急性早幼粒细胞白血病PML/RARα融合基因筛查及实时定量检测平台的建立  

The development of a diagnosis platform for screening and real time quantitation of PML/RARα fusion gene in acute promyelocytic leukemia

作　　者：康志杰[1] 崔嵩[2] 李莉[1] 高贝贝[1] 黄丹[1] 陈雪瑜[1] 高源[1] 杨岩[1] 闫金松[1] 

机构地区：[1]大连医科大学附属第二医院血液科,辽宁大连116021 [2]大连医科大学附属大连市中心医院,辽宁大连116031

出　　处：《现代肿瘤医学》2014年第7期1522-1526,共5页Journal of Modern Oncology

基　　金：大连医科大学附属第二医院青年基金项目(编号:2010-2012)

摘　　要：目的:针对90%以上的急性早幼粒细胞白血病(acute promyelocytic leukemia,APL)患者表达的PML/RARα融合基因,设计引物及探针。对APL患者进行基因筛查,并构建PML/RARα环形质粒作为标准品,建立APL患者PML/RARα融合基因检测及微小残留病变监测的诊断平台,为APL患者的诊治提供分子生物学依据。方法:设计PML/RARα及ABL引物及Taqman探针,对APL患者进行基因筛查。并以PML/RARαL型及S型阳性的APL患者cDNA为模板,应用PCR技术扩增出453bp和550bp基因片段,构建pMD 18TPML/RARα(L)及pMD 18T-PML/RARα(S)标准品。实时荧光定量(real-time quantitative PCR,RQ-PCR)技术对该基因转录本水平的变化情况进行监测。结果:成功构建pMD 18T-PML/RARα质粒标准品,应用RQ-PCR技术,以ABL为内参,应用Taqman探针法,对APL患者标本进行检测,技术可行,数据稳定。结论:成功构建APL融合基因检测及微小残留病变监测的诊断平台,应用于临床病人的基因诊断,为APL的诊治提供了可靠的分子依据。Objective：To improve clinical diagnosis and treatment of acute promyelocytic leukemia （APL）, we aimed to establishe a＇diagnosis platform for detecting PML/RARot fusion gene and mornitoring minimal residual dis- ease by constructing a circular plasmid using PML/RARct fusion gene as a standard. Methods：We designed primers and Taqman probes specific to PML/RARct （L,S） and ABL,and used cDNA of the APL patient as the tamplate to amplify a PML/RARct fragment. The 453bp and 550bp PCR product of PML/RARot was cloned into pMD 18T vector and used as a reference standard. The copy number was then determined and standard curve derived. The transcrip- tional expression of PML/RARot in bone marrow samples was quantitated by real - time quantitative PCR （ RQ - PCR）. Results：We constructed a circular plasmid with PML/RARct （L,S） fusion gene according to the method from cancer groups in Europe. With pMD 18T PML/RARct plasmid as reference standard and ABL as an internal control, we acurately detected PMI_/RARct expression in APL patients using Taqman based RQ - PCR. After further verifica- tion on technical feasibility and data reliability. Conclusion： A diagnosis platform for detecting PML/RARct fusion gene and minimal residual disease in APL patients was established.

关 键 词：PML RARα 标准品 基因筛查 实时荧光定量 诊断平台 急性早幼粒细胞白血病 

分 类 号：R733.71[医药卫生—肿瘤]