均匀设计法在实时定量PCR检测条件优化中的应用研究  

Application of Uniform Design to Optimize Real-time Quantitative PCR Conditions

作　　者：宋莉[1] 叶珏[1] 王林[1] 王慧[1] 孟宪敏[1] 

机构地区：[1]北京协和医学院中国医学科学院阜外心血管病研究所中心实验室,北京100037

出　　处：《中国分子心脏病学杂志》2010年第3期167-170,共4页Molecular Cardiology of China

基　　金：国家自然科学基金(30871057;30940029);阜外心血管病医院院所青年基金(2008F012)

摘　　要：目的探索均匀设计在优化实时定量PCR检测条件中的应用,确立心钠素基因实时定量的最佳条件。方法以乳鼠心肌细胞cDNA为模板,退火温度和引物浓度为影响因素,应用均匀设计法探索影响心钠素基因实时荧光定量PCR的扩增条件;量化不同实验条件下的扩增效果,在最优的扩增条件下建立ANP基因定量扩增的相对标准曲线,并检测扩增效率。结果通过均匀设计法的试验设计,用8个试验点,完成了对温度和引物浓度2因素8水平的实验条件优化,建立了ANP基因实时定量PCR检测的最佳组合,为退火温度59.2℃、引物终浓度200μM。结论均匀设计是一种简便易行、快速经济的实时定量PCR检测条件的优化方法。Objective To investigate the application of uniform design to optimize SYBR Green I detection real-time PCR conditions and apply it for ANP gene.Methods cDNA was obtained from the primary neonatal cardiomyocytes.Annealing temperature and primer quantity were studied as two key factors for ANP gene real-time quantitative PCR conditions.By using uniform design,eight levels for each factor were optimized The standard score and a standard curve of 5 dilution series were used to estimate the amplifi cation conditions.Results By using the uniform design the quantitative PCR conditions for ANP gene are obtained from eight tests in one experiment.The optimized conditions is annealing temperature a 59.2℃ and each primer concentration of 200nM in the fi nal reaction.Conclusion：Uniform design is a high effi cient method to optimize the real-time quantitative PCR conditions.

关 键 词：均匀设计 实时定量 PCR 心钠素 条件优化 实验因素 

分 类 号：R440[医药卫生—诊断学]