11 research outputs found

    Production of Bst polymerase for diagnosis of different infections using loop-mediated isothermal amplification

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    Introduction. The large fragment of DNA polymerase I from Geobacillus stearothermophilus GIM1.543 (Bst DNA polymerase) possesses 5'-3' DNA polymerase activity, 5'-3' displacement activity and high processivity. These properties make it possible to use Bst DNA polymerase in loop-mediated isothermal amplification (LAMP), which provides highly specific amplification of the target sequence and is used for rapid detection of agents causing human infectious diseases. The purpose of the study was to produce a recombinant Bst polymerase enzyme in the bacterial expression system and to assess its properties for LAMP-based diagnostics of infectious diseases. Materials and methods. Expression constructs carrying the Bst polymerase gene were obtained using genetic engineering techniques. Different Escherichia coli strains were used for protein expression. Metal-chelate and gel filtration chromatography techniques were used for protein purification. Catalytic characteristics of the enzyme were assessed in loop-mediated isothermal amplification reactions using AmpliSens SARS-CoV-2-IT, AmpliSens IAV-IT and AmpliSens IBV-IT diagnostic systems designed for high-quality detection of SARS-CoV-2, influenza A virus (IAV) and influenza B virus (IBV) RNA, respectively. Results. The offered protocol for production, extraction and purification of recombinant Bst polymerase makes it possible to produce the enzyme in the bacterial expression system using E. coli cells in a soluble form and reaching the yield up to 20% of the total cell mass. In LAMP reactions, the obtained enzyme demonstrates activity comparable with that of the commercial enzyme Bst 2.0 (NEB). Conclusion. Considering the fast purification and production of the enzyme, the obtained recombinant Bst polymerase can be used in LAMP-based diagnostic kits

    Neutron inspection of moisture in slightly enriched UO2

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    The Level of Meningococcal Carriage and Genotyping of N. meningitidis Strains in the Group of Labor Migrants

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    Relevance. Population migration can play a crucial role in the spread of invasive strains of meningococcus, initiating outbreaks of meningococcal infection, and changing the incidence at the local level.Aim. To assess the prevalence of meningococcal carriage among migrants arriving in Moscow and to characterize the antigenic and genetic properties of carrier strains of meningococcus.Materials and methods. The study was conducted in March 2020 at the bases of the Multifunctional Migration Center of Moscow and the Federal Budget Institution of Science «Central Research Institute of Epidemiology». Samples of nasopharyngeal mucus were collected from 352 people. Nasopharyngeal strains of meningococcus were identified and identified using microbiological, serological, and molecular biological methods.Results. The overall level of the carriage was 5.7%. Of the twenty selected strains, 10 have a serogroup defined: Y – 5 strains, W - 3, A, and B – 1 each. The obtained genetic and antigenic characteristics do not allow talking about the import into the RF of representatives of known hypervirulent clonal complexes. In this study, strains were identified that are part of the clonal complex ST-175 complex, which has not been previously described in the Russian Federation.Conclusion. It seems promising to continue the dynamic monitoring of carriage of meningococcus in various groups, including among people entering the country to obtain a migration patent, as well as identifying risk factors for acquiring carriage. The data obtained will supplement current information on the incidence of the generalized form of meningococcal infection and will be crucial for determining the epidemiology at the country level, the population groups responsible for the transmission of the disease, and the need for targeted vaccination
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