[The development of kit for hybridization extraction of DNA and RNA of agents of hemotransmissive infections from serum and blood plasma].

To decrease dependence of effectiveness of isolation of nucleic acids of composition and amount of applied sample a kit was developed for hybridization extraction of DNA HBV RNA HCV and RNA HIV from blood serum in two formats--using up to 250 mkl and up to 1 ml of sample. This kit, in complex with kits for detection using polymerase chain reaction technique in real-time, forms a test characterized by high analytical sensitivity i.e. HBV50 copies per ml, HCV37.5 copies per ml, HIV 13 copies per ml. The developed kit for extraction of target nucleic acids permits to get rid of total DNA and inhibited effect of heparin. It can be adapted for application wit factors B and automated stations of sample preparation.

[A single-tube real-time RT-PCR assay for the RNA detection and quantification of genetically diverse HIV Including rare non-M group of the HIV-1 and HIV-2].

The RT-PCR method with real-time fluorescence detection was used for development of φ prototype of diagnostic kit for reliable diagnosis of genetic variants of RNA of the HIV-1 of groups M, N, O, P and RNA of the HIV-2 in blood plasma and serum. The kit is stable against nucleotide defects, provides broad linear range of concentration of the HIV RNA, 100% analytical specificity and adequate analytical sensitivity: 42 ME/ml (HIV-1 of group M), 45 copies/ml (HIV-2), 92 copies/ml (HIV-1 of group O), 90 copies/ml (HIV-1 of group N). The kit provided successful detection and measurement of HIV RNA concentration in the samples of the international reference panel of the HIV-1 genotype. The results of the test correlate with results of commercial tests.

[Development of the high-throughput fluorescence assay detecting SNPs in hemostasis and folate metabolism genes for clinical use].

Genetic predisposition of an individual patient should be taken in account to choose the proper treatment. Implementation to clinical practice requires the development of rapid, high-throughput, and easy assays intended to detect single nucleotide polymorphisms. A detection kit intended to identify the hemostasis and folate cycle gene mutations G20210A FII, G1691A FV, G10976A FVII, G103T FXIII, C807T ITGA2, T1565C ITGB3, 5G(-675)4G PAI, G(-455)A FGB, C677T and A1298C MTHFR, A2756G MTR, A66G MTRR was suggested in this work. The method is based on the polymerase chain reaction and subsequent melt curve analysis of the complexes of amplicons with specific probe. Three single nucleotide polymorphisms can be identified in one tube using our detection kit that increases the productivity of the analysis in the clinical use. Different types of biological samples (buccal epithelium, saliva, plasma, serum, and urogenital swabs) can be used as the initial material for DNA isolation and further analysis by the method developed in this work.

[Development of an ultrasensitive mismatch tolerant PCR assay for the qualitative and quantitative determination of HIV-1 RNA].

A real-time fluorescent polymerase chain reaction was used to develop a RealBest HIV RNA kit that was clinically suitable for the detection of HIV-1 RNA and for the estimation of virus load in plasma and serum samples. Due to the selection of a highly conserved target region and to the experimental study of the impact of different primer-template and probe-template mismatches on RT-PCR with subsequent selection of the optimum oligonucleotide set, the developed assay can detect and measure the concentration of all subtypes of HIV-1, group M. The assay provides a high reproducibility and sensitivity and a wide dynamic range of virus loads (20 to 10 million IU/ml of plasma or serum).

[Hepatitis C virus genotyping using 5 nuclease real-time PCR and probes with oligodeoxyinosine linkers].

Hepatitis C virus (HCV) infection is a major cause of severe liver disease including liver cirrhosis and hepatocellular carcinoma. Genotyping became fundamental in the clinical assessment of patients with hepatitis C because the genotype of the hepatitis C virus determines the chance of therapeutic response and duration of treatment. We developed a new real-time PCR assay for genotyping of HCV with increased specificity due to a novel approach to dual-labeled probe design using oligodeoxyinosine linkers. The assay allows genotypes 1, 2, 3 to be distinguished and genotypes 4-6 with high specificity to be blocked. The analytical sensitivity (150 IU/ml) can be implemented. Of the 285 clinical samples genotyped using the developed assay, 45% were genotype 1; 6%, genotype 2; 49%, genotype 3. No discordant results with 5-UTR sequencing and commercial genotyping assays were obtained.

[Development of uncompetitive exogenous internal amplification control for real-time PCR based on UFA method].

An uncompetitive exogenous internal amplification control method (EIAC) was developed on the basis of short synthetic DNA segment, whose amplification can be detected in real time by UFA spectroscopy principle. The EIAC was shown to be useful as internal control in diagnostic test systems based on DNA or RNA detection by multiplex real-time PCR. It can be applied to assess the quality of extracted DNA or RNA, and also to detect and study the factors causing PCR inhibition and earlier plateau effect.