TOLL样受体 骨髓 SYBR Green Ⅰ 荧光定量RT-PCR 

分 类 号：S821.2[农业科学—畜牧学]