荧光定量PCR检测血浆游离DNA方法的建立  

A NOVEL FLUORESCENT QUANTITATIVE PCR METHOD FOR PLASMA FREE DNA DE-TECTION USING TAQMAN PROBE

作　　者：严勇[1,2] 陈新敏[3] 杜晓红[3] 丁显平[1] 

机构地区：[1]四川大学生命科学学院医学遗传研究所,成都610064 [2]四川省产品质量监督检验检测院 [3]四川省妇幼保健院

出　　处：《现代预防医学》2009年第7期1338-1340,共3页Modern Preventive Medicine

摘　　要：[目的]建立TaqMan探针定量PCR技术检测血浆游离DNA的方法。[方法]构建重组质粒pMD-18-T-GAPDH、pMD-18-T-SRY作为标准品,并通过软件设计出TaqMan探针,优化定量PCR体系,并对其特异性进行分析。[结果]建立了1个检测GAPDH和4个检测SRY的荧光定量PCR方法,得到上述5个荧光PCR的扩增动力曲线和定量标准曲线,模板起始浓度与阈值循环数(CT值)之间有很好的相关关系(r分别为0.97、0.99、0.98、0.97、0.99)。[结论]成功建立了定量检测血浆游离DNA的方法,其灵敏度高、特异性强,有望成为一种检测孕母血浆游离DNA含量的快速简便方法。[Objective] To establish a novel real-time fluorescent quantitative PCR method for plasma free DNA detection. [Methods] Two recombinant vector, named pMD-18-T-GAPDH and pMD-18-T-SRY, was used as a standard template. The TaqMan probes were designed according to the gene sequences. Then PCR reaction system was optimized and evaluated, [ Results ] We established 1 FQ-PCR method for detecting the GAPDH and 4 FQ-PCR methods for detecting the SRY. We also got the 5 kinetic curves and quantitative standard curve of FQ-PCR. There was reliable correlation between the template copies and the CT value （r=0.97, 0.99, 0.98, 0.97, 0.99）. [ Conclusion] The real-time fluorescent quantitative PCR method to detect plasma free DNA is successfully established. This method is sensitive and specific, and would be a potential method to detect plasma free DNA from maternal blood.

关 键 词：游离DNA 实时荧光定量PCR SRY基因 

分 类 号：R446.5[医药卫生—诊断学] R734.202[医药卫生—临床医学]