Four-channel asymmetric Real-Time PCR hybridization probe assay: A rapid pre-screening method for critical BCR-ABL kinase domain mutations

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. 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. Assay evaluation performed on 33 samples, previously genotyped by sequentiation, resulted in full concordance of results. This new methodology detects in a few steps the presence of critical mutations associated to Imatinib resistance. ► Four LC channels for the detection of changes within a nucleotide sequence. ► Rapid and easy screening of BCR-ABL mutations. ► Huge advantage for the treatment of CML patients. ► The methodology may be extrapolated for the diagnosis of many other diseases.