鸭Toll样受体3实时荧光定量PCR检测方法的建立

DEVELOPMENT OF REAL-TIME FLUORESCENCE QUANTITATIVE PCR ASSAY FOR DETECTION OF DUCK TOLL-LIKE RECEPTOR 3

根据鸭Toll样受体3（Toll-like receptor 3,TLR3）的基因序列保守区设计特异性引物,以鸭β肌动蛋白（β-actin）mRNA为内参,建立了一种检测鸭TLR3 mRNA表达水平的实时荧光定量PCR方法。该方法的标准曲线在1×102～1×108copies/μL范围内,具有良好的线性关系,相关系数和扩增效率均在0.9以上。熔解曲线显示,PCR产物为单峰,具有高度扩增特异性。重复性结果表明,该方法组内、组间变异系数均小于3%,最低检测浓度达100 copies/μL。利用该方法检测了TLR3在鸭组织器官中的表达和分布情况,结果表明,TLR3在鸭子的各种组织或器官中均有分布,其中以气管的表达水平最高,而在脾脏的表达水平最低。该检测方法的成功建立为研究TLR3在鸭天然免疫过程中的生物学功能提供了良好的技术手段。

The objective of the present study was to develop a real-time fluorescence quantitative PCR（q RT-PCR） assay for detection of duck Toll-like receptor 3（TLR3） mRNA. A pair of specific primers were designed according to the gene sequences of duck TLR3 available in Gen Bank. The β-actin mRNA was used as internal control. The standard curves of this method showed good linear relationships（R20.99） and efficiency about 0.9 in the range of 1×102 to 1×108 copies/μL. The q RT-PCR assay was specific for detecting TLR3 mRNA as analyzed for melting curves. In addition, the minimum detectable concentrations of positive plasmids of TLR3 and β-actin were 100 copies/μL. Both intra-assay and inter-assay coefficients of variation were less than 3%. To verify whether this method was valid, the tissue distribution of TLR3 was examined using the q RT-PCR assay. The results showed that TLR3 was widely expressed in various tissues of duck with the highest expression level in trachea and lowest expression level in spleen.