Bolufer P, Sanz G F, Barragán E, Sanz M A, Cervera J, Lerma E, Senent L, Moreno I, Planelles M D
Department of Clinical Pathology, Hospital Universitario La Fe, Valencia, Spain.
Haematologica. 2000 Dec;85(12):1248-54.
The limited value of qualitative reverse transcription polymerase chain-reaction (RT-PCR) for monitoring chronic myeloid leukemia (CML) patients has prompted the development of quantitative assays. We have developed a quantitative real-time PCR (QC-PCR) method in the LightCycler, based on the use of fluorescently labeled probes (HybProbes), to estimate BCR-ABL fusion gene transcripts in samples from CML patients.
Fifty-two samples (45 peripheral blood, five bone marrow, and two apheresis product samples) from nine patients with CML were analyzed. Seven patients were studied at diagnosis and during follow-up after hematopoietic stem cell transplantation (HSCT), whereas two were evaluated only after HSCT. The PCR reaction was carried out in capillary tubes in a final volume of 10 microL, using 2 microL cDNA, the Mensik et al. primers, and two HybProbes. The results for BCR-ABL were normalized with reference to ABL. The PCR program is completed in only 45 min.
The sensitivity attained allowed the detection of rearrangements at dilutions of between 5-10(-4) and 10(-5) K562 cDNA. The within-assay coefficient of variation was 11% for BCR-ABL, and 9% for ABL. A greater than 2 log reduction in the BCR-ABL/ABL ratio was evident shortly after transplantation in all allografted patients.
We may conclude that the TaqMan probe technology can be easily adapted to HybProbes with equivalent results. Besides, the results of BCR-ABL quantification in the follow-up of patients clearly confirm that real-time PCR with HybProbes is a reliable and sensitive method for monitoring minimal residual leukemia after HSCT in CML patients.
定性逆转录聚合酶链反应（RT-PCR）在监测慢性粒细胞白血病（CML）患者方面价值有限，这促使了定量检测方法的发展。我们基于荧光标记探针（杂交探针）的使用，在LightCycler中开发了一种定量实时PCR（QC-PCR）方法，用于估计CML患者样本中的BCR-ABL融合基因转录本。
分析了9例CML患者的52份样本（45份外周血、5份骨髓和2份单采产品样本）。7例患者在诊断时及造血干细胞移植（HSCT）后的随访期间接受研究，而2例仅在HSCT后进行评估。PCR反应在毛细管中进行，终体积为10微升，使用2微升cDNA、Mensik等人的引物和两种杂交探针。BCR-ABL的结果以ABL为参照进行标准化。PCR程序仅需45分钟即可完成。
所达到的灵敏度能够检测出稀释至5-10^(-4)至10^(-5) K562 cDNA之间的重排。BCR-ABL的批内变异系数为11%，ABL为9%。在所有接受同种异体移植的患者中，移植后不久BCR-ABL/ABL比值明显降低超过2个对数。
我们可以得出结论，TaqMan探针技术可以很容易地应用于杂交探针，且结果相当。此外，对患者随访中BCR-ABL定量的结果清楚地证实，使用杂交探针的实时PCR是监测CML患者HSCT后微小残留白血病的可靠且灵敏的方法。
