四通道非对称实时 PCR 杂交探针检测：一种快速的关键 BCR-ABL 激酶结构域突变预筛选方法。
Four-channel asymmetric Real-Time PCR hybridization probe assay: a rapid pre-screening method for critical BCR-ABL kinase domain mutations.
机构信息
Department of Hematology, University Hospital of Son Espases, Carretera Valldemossa no. 79, Palma de Mallorca, Spain.
出版信息
Clin Biochem. 2012 Mar;45(4-5):345-51. doi: 10.1016/j.clinbiochem.2011.12.026. Epub 2012 Jan 12.
OBJECTIVES
Within the laboratory protocols, used for the study of BCR-ABL resistance mutations in chronic myeloid leukemia patients treated with Imatinib, direct sequencing remains the reference method. Since the incidence of patients with a mutation-related loss of response is not very high, it is very useful in the routine laboratory to perform a fast pre-screening method.
DESIGN AND METHODS
With this in mind, we have designed a new technique, based on a single Real-Time FRET-based PCR, followed by a study of melting peaks. This new tool, developed in a LightCycler 2.0, combines four different fluorescence channels for the simultaneous detection, in a single close tube, of critical mutations within the ABL kinase domain.
RESULTS
Assay evaluation performed on 33 samples, previously genotyped by sequentiation, resulted in full concordance of results.
CONCLUSIONS
This new methodology detects in a few steps the presence of critical mutations associated to Imatinib resistance.
目的
在研究接受伊马替尼治疗的慢性髓性白血病患者 BCR-ABL 耐药突变的实验室方案中，直接测序仍然是参考方法。由于与突变相关的无应答患者的发生率并不是很高，因此在常规实验室中进行快速预筛选方法非常有用。
设计与方法
考虑到这一点，我们设计了一种新的技术，基于单个实时荧光共振能量转移（FRET）PCR，随后进行熔解峰研究。该新工具在 LightCycler 2.0 上开发，结合了四个不同的荧光通道，用于在单个封闭管中同时检测 ABL 激酶结构域内的关键突变。
结果
对 33 个先前通过测序进行基因分型的样本进行了检测评估，结果完全一致。
结论
该新方法可在几个步骤中检测出与伊马替尼耐药相关的关键突变。
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