SYBR Green Ⅰ实时荧光PCR检测survivin甲基化状态

Detection of survivin gene methylation with SYBR Green Ⅰ real-time fluorescent PCR

目的建立SYBR Green Ⅰ实时荧光聚合酶链反应(PCR)检测survivin甲基化的方法。方法25例胃癌组织标本及与其配对的正常胃组织经甲基化敏感性限制性内切酶HpaⅡ和MspⅠ处理后,再用SYBR Green Ⅰ实时荧光PCR对survivin外显子1进行检测。实时荧光定量PCR检测survivin基因内含子2,用于监测经限制性酶消化的基因组DNA浓度变化,10倍系列稀释的基因组标准物用于检验实时PCR的敏感度,PCR产物通过凝胶电泳分析证实。结果经HpaⅡ酶切后,甲基化的目的基因PCR产物在熔解曲线上有Tm值为(91.5±0.5)℃的峰,电泳证实为338bp的条带。所有经酶消化的样本都能扩增出以Tm值(79.5±0.5)℃为特征的对照基因(survivin基因内含子2),表明基因组DNA未产生严重的非特异性降解。SYBR Green Ⅰ实时荧光PCR检测甲基化目的基因的灵敏度为100拷贝/μL。用以上新建的体系检测25例胃癌标本,发现其survivin基因外显子1的去甲基化频率为96%。结论SYBR Green Ⅰ荧光PCR法具有快速、准确、敏感、实时、简单和敏感的特点,是检测survivin外显子1甲基化状态的一种可靠的新方法。

Objective To establish a real time PCR based method for detection of survivin gene samples methylation with dye SYBR Green Ⅰ. Methods DNA samples from 25 pairs of gastric cancer tissue and matched normal gastric tissues were digested with mCpG-sensitive Hpa Ⅱ and Msp Ⅰ , and detected by SYBR Green Ⅰ real-time PCR for survivin exon 1 specific primers. The concentration of digested DNA was monitored by real time quantitative PCR with survivin intron 2 specific primers. A normal genomic DNA which served as a standard was diluted serially 10-fold to determine the sensitivi ty of real-time PCR. The products were further verified by agarose gel electrophoresis analysis. Results After Hpa Ⅱ enzyme digestion, a peak of PCR product derived from methylated targeted gene was found in the melting curve at Tm （91.5±0.5） ℃, which was verified as 338 bp strap. The control gene （survivin intron 2） characteristic of Tm （79.5±0.5）℃ was amplified in all digested samples, indicating no non-specific degradation occurred in the genome DNA. The sensitivity of this method was 100 copies/μL. The new established PCR system was applied to detecting 25 cases of lung cancer tissue samples. As a result, the frequency of survivin exon 1 demethylation was 96%. Conclusion As a reliable new method for detection of survivin exon 1 methylation, SYBR Green Ⅰ real-time fluorescent PCR assay is rapid, accurate, sensitive, timing and simple.