Abstract
The detection of BCR-ABL1 mRNA transcripts is essential to molecular chronic myeloid leukemia (CML) diagnosis. In most cases, the RT-qPCR technique is performed as the gold standard diagnosis tool for clinical cases. However, this method requires expensive reagents and equipment, such as a real-time thermal cycler, probes and master mix. Consequently, the development and validation of simple and low-cost methods are essential for a rapid CML diagnosis in less specialized and equipped centers. In this study, we develop and demonstrate an accessible, rapid, and low-cost method using RT-LAMP for BCR-ABL1 detection in both cell lines and CML clinical samples, using fluorescent and colorimetric assays. Both methods demonstrated diagnostic specificity of 100% and while diagnostic sensitivity reaches more than 90% in samples with RT-qPCR cycle threshold above 31. The obtained data indicates that the proposed method here described is robust, specific and rapid approach for CML diagnosis with outstanding performance, especially for CML diagnostic procedure where present high BCR-ABL1 expression.
Highlights
Chronic myeloid leukemia (CML) is a clonal myeloproliferative disorder characterized by the translocation t(9;22)(q34;q11), that generates BCR-ABL1 fusion gene and protein, the hallmarks of CML 1
In this study we propose a fluorescent and colorimetric RT-Loop-Mediated Isothermal Amplification (LAMP) for detection of BCR-ABL1 b2a2 and b3a2 isoforms in cell lines and in CML clinical samples, as a fast and specific approach in clinical practice for CML diagnosis
BCR-ABL1 p210 transcript was detected in the assays at [125, 12,5] and [1,25] ng per well, while the internal control was detected in all concentrations (Fig. 2)
Summary
Chronic myeloid leukemia (CML) is a clonal myeloproliferative disorder characterized by the translocation t(9;22)(q34;q11), that generates BCR-ABL1 fusion gene and protein, the hallmarks of CML 1. This agerelated neoplasm has an average diagnosis age estimated around 60 years old in western countries, studies indicate that in Africa and Asia the diagnostic age is about ten years younger when compared to other countries, in which the higher frequency occurs in men [2,3]. CML patients are treated with tyrosine kinase inhibitors (TKIs), one of the most effective oncological targeted therapies This class of drugs targets the BCR-ABL1 positive cells and induce remission in most cases of CML. Resistance and tolerance to TKIs can occur, the treatment response needs to be monitored in the determined time-points in order to define the appropriate drug therapy, the effective response to the treatment and to select patients who are eligible for TKI discontinuation 7
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