鸡IL-2 mRNA SYBR GreenⅠ实时荧光定量RT-PCR检测方法的建立与应用

Establishment and application of SYBR GreenⅠ real-time fluoresent quantitative RT-PCR assay for detection chicken interluekin-2 mRNA

根据GenBank提供的ChIL-2参考序列设计了特异性引物,并以鸡3-磷酸甘油脱氢酶(GAPDH)基因为内参,以二者标准质粒为模板,应用SYBR GreenⅠ实时荧光定量PCR技术,并采用双标准曲线相对定量方法建立了ChIL-2mRNA荧光定量RT-PCR检测方法。结果显示,该方法该可以在3h左右对低拷贝量的模板(200copy/μL)进行检测,同时该检测方法具有很好的特异性和重复性。雏鸡免疫试验IL-2mRNA检测结果显示,该方法可有效应用于鸡体内IL-2在核酸水平的检测,并为今后IL-2相对定量的检测研究提供参考依据。

For establishing a method to detect chicken interleukin 2 （ChlL-2） mRNA expression, the primers were desigend for developing a SYBR Green I real-time fluoresence quantitative RT- PCR method,according to the chicken ChlL-2 gene and chicken glyceraldehyde-3-phosphate dehy- drogenase （ChGAPDH） gene sequences available in GenBank of which plasmids were used as the templates. ChGAPDH was used as the internal control and ＂two standard curve relative quantita- tive＂ method was applied to compute the value. The results of this expriment revealed that this method could detect the template of low copies （200 copy//;L） in short time （about 3 hours） ,and also had a good specificity and repeatability. This method was used to detect the dynamic change of IL-2 mRNA in chicks which were inoculated with IBV S1 gene vaccine. The results of signifi- cant change between inoculated group and control group showed that this method could be effec- tively applied to detect the nucleic acid level of IL-2 in the body of chicken and provided a suitable reference approach for further research on relative quantification of ChlL-2.