FIELD: biotechnology; medicine.
SUBSTANCE: group of inventions relates to biotechnology and medicine, namely to immunodiagnostics, in particular to the problem of assessing the immune status of newborns. The diagnostics is carried out using DNA isolated from whole blood or dry blood spots (dry blood drop). Diagnostics is carried out in two stages using oligodeoxyribonucleotide primers and corresponding fluorescently labeled probes complementary to sites of detected fragments. The analysis of the results is carried out using the software of a device used for conducting PCR with hybridization-fluorescence detection in “real time” mode, the analysis of TREC and KREC is carried out with normalization to reference genes, the absolute number of TREC, KREC target molecules and normalization genes in analyzed samples is determined using calibration curves built during amplification of a series of sequential dilutions of standard DNA samples with known concentrations of required products (TREC, KREC) and reference genes (GAPDH, HPRT, RPP30) or a known number of cells, based on the results of determining TREC and KREC, conclusions are drawn about the immunity condition of newborns. At the first stage, the isolated DNA is used for simultaneous amplification in one container of sites of TREC, KREC DNA molecules and two reference normalization genes GAPDH and HPRT. A “normalization coefficient” is calculated as geometric mean or arithmetic mean of a value of all reference normalization genes. If, according to the results of the first stage, the absence or low level of target TREC, KREC DNA molecules is detected for detected samples, the second stage is carried out, including two multiplex formulations from one sample of extracted DNA: in one (target) container, sites of TREC, KREC DNA molecules and one reference normalization gene GAPDH are amplified, in the second (normalization) container, sites of DNA molecules of three reference genes – GAPDH, HPRT and RPP30 are amplified. When analyzing the results, the correlation recalculation of data obtained for the multiplex formulation of reference genes is performed on data obtained for the reference gene in the target container, after which the “normalization coefficient” is entered, calculated as the geometric mean or arithmetic mean of the value of all reference normalization genes. Next, data obtained for TREC and KREC DNA molecules is calculated.
EFFECT: invention provides an increase in the sensitivity and reliability of diagnostics, a decrease in false-positive (absence and/or low levels of TREC, KREC DNA molecules) results.
5 cl, 9 dwg, 4 ex
