摘要
目的 建立一种用于肝癌早期诊断和监测的血清miRNA-125a Taqman实时荧光定量PCR检测方法。方法 采用Trizol法提取肝癌患者血清中总RNA,以肝癌发生具有代表性的miRNA-125a基因为检测序列,分别设计逆转录引物、扩增引物和Taqman探针; 构建和制备miRNA-125a重组质粒标准品,建立血清miRNA-125a Taqman 实时荧光定量PCR检测方法。结果 成功构建了miRNA-125a重组质粒载体,制备了miRNA-125a重组质粒标准品,建立了血清miRNA-125a Taqman实时荧光定量PCR扩增体系。经实验验证,所设计引物具有较好的特异性,引物的最低检测限为78.125 copies/μl; 对17例肝癌患者血清miRNA-125a的检测结果在（1.45×10^2-3.52×10^5）copies/μl之间。结论 血清miRNA-125a Taqman实时荧光定量PCR检测方法的建立,为原发性肝癌早期诊断和监测提供一种新的分子诊断定量方法。
Objective Tobuild the method of real-time fluorescence quota PCR examination of serum miRNA 125a Taqman for diagnosing and monitoring liver cancer early. Methods 1st, based on specific miRNA-125a geneassociated with liver cancer, to design reverse transcriptase primer,amplification primer and Taqman probeindividually through the Trizaol meth- od to extract patients＇ total RNA from serum. 2nd,to prepare the miRNA-125a recombinant plasmid standard substance and buildthe method of real-time fluorescence quota PCR examination of serum miRNA-125a Taqman. Results To form the miRNA-125a recombinant plasmid carriers successfully. To prepare the miRNA-125a recombinant plasmid standard sub- stance successfully. To buildamplification system of real-time fluorescence quota PCR of serum miRNA-125a Taqman. It had been proved that the designed primer whose minimum detection limit was 78. 125 copies/μl had proper specificity. The ex-amination results of serum miRNA-125a of 17 patients with liver cancer vary between 1.45×10^2-3.52×10^5 copies/μl. Conclusion To set up the method of real-time fluorescence quota PCR examination of serum miRNA-125a Taqman can offer an efficientmolecular diagnosis way to diagnose and monitor the primary liver cancer early.
出处
《现代检验医学杂志》
CAS
2016年第4期10-13,共4页
Journal of Modern Laboratory Medicine
基金
陕西省科技计划项目(2013K12-05-12)
西安市卫生局科技计划项目(2013)资助
