慢性粒细胞白血病BCR-ABL融合基因实时定量检测平台的建立

Development of a diagnosis platform for real time quantitation of BCR-ABL fusion gene in chronic myelogenous leukemia

目的建立慢性粒细胞白血病(chronic myelogenous leukemia,CML)患者BCR-ABL融合基因检测及微小残留病变实时定量监测的诊断平台。方法依据GenBank中编码P210蛋白的融合基因M(b2a2,b3a3)及ABL的基因序列,分别设计引物及Taqman探针,以BCR-ABL阳性的CML患者cDNA为模板,通过PCR扩增出587 bp的基因片段,连入pMD 18-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(real-time quantitative PCR,RQ-PCR)技术监测BCR-ABL转录水平的变化。结果成功构建BCR-ABL标准品。应用RQ-PCR探针法,以ABL为内参,对CML患者进行BCR-ABL融合基因的检测,得到比较稳定的定量数据,与定性结果一致。结论自行构建BCR-ABL质粒为标准品,运用RQ-PCR实时监测BCR-ABL融合基因的表达变化,敏感性好,稳定性高,对临床检验具有普遍意义。

Objective To improve clinical diagnosis and treatment of chronic myeloid leukemia （ CML） , we aim to establish a diagnosis platform for detecting BCR -ABL fusion gene and mornitoring minimal residual disease by constructing a circular plasmid using BCR-ABL fusion gene as a standard .Methods We designed primers and Taqman probes specific to BCR -ABL（b2-a2,b3-a2）and ABL, and used cDNA of the CML patient as the tamplate to amplify a BCR -ABL fragment.The 587bp PCR product of BCR-ABL was cloned into pMD 18T vector and used as a reference standard .The copy numbers was then determined and standard curve derived .The transcriptional expression of BCR/ABL in bone marrow samples from pa-tients was quantitated by real -time quantitative PCR （ RQ-PCR） .Results We constructed a circular plasmid with BCR -ABL fusion gene according to the method from cancer groups in Europe .With pMD 18T BCR-ABL plasmid as reference standard and ABL as an internal control , we accurately detected BCR -ABL expression in CML patients using Taqman based RQ-PCR.After further verification on technical feasibility and data reliability , a diagnosis platform for detecting BCR -ABL fusion gene and minimal residual disease in CML patients was then established .Conclusion The Taqman based RQ -PCR can greatly improve the sensitivity and reliability in detection of BCR -ABL fusion gene expression .With the technical feasibility, the platform could be helpful for the clinical gene diagnosis and provide guidance for CML treatment .