实时定量逆转录PCR在白血病标志物检测中的应用  

Detection of molecular markers in leukemias using real-time quantitative RT-PCR

作　　者：姚利[1] 陈子兴[1] 岑建农[1] 何军[1] 邱桥成[1] 鲍晓晶[1] 袁晓妮[1] 

机构地区：[1]苏州大学附属第一医院,江苏省血液研究所,江苏苏州215006

出　　处：《临床检验杂志》2007年第3期212-214,共3页Chinese Journal of Clinical Laboratory Science

摘　　要：目的 建立实时定量逆转录PCR（RQ-RT-PCR）检测各种急慢性白血病的特征性分子生物学标志物,评价其在疾病诊断和微小残留病（MRD）监测中的意义.方法 设计TaqMan探针和引物,建立RQ-RT-PCR法对各种融合基因转录本和ABL阳性模板进行扩增,并检测177份白血病标本的转录本含量,同时做细胞遗传学检查.结果 RQ-RT-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（10^8～10^2）拷贝/μl的重复性良好,正常对照无扩增信号.与细胞遗传学相比,RQRT-PCR的敏感性和特异性更强,对白血病标志物的阳性检出率更高.对12例不同类型白血病患者的MRD随访监测表明：患者体内融合基因转录本的含量随病情进展逐渐升高,随病情好转逐渐下降,并且早于细胞遗传学预测疾病复发.结论 RQ-RT-PCR方法更敏感、可靠,与疾病的关系更密切,可用于白血病的诊断和MRD随访.Objective To establish a real-time quantitative reverse transcriptase polymerase chain reaction （RQ-RT-PCR） approach for quantitative detection of various characteristic biomolecular markers in the patients with acute or chronic leukemia, and evaluate its significance for diagnosis of leukemias and monitoring minimal residual disease （MRD）. Methods Primers and TaqMan probes were designed for detecting various fusion transcripts, and normal ABL gene was used as the internal control. The conditions of RQ-RT-PCR were established for detection of various fusion transcripts and ABL positive templates with a series of dilutions. The levels of fusion transcripts in 177 bone marrow samples from various leukemias were detected, and the cytogenetics of the samples was analyzed simultaneity. Results A fine reproducibility was obtained between 108 and 102copies/ml although maximal sensitivity of 10 copies/ml was achieved, and no amplified fluorescent signals were detectable in normal controls. The sensitivity and specificity of RQ-RT-PCR assay were significantly higher than those of cytogenetics, so that the positive rate of molecular markers detected in leukemias was higher than that of cytogenetical method. MRD of twelve leukemia patients with the different type was monitored and the results indicated that the expression level of fusion transcripts persistently increased with the progress of the disease, but steadily reduced with the improvement of the disease condition. In comparison with the results of cytogenetics, RQ-RT-PCR can predict early relapse of diseases. Conclusion RQ-RT-PCR is a more sensitive and reliable quantitative assay and correlated strongly with the clinical change of leukemias. So it can be used in the diagnosis of leukemias and monitoring MRD.

关 键 词：实时定量RT-PCR 白血病 融合基因转录本 

分 类 号：R733.7[医药卫生—肿瘤]