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.
(Copyright © 2012 Elsevier Inc. All rights reserved.)