草鱼TBK1基因荧光定量PCR检测方法的建立

Establishment and application of Quantitative Realtime PCR assay for Ctenopharyngodon idella TBK1 Gene

TANK结合激酶1(TANK-binding kinase 1,TBK1)基因在天然抗菌免疫中发挥重要作用,为研究该基因在草鱼(Ctenopharyngodon idella)抗病天然免疫中的表达水平,本研究根据已报道的草鱼TBK1基因(ciTBK1)序列,设计特异性引物2对,基于ciTBK1和18S rRNA双标准曲线建立了荧光定量PCR(Quantitative Real-time PCR,qRT-PCR)检测方法。结果表明,建立的标准曲线Ct(Cycle thrshold)值与模板浓度的对数呈现良好的线性关系(R^(2)均大于0.99),建立的qRT-PCR检测方法能特异地定量检测ciTBK1基因,检测灵敏度可达50拷贝/μL,且组内及组间变异系数均小于5%。所建立的qRT-PCR检测方法具有良好的特异性、灵敏性和重复性。

TBKl gene plays an important role in natural antibacterial immunity.In order to study the expression level of this gene in antibacterial immunity of grass carp(Ctenopharyngodon idella),this study designed two pairs of specific primers according to the reported TBK1 gene(ciTBK1)sequence of grass carp.A quantitative realtime PCR(qRT-PCR)method was established based on ciTBK1 and 18S rRNA double standard curves.The results showed that the standard curve had a good linear relationship between Ct values and the logarithm of template concentration,with an excellent correlation coefficient(R^(2)>0.99).The qRT-PCR method could detect ciTBKl gene quantitatively with a sensitivity of 50 copies/μL and the intra and inter group variation coefficients were less than 5%.The qRT-PCR method has good specificity,sensitivity and repeatability.