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

关键词:  慢性粒细胞白血病  BCR-ABL  标准品  实时荧光定量PCR  诊断平台

Key words:  [Key words]　chronic myeloid leukemia  BCR-ABL  standard reference  RQ-PCR  diagnosis platform

摘要:

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。

Abstract:

[Abstract] 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 patients 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.