实时荧光定量PCR检测人类EZH2基因方法的建立  

Quantification of human EZH2 by using 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学院附属协和医院检验科,武汉430022

出　　处：《临床血液学杂志（输血与检验）》2007年第6期243-246,共4页Journal of Clinical Hematology(Blood Transfusion & Laboratory Medicine)

基　　金：湖北省科技攻关项目(No:2005AA304B08)

摘　　要：目的:建立SYBR GreenI实时荧光PCR定量检测人类EZH2基因的方法。方法:将EZH2 RT-PCR扩增片段克隆入载体pGEM-T后,经测序鉴定正确后,进行纯化和定量及系列稀释,应用SYBR GreenI实时荧光定量PCR检测EZH2,建立标准曲线,熔解曲线分析产物的特异性。结果:该法检测的最低拷贝数为10,线性范围为101~108拷贝,相关系数r为-1.00,104copies/μl标本的批内变异系数(CV)和日间CV分别为1.0%和2.7%。熔解曲线分析显示单一的峰,熔解温度(Tm)为(83.42±0.13)℃。结论:实时荧光定量PCR方法检测EZH2基因,具有高敏感性、高特异性和高精确性等优点,可作为进一步研究EZH2的方法。Objective:To establish a real-time fluorescent polymerase chain reaction method for quantifying human EZH2.Method:The EZH2 fragment in pure form from classical RT-PCR was cloned to pGEM-T vector, and recombinant plasmid pGEM-EZH2 was purified and quantified spectrophotometrically.Standard curve was established using a series dilution of quantified plasmids to measure EZH2 using SYBR Green I real-time fluorescent polymerase chain reaction and the characteristic of specific EZH2 amplicon was analysed by melting curve.Result:The method can detect as low as 10 copies. A good linearity was found from 101 to 108 copies/reaction and the correlation coeffecient was -1.0.The intraassay and interassay variation of 104 copies/reaction was 1.0% and 2.7%, respectively. Melting curve analysis showed a single peak, and Tm was(83.42±0.13)℃.Conclusion:SYBR Green I real-time fluorescent quantitative polymerase chain reaction which is specific, sensitive and accurate can be used to further research on human EZH2.

关 键 词：EZH2基因 SYBR GreenI 实时荧光定量PCR 

分 类 号：Q987-3[生物学—遗传学]