实时荧光定量RT-PCR检测白血病细胞WT1 mRNA方法的建立

目的 建立实时荧光定量RT-PCR检测白血病细胞WT1 mRNA的方法。方法 在PCR反应体系中加入了一条与靶基因序列互补的寡核苷酸双荧光标记探针(5′端为荧光报告基团，3′端为荧光淬灭基团)，建立实时荧光定量RT—PCR检测白血病细胞WT1 mRNA的方法，并测试其特异性及敏感性。结果 所建立的实时荧光定量RT—PCR检测WT1 mRNA的方法具有很好的准确性，检测WT1 mRNA的灵敏度达10^-4。方法的变异系数为1．93％，稳定性较好。循环阈值与PCR体系中起始模板量的对数值之问有着良好的线性关系(斜率为-3．5，线性相关系数，-0．997)，可对WT1 mRNA的表达进行准确定量分析。结论 实时荧光定量RT-PCR检测白血病细胞WT1 mRNA的方法具有很好的特异性和敏感性，且操作简便、安全、快速。

Establishment of a real-time fluorescence quantitative RT-PCR for detecting WT1 mRNA in leukemia cells

Objective To establish a real-time fluorescence quantitative RT-PCR(RQ-PCR) method for detecting the expression of WT1 gene in leukemia cells. Methods A specific fluorescence-labeled probe capable of binding to cDNA target was i ncorporated into the reaction system of a regular RT-PCR. The RQ-PCR method fo r WT1 mRNA was set up and the specificity and sensitivity were tested. Res ults The specificity and sensitivity of this RQ-PCR were ideal. Sensit ive experiments revealed that 10 -4 WT1 mRNA could be detected. The CV of t his method was 1 93%. The standard curve indicated that there was a linear rela tionship between CT value and the logarithmic value of template concentration (s lope=-3 5, r=0 997). With this method the accurate quantitative assessment of initial amount of WT1 mRNA was performed. Conclusion RQ-PC R is proved to be a sensitive, specific, reliable and convenient method for the detection of WT1 mRNA in leukemia cells.