分子信标实时定量PCR法检测胃癌中Survivin基因mRNA的表达(精).docVIP

分子信标实时定量PCR法检测胃癌中Survivin基因mRNA的表达 ????作者:郑红，孙保存，王立梅，李希，王家仓 【摘要】 目的 采用分子信标实时定量PCR方法检测胃癌样本中Survivin基因mRNA的表达量，并分析其与临床资料之间的关系。方法 设计Survivin基因特异性分子信标探针和引物，以cDNA克隆重组质粒为标准品，建立Survivin基因实时定量检测方法；并检测胃癌样本中Survivin基因mRNA的模板拷贝数。结果 Survivin基因mRNA分子信标实时定量检测方法的线性范围为103～1010拷贝数，批间变异为28.16%，批内变异为13.34%，灵敏度为42个拷贝数，平均回收率为109.83%；胃癌组织、癌旁组织和正常组织以及胃良性病变组织间比较，mRNA模板拷贝数差异有显著性（P＜0.05）；胃癌样本中mRNA模板拷贝数与淋巴结转移和2年生存期以及病理类型相关（P＜0.05）。结论 成功建立Survivin基因分子信标实时定量检测方法。胃癌中该基因表达的模板拷贝数可作为预测淋巴结转移、评价预后的一个重要指标。????【关键词】 分子信标；实时定量PCR；Survivin；胃癌????分子信标；实时定量PCR；Survivin；胃癌????Real Time PCR detection of Survivin expression by molecular beacon in gastric carcinoma????【Abstract】 Objective To develop a real time fluorescent quantitative method based on molecular beacon technique and quantification of mRNA expression of survivin gene in gastric carcinoma.Methods A molecular beacon probe and primers were designed and applied in the detection of survivin gene，while recombination plasmid containing the sequence of survivin was standard. The copies of survivin expression were detected in gastric carcinoma，and relationship between copies and clinical data was analyzed. Results A linear standard curve was obtained between 103 and 1010 copies. The inter-and intra-assay coefficient variation (CV) was 28.16% and 13.34%，respectively. The sensitivity of this assay was 42 copies. The average recovery was 109.83%. The copies of survivin expression were significantly higher in gastric carcinoma than in the other groups (P＜0.05). There was a relationship between copies of survivin gene with lymph node metastasis，poor survival and histological types. Conclusion The method can detect copies of survivin expression. It might be an important indicator in predicting lymph nodes metastasis and evaluating the prognosis.????【Key words】 molecular beacon；real time PCR；survivin；gastric carcinoma????实时定量PCR方法是近年发展起来的一种新的PCR定量技术，由于在PCR反应中引入了荧光探针，可以通过检测扩增过程中荧光信号的有无和强弱来判定目的基因的有无及表达量多少。分子信标（molecular beacon）探针就是其中一种荧光探针，它是在核酸杂交原理和荧光共振能量转