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目的建立分子信标定量检测Survivin基因mRNA表达的方法,对方法进行评估,并应用于临床。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基因分子信标定量检测方法;用该方法检测结直肠癌样本中Survivin基因mR- NA的表达量,分析mRNA表达量与临床资料之间的关系。结果所建立的分子信标定量检测方法,线性范围为1×103~1×1010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%;肠癌组织、癌旁组织和正常组织以及肠良性病变组织间比较,mR- NA表达量差异有统计学意义(P<0.05),肠癌样本中mRNA表达量与病理分型和淋巴结转移有关(P<0.05)。结论成功建立Survivin基因分子信标定量检测方法,为基因诊断提供新的定量检测方法。
Objective To establish a method for quantitating the mRNA expression of Survivin gene by molecular beacons, to evaluate the method and to apply it in clinic. Methods Survivin gene-specific molecular beacon probes and primers were designed. The recombinant plasmid of cDNA clone was used as a standard to establish a quantitative detection method of Survivin gene molecular beacon. The expression of Survivin gene mR-NA in colorectal cancer samples , Analysis of the relationship between mRNA expression and clinical data. Results The quantitative detection method of molecular beacons established by this method was linear in the range of 1 × 10 3 to 1 × 10 10 copies with an intra-assay variation of 28.16%, an intra-assay variation of 13.34% and a sensitivity of 42 copies, with an average of The recovery rate was 109.83%. The expression of mR-NA in colorectal cancer tissues, adjacent non-cancerous tissues and normal tissues as well as benign intestinal lesions was significantly different (P <0.05). The mRNA expression of mR- The quantity and pathological type and lymph node metastasis (P <0.05). Conclusion Survivin gene molecular beacon quantitative detection method was successfully established to provide a new quantitative detection method for gene diagnosis.