SYBR Green Ⅰ实时定量PCR检测生存素基因的方法建立及条件优化  

Establishment and optimization of real-time PCR with SYBR Green Ⅰ for quantification of survivin

作　　者：胡丽华[1] 程正江[1] 李一荣[1] 黄少军[2] 

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 [2]华中科技大学同济医学院附属襄樊医院检验科

出　　处：《中华检验医学杂志》2007年第12期1364-1367,共4页Chinese Journal of Laboratory Medicine

摘　　要：目的建立SYBRGreenⅠ染料实时定量PCR检测生存素（Survivin）基因定量的方法。方法根据PCR产物荧光强度、循环阈值（Ct值）、标准曲线斜率、相关系数和熔解曲线优化反应体系中各组分的量及反应条件，对引物二聚体消除策略和Ct值获取方式进行评价。并用该方法检测43份胃癌组织Survivin基因扩增情况。结果SYBRGreenⅠ实时荧光定量PCR体系扩增Survivin的最佳组成和条件是：Taq酶2．5U／100μl、MsCl2 2mmol／L、引物浓度0．2μmol／L和退火温度58℃；在PCR循环延伸结束后设置1个低于特异产物退火温度值2℃的荧光读取温度，能有效消除引物二聚体对定量检测的影响；以二次倒数最大值方式确定Ct值，能避免主观因素所致误差。研究建立的SYBRGreenⅠ染料实时定量PCR检测Survivin基因含量方法的灵敏度为10拷贝／μl，线性范围10^1～10^4拷贝μl（r=0．9997），批内变异系数（CV）1．13％-1．91％，批间CV3．31％-4．50％；胃癌组织中Sttrvivin基因扩增率为13．9％（6／43）。结论优化后的SYBRGreenI实时定量PCR方法具有方便、经济、灵敏度高和重复好等特性，可用于Survivin基因含量分析。Objective To establish real-time PCR with SYBR Green Ⅰ for quantification the gene of survivin. Methods The components and conditions of PCR system were optimally determined by fluorescence intensity, cycle threshold （Ct）, melting curve, coefficiency and slope of the standard curve. The means to eliminate the contaminating fluorescence of primer-dimers and the mode for Ct value determination were also optimized. Using the developed PCR system, we quantifieated the survivin gene in 43 patients with gastric carcinoma. Results The optimized condition for PCR amplification of survivin were 2 mmol/L of MgC12 ,2. 5 U/100μl of Taq DNA polymerase, 0. 2 μmol/L of primers, and the optimized annealing temperatures for PCR were 58℃. The influence of primer dimmer can be eliminated by setting the fluorescence collecting temperature below the Tm of the specific amplicon by 2℃. The second derivative maximum mode, instead of fit point mode, was a feasible method to determine the Ct value for quantification. The sensitivity of this method was 10 copies/μl ,and a good linearity was found from 101 to 104 copies/μl （r = 0. 999 7 ）. The inter-experimental coefficient of variation was 1.13% - 1.91%, whereas the coefficient of variation between runs was 3.31% -4.50%. Using the optimized PCR system,we quantificated the gene of survivin, the result indicated that survivin gene was amplified in 13. 9% of gastric carcinomas. Conclusions The optimal real-time PCR with SYBR Green Ⅰ,as a cost-effective and feasible DNA quantitative method, is fit for quantification of the survivin with satisfactory repeatability and high sensitivity.

关 键 词：聚合酶链反应 基因 生存素 

分 类 号：R450[医药卫生—治疗学]