SYBR Green I实时荧光定量PCR检测BMI-1 mRNA方法的建立  

Quantification of human BMI-1 mRNA with SYBR Green I real-time fluorescent polymerase chain reaction

作　　者：陈凤花[1] 胡丽华[1] 王琳[1] 李一荣[1] 周志明[1] 

机构地区：[1]华中科技大学同济医院附属协和医院,湖北武汉430022

出　　处：《山东医药》2007年第29期10-12,共3页Shandong Medical Journal

基　　金：湖北省科技攻关项目(2005AA304B08)。

摘　　要：目的 建立SYBR Green I实时荧光PCR定量检测人类BMI-1 mRNA的方法。方法 以含有目的基因BMI-1 cDNA的质粒pEGFP-BMI-1为阳性模板,进行纯化和定量及系列稀释,应用SYBR Green I实时荧光定量PCR检测BMI-1,建立标准曲线,熔解曲线分析产物的特异性。结果 该法检测的线性范围为6.4×10^1～6.4×10^7拷贝,相关系数r为-1.00,6.4×10^3拷贝/反应的标本批内变异系数（CV）和日间CV分别为1.6%和2.8%。熔解曲线分析显示单一的峰,Tm为（80.40±0.18）℃。结论 SYBR Green I实时荧光PCR定量检测BMI-1 mRNA是快速有效、灵敏度高、特异性好的方法,可为BMI-1的进一步研究奠定基础。[ Objective] To establish a real-time fluorescent polymerase chain reaction method for quantifying human BMI-1 mRNA. [ Methods] The recombinant plasmid pEGFP-BMI-1 was purified and quantified spectrophotometricaUy. Standard curve was established by using a series dilution of quantified plasmids to measure BMI-1 using SYBR Green I real- time fluorescent polymerase chain reaction and the characteristic of specific BMI-1 amplicon was analyzed by melting curve. [Results] A good linearity was found from 6.4 × 10^1 to 6.4 × 10^7 copies/reaction, there was a good correlation between template concentration and cycle threshold, and the correlation coefficient was - 1.00. The intraassay and interassay variation of 6.4 × 10^3 copies/reaction was 1.6% and 2.8% respectively. Melting curve analysis showed a single peak, and Tm was （80.40 ± 0.18）℃. [ Conclusions ] SYBR Green I real-time fluorescent quantitative polymerase chain reaction is a rapid, specific, sensitive and accurate method,it can be used to further research on human BMI-1.

关 键 词：BMI-1 SYBRGreenI 荧光光度测定法 

分 类 号：R392[医药卫生—免疫学]