Four-channel Asymmetric Real-Time PCR Hybridization Probe Assay: A Rapid Pre-Screening Method for Critical BCR-ABL Kinase Domain Mutations

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. (C) 2012 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.