REAGENT SET FOR ESTIMATING EFFICIENCY OF THERAPY WITH TYROSINE KINASE INHIBITORS AT Ph-POSITIVE NEOPLASMS AND ITS APPLICATION METHOD Russian patent published in 2021 - IPC C12Q1/68 

FIELD: molecular biology; biotechnology; medicine.

SUBSTANCE: invention relates to molecular biology, biotechnology and medicine, in particular to hematology, oncology; it can be used for the estimation of the efficiency of the therapy of Ph-positive oncohematological diseases. A set for a multiplex PCR analysis of an expression level of chimeric RNA molecules with simultaneous detection of mutations of resistance to tyrosine kinase inhibitor (hereinafter – TKI) therapy, as well as an accelerated and economical method for the diagnostics of Ph-positive oncohematological diseases based on it, contains a buffer solution, deoxynucleotide triphosphates, specific primers, TaqMan probes, Taq-polymerase, MgCI₂ and water in optimal ratios providing an optimal enzyme work. A method for the estimation of the efficiency of the therapy with tyrosine kinase inhibitors at Ph-positive neoplasms using the claimed set also includes the amplification in the following temperature mode: preliminary denaturation – 95°C – 5 minutes; 50 reaction cycles in the mode of 95°C – 15 s, 60°C – 40 s. A quantitative assessment of a chimeric DNA fraction is calculated based on values of threshold Ct cycles in amplification reactions of BCR-ABL1 and ABL1 fragments, where a number of ABL1 copies is calculated according to the formula: IS(%)=(N(BCR-ABL1)/N(ABL1))·1.265·100. when BCR-ABL1 level of more than 1% is determined, it is concluded that a patient has resistance to used TKI preparations. In the case if simultaneously detected difference of values of Ct (mutation under study) and Ct (ABL1) for any of detected mutations is less than the set critical threshold, it is concluded that the detected mutation or mutation combination is a cause of this resistance.

EFFECT: manufacture of the reagent set for multiplex PCR in real time, allowing, in one analytical setting, for quantitative assessment of the level of chimeric BCR-ABL1 transcript in the blood sample, as well as detection of 8 mutations of TKI resistance – G250E, Y253H, E244K, E255V, T315I, F317L, F359I, F359V – for the estimation of the therapy efficiency, which allows, while reducing research time and material costs, for obtaining more complete information about the disease course.

2 cl, 9 tbl, 4 dwg, 3 ex