白血病融合基因BCR-ABL和PML-RARα多重实时定量检测方法的建立

Development of a diagnosis method for real time quantitation of BCR-ABL and PML-RARα fusion gene in leukemia

目的建立白血病常见融合基因BCR-ABL和PML-RARα多重实时定量检测方法。方法针对BCR-ABL和PML-RARα融合基因序列分别设计引物及不同荧光信号的Taqman探针,同时以肿瘤细胞K562、NB4总RNA所逆转录的c DNA为模板,通过PCR扩增出BCR-ABL599 bp和PML-RARα496 bp的基因片段,定向插入p MD18-T载体,制备成标准品并绘制标准曲线,运用实时荧光定量PCR扩增曲线判定是否具有融合基因,同时定量融合基因拷贝数。结果成功构建BCR-ABL和PML-RARα质粒标准品。应用RQ-PCR法,以Actin为内参,对临床血液样本进行检测,分析样本是否具有这两类融合基因及融合基因拷贝数。结论本研究以探针荧光信号及其信号类型来判断是否具有融合基因及融合基因类型,同时通过BCR-ABL和PML-RARα质粒标准品所绘制的标准曲线来定量融合基因初始拷贝数。在同一体系中同时检测两种常见的融合基因有利于防止漏检、节约成本,对临床检验具有普遍意义。

Objective To establish a diagnosis method for detecting BCR-ABL and PML-RARct fusion gene and mornitoring minimal residual disease by constructing a circular plasmid using BCR-ABL and PML-RARα fusion gene as standards. Methods We designed primers and Taqman probes specific to BCR-ABL, PML-RARα and Actin. To amplify BCR-ABL（599） and PML-RARa （496） fragments by templates of cDNAs which reversed by tumor cell K562 and NB4＇ total RNAs. Then cloned this two fragments into pMD-18T vector and used as a reference standard. We can know samples that whether has the fusion gene and quantitative fusion gene copy number due to the real-time quantitative PCR amplification curve. Results We constructed two circular plasmids with BCR-ABL and PML-RARα fusion gene. With the two plasmids as reference standards and Actin as an internal control, we accurately detected fusion gene expression and copy number in Clinical blood sample using Taqman based RQ PCR. Conclusion This study use probe fluorescence signal and signal types to determine samples whether has the fusion gene and fusion gene types. Because of the standard curve,we also can detected fusion gene expression. Simultaneous detection of 2 fusion genes in a same system can helps prevent false negative sample and save the cost. With the technical feasibility, the method could be helpful for the clinical gene diagnosis and provide guidance for leukemia treatment.