鸡IL-6、IL-17和IFN-γ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 chicken IL-6, IL-17 and interferon-γ genes

【目的】建立针对鸡白介素6(IL-6)、白介素17(IL-17)和γ干扰素(IFN-γ)的实时荧光定量PCR检测方法,为细胞因子的定量检测及病毒致病机制的研究奠定基础。【方法】根据GenBank已发表的鸡IL-6、IL-17、IFN-γ和3-磷酸甘油醛脱氢酶(GAPDH)基因保守序列,设计并合成4对相应的特异性引物,以鸡胚成纤维细胞的cDNA为模板构建重组质粒,对退火温度、引物浓度等SYBR Green I实时荧光定量PCR反应条件进行优化,建立各基因的标准曲线,并进行特异性、敏感性、重复性试验。【结果】GAPDH、IL-6、IL-17和IFN-γ的SYBR Green I实时荧光定量PCR扩增效率分别是98.2%、99.2%、102.0%和100.8%,相应的标准误差为0.00666、0.00813、0.00365和0.00458。特异性试验结果显示各基因的溶解曲线均呈单一溶解峰,敏感性试验结果表明各基因的检测下限均为100拷贝,重复性试验结果表明组内变异系数均小于2.00%。【结论】建立的SYBR Green I实时荧光定量PCR具有敏感性高、重复性好、特异性强等特点,为快速检测和定量鸡源IL-6、IL-17和IFN-γ的表达水平提供了技术平台。

[Objective]A real-time PCR method for detection of chicken interleukin-6 （IL-6）,interleukin-17 （IL-17） and interferon-γ （IFN-γ） mRNA was established to provide references for quantitative detection of cytokines and virus pathogenic mechanism.[Method]Four specific pairs were designed according to the chicken＇s IL-6,IL-17,IFN-γ and 3 -glyceraldehyde phosphate dehydrogenase （GAPDH） gene available sequences in GenBank.The positive recombinant plasmids which were constructed from cDNA of chicken embryo fibroblast were used for the development of standard curve of SYBR Green Ⅰ real-time PCR.The conditions were optimized such as the annealing temperature,and the concentration of primer.The specificity,sensitivity,and repeatability were tested.[Result]The results showed that each gene melting curve had a single peak.Each gene amplification efficiency was 98.2%,99.2%,102.0% and 100.8%,and corresponoling Standard error was 0.00666,0.00813,0.00365 and 0.00458.Sensitivity tests results showed that lower limit of detection for each gene was 100 DNA copy number.The repeatability tests results showed that the coefficient of variation within the group was less than 2.00%.[Conclusion]The established SYBR Green Ⅰ real-time PCR assay was specific,sensitive and repeatable,which provided a rapid and quantitative detection method for chicken IL-6,IL-17 and IFN-γ mRNA transcription.