ТЕСТ-СИСТЕМА ДЛЯ ПРОВЕДЕНИЯ ПЦР В РЕЖИМЕ РЕАЛЬНОГО ВРЕМЕНИ ДЛЯ ОБНАРУЖЕНИЯ ДНК ТТV В БИОЛОГИЧЕСКОМ МАТЕРИАЛЕ

Abstract

Background. The study of biological material for the presence of TTV DNA using the PCR method allows for a timely assessment of the functional state of the human liver and immune system. Objective. To develop components for real-time PCR for TTV DNA detection in biological material. Material and methods. The design and selection of optimal primers and probes (taking into account the size (length) of the amplicon, annealing temperature, nucleotide composition, distribution of nucleotides along the length of the primer, length of primers, the possibility of formation of hairpins and dimers by primers) were performed using the Primer-BLAST/Primer3, FastPCR programs. Since primers, even absolutely unique for certain DNA sequences, could anneal at nonspecific sites, not related to the gene analyzed, we checked the correspondence of the primers to the sequences of the target gene. For this purpose, we used the NCBI Primer BLAST online service and assessed the local pairwise alignment of each primer with all nucleotide sequences of the Refseq databases. Results. As the result of studies carried out on the selection of the optimal primer annealing temperature, primer concentrations, as well as the selection of the optimal nucleotide pair, the main parameters of the designed primers were determined. Conclusions. A kit for the detection and quantification of TTV DNA using the polymerase chain reaction method with hybridization-fluorescent detection in real time was created and became the basis for the development of a commercial test system.