【摘要】 根据GenBank上鸡β-actin基因的序列,在保守区域设计并合成一对引物,采用SYBR Green I染料建立了荧光定量PCR法(real-time PCR)。以常规PCR产物为标准品,建立了标准曲线,并进行了熔解曲线分析。结果表明,标准曲线Ct值检测范围为12~31,扩增效率为95.1%;熔解曲线分析结果显示产物特异的单个峰,其Tm为88±0℃,检测周期从RNA提取到荧光定量PCR结束只需4 h。本试验建立的鸡β-actin基因实时荧光定量PCR法扩增效率高、检测范围广、检测周期短,为β-actin基因作为内参基因进行鸡功能基因与病原基因表达的定量分析奠定了基础。更多还原

【Abstract】 According to the chicken β-actin gene sequences available in GenBank,a pair of primers was designed for establish a SYBR Green I quantitative real-time PCR method for β-actin gene of chicken.To establish the standard curve,the product of conventional PCR served as a standard. The analysis of melting curve was also carried out.The results show the linear range of Ct value was from 12 to 31 with a good correlation coefficient(r=0.996).The melting curve show a single peak with a 88±0℃ Tm value.The real-time PCR assay developed in this study can detect β-actin in expand range with high efficiency and less time.The assay provide the basis for β-actin gene of chicken as a reference gene in quantitative analysis of mRNA expression.更多还原