实时定量荧光PCR检测白血病融合基因

Detection and Quantification of Aberrant Leukemia Fusion Gene by Real-time RT-PCR

本研究旨在建立实时定量荧光PCR的方法并检测CML、ALL、APL患者中融合基因的拷贝数,观察患者体内融合基因转录水平的变化。构建质粒标准品,制作标准曲线。检测49例患者的骨髓及外周血标本130份,对个别患者连续监测融合基因转录表达水平。结果表明:在82.4%(28/34)的CML患者中检测到BCR-ABLP210阳性(BCR-ABLP210/ABL比值为0.01～3.19),其中在1例CML急淋变患者检测到BCR-ABLP210和BCR-ABLP190双阳性;在66.7%(4/6)的ALL患者中检测到BCR-ABL阳性,其中2例BCR-ABLP210阳性,2例BCR-ABLP190阳性;在77.8%(7/9)的APL患者中检测到PML-RARa融合基因阳性(PML-RARa/ABL比值为0.0014～3.199),其中3例为长型,3例为短型,1例为变异型,检测结果为阴性的患者处于缓解期;连续监测患者融合基因转录表达水平与临床缓解和复发的变化情况相吻合。结论:实时定量荧光PCR技术成熟、操作简便,检测白血病融合基因结果准确稳定,对于临床明确诊断、具体分型、动态观测肿瘤负荷、选择治疗方案、评估治疗效果和预后都有较大价值,值得推广应用。

This study was purposed to set up real-time quantitative RT-PCR technique and to measure leukemia fusion gene transcripts in patients with chronic myeloid leukemia （CML） , acute lymphoblastic leukemia （ALL） and acute promyelocytic leukemia（APL）. All plasmids containing the target gene sequences were constructed to establish the standard curves. A TaqMan based real-time quantitative RT-PCR was performed to measure aberrant fusion gene transcripts in 130 samples of peripheral blood （PB） or bone marrow （BM） from 49 patients with leukemia. The results showd that the BCR-ABL^p210 transcripts were detected in 28 （82.4%） out of 34 CML patients （ the ratios of BCR- ABL^p210/ABL varied from 0.01 to 3.19）and also in 2 （33.3%） out of 6 ALL patients. The BCR-ABL^p190 transcripts were detected in 2 （33.3%） out of 6 ALL patients. The BCR-ABL^p210 and BCR-ABL^p190 transcripts were both detected in 1 （2.9%） CML patients. The PML/RARα transcripts were detected in 7 （ 77.8% ） out of 9 APL patients（ the ratio of PML-RARa/ABL varied from 0.0014 to 3. 199）. The relative frequency of both bcrl and bcr3 was 42.9%, while that of bcr2 was 14.3%. The transcript level of aberrant fusion gene varied from the clinical situation of patient. It is concluded that real-time quantitative PCR is a reliable, innovative and promising technology with high sensitivity and speciality. It has potential clinical value for defining diagnosis, typing tumor, selecting treatment, measuring the tumor load, monitoring fusion gene expression level and evaluating therapeutic strategies, which is worthy to be popularized.