建鲤IL–1β和IL–8基因实时荧光定量RT–PCR检测方法的建立

Establishment of a real-time fluorescent quantitative RT–PCR for detection of IL–1β and IL–8 genes of Cyprinus carpio var. Jian

根据Gen Bank上的基因序列,在保守区设定并合成特异性引物,选择β–actin作为内参基因,采用SYBR Green I染料法,进行熔解曲线分析和标准曲线的制作。熔解曲线表明,β–actin、IL–1β、IL–8的基因产物均为特异性产物,其Tm值分别为86、82.5和84℃;标准曲线表明,各基因Ct值的检测范围为12～32,扩增效率分别为96.3%,103.2%和102.6%,具有良好的线性关系,且r2均大于0.990;组内变异系数分别为0.14%～0.86%,0.18%～0.93%和0.13%~0.86%;用建立的方法检测健康建鲤头、肾组织中IL–1β、IL–8的表达情况,相对表达量分别为1.09和1.71。综合分析,建立的建鲤IL–1β、IL–8基因实时荧光定量PCR方法具有检测范围广,扩增效率高,特异性强,重复性高的特点,可用于建鲤IL–1β、IL–8基因的表达测定。

To establish a real-time fluorescent quantitative PCR assay for detection oflL-1β and β-8 genes of Cyprinus carpio var. Jian, specific primers were designed according to the gene sequences available in GenBank, and the β-actin gene was used as a reference gene. The standard curve and the melting curve were analyzed through SYBR Green I method. Melting curve showed specific PCR products of β-actin, β-1β and β-8 gene were obtained with Tm 86, 82.5 and 84 ℃, respectively. Standard curve there were good linear relationship for ℃-actin, β-1β and β-8 （r2〉 0. 990） under detecting Ctrange of 12 to 32. The amplification efficiency were 96.3%, 103.2%, and 102.6%, respectively, and the coefficients of variation （CV） of using this developed assay to detect these genes are 0.14%-0.86%, 0.18%-0.93% and 0.13%-0.86%, respectively. Relative expression value of β-1β and β-8 in healthy Cyprinus carpio var. Jian was 1.07 and 1.71, respectively. Conclusion： The developed real-time fluorescent quantitative PCR assay for β-1β and β-8 of Cyprinus carpio var. Jian, which showed broad range, high efficiency, specificity and reliability, could be used to detect β-1β and β-8 expression at mRNA level.