RESUMO
Globally, about 70 million people are infected with the hepatitis C virus (HCV), and about 400 thousand people die annually from chronic hepatitis C complications. The management of patients with chronic hepatitis C may require HCV genotyping, since the efficiency of some widely used antiviral drugs strongly depend on the viral genotype and/or subtype. The most prevalent HCV circulating recombinant form, RF1_2k/1b, is misclassified as genotype 2 by many commercial HCV genotyping kits, based on the RT-PCR analysis of the 5' untranslated region of the HCV genome. This leads to inappropriate patient treatment, since the accepted treatment schemes for HCV genotype 2 are ineffective for the RF1_2k/1b. Here we describe a method for detecting the RNA HCV RF1_2k/1b in blood samples by RT-PCR analysis of two regions in HCV genome (5'UTR and NS5b). The method was tested on 240 blood serum samples from HCV infected patients, in which HCV genotype was defined as 2 or mixed (2+1 or 2+3) by the two commercial genotyping kits "OT-Hepatogen-C genotype" ("DNA-Technology", Moscow) and "RealBest RNA HCV-1/2/3" ("Vector- Best ", Novosibirsk). 50 (20.8%) RF1_2k/1b cases were revealed, including three mixed infections: RF1_2k/1b + 1a, RF1_2k/1b + 3a, RF1_2k/1b + 1b. In all cases, the accuracy of HCV typing by the proposed method was confirmed by Sanger sequencing and phylogenetic analysis. The method is easy to implement into clinical practice and may be used in clinical settings equipped for RT-PCR analysis to correctly identify the recombinant variant RF1_2k/1b.
Assuntos
Hepacivirus , Hepatite C , Genótipo , Hepacivirus/genética , Hepatite C/diagnóstico , Hepatite C/genética , Humanos , Moscou , Filogenia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNA , SoroRESUMO
The evaluation of content of DNA of lactobaccilli and particular types of aerobic anaerobic opportunistic bacteria in sampling of scrapes from urogenital tract offemale patients of the network laboratory INVITRO was implemented. The technique of polymerase chain reaction in real-time was implemented. It is demonstrated that decreasing of content of lactobaccilli in total bacterial mass isfollowed by increasing of occurrence, concentration and relative content of all types of opportunistic pathogens except ureaplasmna. These changes are expressed in different degree for different types of opportunistic pathogens. The increasing of varieties of types of microflora of urogenital tract under decreasing of content of lactobaccilli is noted.
Assuntos
Bactérias Aeróbias/isolamento & purificação , Bactérias Anaeróbias/isolamento & purificação , Sistema Urogenital/microbiologia , Bactérias Aeróbias/classificação , Bactérias Aeróbias/patogenicidade , Bactérias Anaeróbias/classificação , Bactérias Anaeróbias/patogenicidade , Feminino , Humanos , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Sistema Urogenital/patologiaRESUMO
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.
Assuntos
Infecções por HIV/genética , HIV-1/genética , HIV-2/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Variação Genética , Infecções por HIV/diagnóstico , Soropositividade para HIV/genética , HIV-1/isolamento & purificação , HIV-2/isolamento & purificação , Humanos , RNA Viral/genética , RNA Viral/isolamento & purificaçãoRESUMO
Microtine rodents were captured in two disconnected sampling sites in Omsk region where Ixodes pesulrcatus and Ixodes trianguliceps are sympatric. In blood samples of rodents the DNA was revealed belonging to several ixodid-transmitted pathogens: Borrelia burgdorferi sensu lato (prevalence 20.0 and 6.0%, here and further values are given for the first and second site, respectively), Borrelia miyamotoi (8.3 and 2.0%), Anlaplasnma phagocytophilum (33.3 and 48.0%), Ehrlichia muris (30.0 and 2.0%) and Babesia microti (33.3 and 42.0%). Three genetic groups of A. phagocytophilhm based on 16S rRNA gene and groESL operon, as well as two genetic groups of B. microti, B. microti 'US'-type and B. microti 'Munich'-type, were detected.
Assuntos
Babesia microti/genética , DNA Bacteriano/genética , DNA de Protozoário/genética , DNA Ribossômico/genética , Bactérias Gram-Negativas/genética , Ixodes/microbiologia , Animais , Humanos , RNA Bacteriano/genética , RNA de Protozoário/genética , RNA Ribossômico 16S/genética , RNA Ribossômico 18S/genéticaRESUMO
MicroRNAs are known as a posttranscriptional negative regulators of gene expression by binding to the 3'UTP of target mRNAs in cytoplasm. More than 1600 microRNAs expressed in human cells, are involved in the regulation of embryogenesis, differentiation, cell cycle, apoptosis, senescence, thus determining cell fate. Up to 60 % of protein coding genes are under their control. Various sets of microRNAs found in different human tissues under normal and pathological conditions, including cancer, suggest that miRNAs are involved in most cellular pathways. To date, there is no doubt that regulatory potential of the genome is largely determined by miRNAs. In our study, we performed a comparative phylogenetic analysis of the origin and evolution of the total set of 1048 miRNAs in the human genome and investigated the role of certain miRNAs in carcinogenesis of thyroid and mammary glands, as potential diagnostic and prognostic biomarkers of malignancy. Analysis of phylogenetic distribution of miRNAs in the human genome has shown four peaks of appearance of new miRNA genes in the evolution from Methazoa to H. sapiens. The highest amount of new miRNA genes appeared after divergence of H. s. from common ancestor with P. t. Expansion of transposable elements in genome was accompanied by the origin of new miRNA genes on the basis of their sequences. More than 14 % from 1600 miRNAs of human genome originated from mobile elements and still remain. Profiles of expression of 5 miRNAs, pertaining to oncomicroRNAs - miR-21, -221, -222, -155 and -205 - allow distinguishing ductal invasive carcinoma of mammary gland and thyroid papillary carcinoma. The data obtained suggest different ways and roles of participation of the same miRNAs in carcinogenesis of thyroid and mammary glands. So, these miRNAs and profiles of their expression might be used in the diagnosis and prognosis of cancer.
Assuntos
Elementos de DNA Transponíveis/genética , MicroRNAs/genética , Neoplasias/genética , Animais , Biomarcadores Tumorais , Regulação Neoplásica da Expressão Gênica , Humanos , FilogeniaRESUMO
The analysis was applied to detect DNA of agents of human granulocytic anaplasmosis and monocytic erlychiosis. The sampling included 109 ticks of Ixodes species from Novosibirsk oblast and Khabarovsk kray and blood samples of 111 mouse-like rodents from Omsk oblast. The used techniques included polymerase chain reaction in real-time operation mode with set of reagents "RealBest DNA Anaplasma phagocytophilum/Ehrlichia muris, ehrlichia chaffeensis" ("Vector-Best" Novosibirsk) and double round polymerase chain reaction. The DNA of A. phagocytophilum, agent of granulocytic anaplasmosis and/or DNA of E. muris, agent of monocytic erlychiosis was detected in 21 ticks and in blood samples of 52 voles. Both techniques were applied. The DNA of A. phagocytophilum was detected in samples of 2 voles and in 1 tick only after polymerase chain reaction in real-time operation mode was applied. It demonstrated that the set of reagents "RealBest DNA Anaplasma phagocytophilum/Ehrlichia muris, ehrlichia chaffeensis" permits to detect the DNA of isolates of A. phagocytophilum subsumed to different genetic groups. The set can be used for fast and effective detection of the DNA of agents of agents of human granulocytic anaplasmosis and monocytic erlychiosis in suspensions of analyzed ticks and blood samples.
Assuntos
Anaplasmataceae/isolamento & purificação , Anaplasmose/microbiologia , DNA Bacteriano/análise , Ehrlichiose/microbiologia , Ixodes/microbiologia , Reação em Cadeia da Polimerase em Tempo Real/métodos , Anaplasma phagocytophilum/genética , Anaplasma phagocytophilum/isolamento & purificação , Anaplasmataceae/genética , Anaplasmose/sangue , Animais , Arvicolinae/sangue , DNA Bacteriano/sangue , Ehrlichia/genética , Ehrlichia/isolamento & purificação , Ehrlichiose/sangue , Humanos , Murinae/sangue , RNA Ribossômico 16S/genética , Sensibilidade e Especificidade , SibériaRESUMO
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).
Assuntos
Infecções por HIV/diagnóstico , HIV-1/genética , Reação em Cadeia da Polimerase/métodos , RNA Viral , Kit de Reagentes para Diagnóstico/virologia , Pareamento Incorreto de Bases , Primers do DNA/química , Fluorescência , Variação Genética , Infecções por HIV/sangue , Infecções por HIV/genética , Infecções por HIV/virologia , Humanos , Oligonucleotídeos/química , RNA Viral/sangue , RNA Viral/genética , Reprodutibilidade dos Testes , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sensibilidade e Especificidade , Carga ViralRESUMO
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.
Assuntos
Primers do DNA/química , Hepacivirus/genética , Hepatite C/genética , RNA Viral/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Primers do DNA/genética , Genótipo , Hepacivirus/química , Humanos , RNA Viral/químicaRESUMO
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.
Assuntos
Oligonucleotídeos/química , Reação em Cadeia da Polimerase/métodos , Fluorescência , Kit de Reagentes para DiagnósticoRESUMO
A new method is proposed for estimation of polymerase activities using fluorescence detection during isothermal reaction. The method allows simultaneous determination of DNA-dependent DNA polymerase and 5'-3'-exonuclease activities using amplifiers supplied with an optical module for fluorescence detection under real-time conditions. Different primer-template combinations used as polymerase substrates were compared. Primer elongation (polymerase reaction) is detected by changes in SYBR Green I fluorescence upon binding to dsDNA during reaction; nuclease activities are detected by changes in fluorescence due to cleavage of the probe, containing the reporter fluorophore and fluorescence quencher, and hybridized in advance to the template single-stranded region. It was also shown that the method can be used for determination of relative activities of DNA polymerase preparations, estimation of temperature-time dissociation parameters of polymerase complexes with specific antibodies to its active center, and analysis of effects of inhibitors and activators of different nature on reaction rates of dsDNA polymerization and 5'-3'-exonuclease cleavage by polymerase. The method can be also used for estimation of endonuclease activities of DNA polymerases.
Assuntos
Bioquímica/métodos , DNA Polimerase Dirigida por DNA/isolamento & purificação , Exonucleases/isolamento & purificação , Primers do DNA/química , Primers do DNA/metabolismo , DNA Polimerase Dirigida por DNA/química , DNA Polimerase Dirigida por DNA/metabolismo , Endonucleases/química , Endonucleases/isolamento & purificação , Endonucleases/metabolismo , Exonucleases/química , Exonucleases/metabolismo , Fluorescência , Corantes Fluorescentes/química , Corantes Fluorescentes/metabolismo , Taq Polimerase , Temperatura , Moldes Genéticos , Fatores de TempoRESUMO
The review deals with cytoplasmic male sterility (CMS) in higher plants: impairment of the pollen formation resulting from interaction of the nuclear and mitochondrial genomes. The information on the known nuclear restorer-of-fertility genes and their effects on the expression of CMS-associated mitochondrial loci are considered. Heteroplasmy of mtDNA in plants and its potential association with CMS inheritance, as well as possible mechanisms of the observed direct and reverse association between altered expression of the CMS-inducing mitochondrial genome, metabolic defects, and pollen sterility are discussed.
Assuntos
Citoplasma/genética , DNA Mitocondrial/genética , DNA de Plantas/genética , Genes de Plantas , Plantas/genética , Pólen/genética , Fertilidade/genéticaRESUMO
The cytoplasm status according to mitochondrial sequence tags was determined in agamospermous sugar beet progenies characterized by unstable manifestation of cytoplasmic male sterility. The detected variations in the ratios of homologous sequences related to the N and S mtDNA types did not correlate with pollen phenotypes of the plants. Polymerase chain reaction allowed semiquantitative evaluation of these variations and their detection. A cDNA corresponding to a hitherto unknown minor RNA and containing a consensus sequence of dicotyledonous plant promoters was detected by mtRNA display. Probable sources of CMS variability in agamospermous sugar beet progenies are discussed.
Assuntos
Beta vulgaris/genética , DNA Mitocondrial/genética , Genes de Plantas/genética , Variação Genética , Infertilidade das Plantas , Pólen , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas/genética , RNA de Plantas/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodosRESUMO
The multistep regulation of mitochondrial gene expression in higher plants is considered. Data are summarized that concern the structure and function of the transcription system, posttranscriptional changes in the mRNA structure, and other levels of expression regulation.
Assuntos
DNA Mitocondrial , Regulação da Expressão Gênica de Plantas , Genoma de Planta , Plantas/genética , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Poliadenilação , Regiões Promotoras Genéticas , Processamento Pós-Transcricional do RNA , Splicing de RNA , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
Several 5'-degenerate primers were selected by computer analysis and used for mtDNA typing in sugar beet cultivars with cytoplasms of the S (typical for cytoplasmic male sterility) or N (normal) type. A number of N- or S-specific markers were found to correspond to transcribed mitochondrial genes. One was from the orf215 region of the N-type mtDNA. A physical map of the corresponding region was constructed for the S-type mtDNA, and a substantial difference observed for the two genome types. One N-specific marker proved to contain a rearranged rps3 region and a truncated atp9 copy. With the known nucleotide sequence of this marker, three-primer PCR was designed and showed that both variants of the rps3 region simultaneously take place in the mtDNA pool, the new one occurring in a substochiometric proportion.
Assuntos
Beta vulgaris/genética , DNA Mitocondrial/genética , Genoma de Planta , Proteínas de Plantas/genética , Sequência de Bases , Beta vulgaris/fisiologia , Primers do DNA , Reação em Cadeia da Polimerase , Técnica de Amplificação ao Acaso de DNA Polimórfico , Mapeamento por RestriçãoRESUMO
Physical maps of the cob mtDNA region were constructed and compared between sugar beet Beta vulgaris L. plants with normal fertility and with cytoplasmic male sterility (CMS). A CMS-associated rearrangement did not affect the coding region of cob and combined two mtDNA regions which are normally about 150 kb apart. Two point substitutions were found in the 3'-untranslated region of cob.
Assuntos
Beta vulgaris/genética , Citoplasma/metabolismo , DNA Mitocondrial/genética , Genoma de Planta , Proteínas de Plantas/genética , Regiões 3' não Traduzidas , Sequência de Bases , Primers do DNA , Mutação PuntualRESUMO
Morbillivirus of Baikal seal (BSM) was isolated from organs of a dead animal during 1987-1988 epizootic of Baikal seal (Phoca sibirica). A method of cellular enzyme immunoassay for testing for virus-specific antibodies was developed using BSM. The method was used for antibody detection in sera of 115 apparently normal seals collected in the spring of 1989. Antibody to BSM were found in sera from 75 animals. Examinations of seropositive animals of different age and sex were carried out. The results obtained indicate a possible role of BSM in the epizootic.