用实时定量PCR检测慢性髓系白血病患者的bcr/abl^(P210)融合基因转录本

Quantification of p210 bcr/abl fusion transcripts in patients with chronic myeloid leukemia by using real-time quantitative PCR

目的 建立实时定量PCR(RQ PCR)检测慢性髓系白血病(CML)bcr/ablP210融合基因转录本,评价其在CML诊断和微小残留病(MRD)监测中的意义。方法 设计TaqMan探针和引物,建立RQ PCR法对b3a2和ABL阳性模板进行扩增,并检测22例CML患者bcr/ablP210转录本含量。结果 RQ PCR法最低可检测到 10个拷贝 /μl的阳性模板,但重复性较差,而 108 ~102 拷贝 /μl的重复性良好,正常对照无扩增信号。19例初诊CML患者bcr/ablP210转录本绝对含量为 1. 42×102 ~2. 24×105拷贝 /50ng(中位数量 3. 06×104 拷贝 /50ng),ABL纠正后的相对含量为 3% ~687% (中位数 147% )。2例患者骨髓移植后bcr/ablP210转录本下降, 1例患者由慢性期进展为加速期时含量显著增高。结论 RQ PCR方法敏感、可靠,可用于CML患者的诊断和MRD随访监测。

Objective To establish a real-time quantitative PCR (RQ-PCR) method for quantitative detection of p210 bcr/abl fusion transcripts in the patients with chronic myeloid leukemia (CML) and evaluate its significance for diagnosis of CML and monitoring minimal residual disease (MRD).Methods A pair of primers and TaqMan probe were designed for detecting the most frequent p210 bcr/abl transcripts and normal ABL gene was used as the internal control.The conditions of real-time PCR were established for detection of p210 bcr/abl and ABL positive templates with a series of dilutions.The level of p210 bcr/abl fusion transcripts in bone marrow samples from 22 CML patients were detected.Results A fine reproducibility was obtained between 102 and 108 copies/μl although maximal sensitivity of 10 copies/μl was achieved,and no amplified fluorescent signals were detectable in the normal controls.In 19 CML patients the absolute amount of p210 transcripts was from 1.42×102 to 2.24×105 copies/50ng (median 3.06×104 copies/50ng) and calibrated relative amount was from 3% to 687% (median 147%).In two cases p210 bcr/abl transcripts significantly decreased after bone marrow transplantation.In another CML patient the fusion transcripts significantly increased during the progressive course from chronic phase to accelerated phase.Conclusion RQ-PCR is a sensitive,reliable quantitative assay and can be used in the diagnosis of CML and monitoring MRD.