人血浆DNA双重实时荧光定量PCR检测法的建立  

A novel duplex real-time PCR assay for quantification of plasma DNA

作　　者：陈丹[1] 潘世扬[1] 张丽霞[1] 高丽[1] 谢而付[1] 黄? 戎国栋[1] 

机构地区：[1]南京医科大学第一附属医院临床检验中心,南京210029

出　　处：《临床检验杂志》2007年第3期177-179,共3页Chinese Journal of Clinical Laboratory Science

基　　金：江苏省抗病毒药物临床试验服务中心(BM2006113);江苏省政府"135"重点实验室科研基金(SK200205);江苏省卫生厅指导课题(Z200601)。

摘　　要：目的建立带有内参照的双重实时荧光定量PCR方法检测血浆DNA含量。方法构建重组质粒DNA作为内参照物,采用共用下游引物的双重实时荧光定量PCR技术同步扩增人看家基因β-actin和重组质粒载体中人工合成DNA序列,定量检测健康成年人血浆DNA含量。结果本法能在同一个反应管中对目的基因和内参照进行同步扩增,两者的扩增无相互干扰,特异性好;重组质粒DNA的平均扩增效率达90%,β-actin基因的平均扩增效率接近100%;本法批内变异系数(CV)11%,批间CV17%。结论成功建立含有内参照的双重实时荧光定量PCR方法,可对血浆DNA进行定量检测。Objective To develop a novel duplex real-time PCR assay with internal control for the quantification of plasma DNA. Methods Recombined plasmid was quantified and added into plasma as internal control before DNA extracting. The concentration of plasma DNA in healthy adults was determined using a duplex real-time PCR assay for simultaneous amplifications of plasmid and target β-actin gene in a same reaction system. Results No cross-species amplification was observed in the duplex PCR reaction. The amplification efficiency was 90% for plasmid DNA and close to 100% for β-actin gene. The experimental variability of assay for inter-day of and intraday were 11% and 17% respectively. Conclusion A novel duplex real-time PCR assay with internal control was developed and allowed the quantification of olasma DNA.

关 键 词：血浆DNA 实时荧光定量PCR 双重PCR 

分 类 号：Q503[生物学—生物化学]