牛Toll样受体实时荧光定量PCR检测方法的建立  

Establishment of a Real-time Fluorescent Quantitative PCR Assay for Detection of Bovine Tol1-like Receptors Genes

作　　者：韩猛立[1,2] 黄新[1] 何延华[1] 宋天增[1,2] 薄新文[1] 钟发刚[1] 

机构地区：[1]新疆生产建设兵团绵羊繁育生物技术重点实验室,石河子832000 [2]石河子大学动物科技学院,石河子832003

出　　处：《农业生物技术学报》2011年第3期521-529,共9页Journal of Agricultural Biotechnology

基　　金：国家重点基础研究发展规划(973)前期研究专项(No.2008CB117017);国家自然基金项目(No.30960286);兵团博士资金项目(No.2007JC15);新疆农垦科学院青年科学基金项目(No.YQJ201113)共同资助

摘　　要：建立检测牛Toll样受体(TLRs)mRNA表达水平的SYBR GreenΙ实时荧光定量PCR方法。根据GenBank中BoTLR-2、3、4、5、7、8和10的基因序列,在其保守区设计并合成各自特异性引物,以牛3-磷酸甘油脱氢酶基因(GAPDH)为内参,建立实时荧光定量PCR方法。结果表明,在1×102～1×109copies/μL范围内,BoTLRs和GAPDH基因的Ct值与阳性质粒的浓度均呈良好的线性关系,相关系数均大于0.991。溶解曲线分析表明,产物为特异的单峰,具有较高的灵敏度和特异性。重复性结果表明,TLRs和GAPDH基因阳性质粒,最小检出浓度达到100和10copies/μL,组内、组间变异系数值均保持在3.5%以内。临床样品检测结果表明,TLR3和TLR8mRNA水平在诱导培养早期较高,在4h达到高峰,而TLR4和TLR7水平与之相反,在诱导培养后期较高,在24h达到高峰,表明本研究所建立的检测方法成功用于临床样品的检测,为在mRNA水平对BoTLRs的定量分析提供技术平台。This work aimed to develop a SYBR Green Ι Real-time fluorescent quantitative PCR assay for detection of bovine toll-like receptors mRNA.According to the gene sequences of bovine＇s Tol1-like receptors（BoTLR-2,3,4,5,7,8 and 10） available in GenBank,eight pairs of primers based on their conserved regions were designed with bovine glyceraldehyde-3-phosphate dehydrogenase（BoGAPDH） as an interna control to construct Real-time fluorescent quantitative PCR assay.The results showed a good linear relationships（r20.991） between the Ct value and the concentration of positive plasmid for each gene on the condition that the concentration of positive plasmids were limited from 1×102 to 1×109 copies /μL.The melting curve analysis showed the product was specific to a single peak,with high sensitivity and specificity.Reproducibility showed that the minimum detectable concentration of positive plasmids of TLRs and GAPDH gene were 100 copies /μL and 10 copies /μL.And the intra-assay and inter-assay coefficient of variation values were maintained at less than 3.5%.The clinical sample test showed that TLR3 and TLR8 mRNA levels in the induction of early high peak at 4h,while the level of TLR4 and TLR7 contrast,higher late in the induction,peaked at 24 h.These data showed that the detection method in this study successfully can be use for the detection of clinical samples and provids a technical platform in the quantitative analysis of BoTLRs on the mRNA levels.

关 键 词：牛 TOLL样受体 外周血单核细胞 实时荧光定量RT-PCR SYBR-GreenⅠ 

分 类 号：S858.23[农业科学—临床兽医学]