鸡Ⅰ型IFN及TLR-3和TLR-7实时荧光定量RT-PCR检测方法的建立

Development of real-time quantitative PCR for chicken IFN type Ⅰ,TLR-3 and TLR-7

为检测鸡抗病毒相关基因在病毒感染过程中的动态变化规律,研究建立了鸡IFN-a、IFN-β、TLR-3和TLR-7的实时荧光定量RT-PCR的检测方法。根据GenBank提供的鸡IFN-α、IFN-β、FLR-3和TLR-7参考序列设计了相应的特异引物,用常规RT-PCR方法从鸡脾脏总RNA中扩增得到鸡相应基因。将其cDNA分别克隆到pMD19-T载体中进行测序鉴定,采用SYBR Green I染料法,建立标准曲线并分析其溶解曲线。结果表明,这4种基因的检测灵敏度可高达46 copies/μL,且具有良好的特异性和重复性。此检测方法的建立为动态定量检测及研究鸡IFN-α、IFN-β、TLR-3和TLR-7基因的表达变化提供了有效的手段。

To study the dynamic variety of chicken genes correlated with the innate immunity after infected with viruses,the real- time quantitative RT-PCR for IFN-α,IFN- β,TLR-3,and TLR-7 was developed in the study.Four pairs of primers were designed for these cytokines according to the gene sequences available in CenBank,and these four genes were amplified from total RNA from the in chicken spleen.Then the cDNA of these four genes were cloned into the pMD19-T vector which are used as the standards after sequencing,and standard curve was established and melting curve was analyzed.The results showed that the method with high detection sensitive,good specificity and repeatability.The results suggested that real- time quantitative RT-PCR developed in the study will provide an efficient method for the quantitative analysis of chicken IFN-α,IFN-β,TLR-3,and TLR-7gene expression.