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

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

Influence of the evenness of seeding using a disk-cell seeding mechanism on the productivity of scarlet amaranth

The paper presents the results of a field study of the effect of the evenness of seeding using a disk-cell seeding mechanism on the fresh yield and Scarlet amaranth grain of Kinelsky 254 variety. The problem of the amaranth evenness of seeding was solved by developing an experimental seeder of precision sowing for small-seeded crops. Field experiments were carried out on the fields of the Volga Research Institute of Selection and Seed Production named after P. N. Konstantinovin 2019-2021. To control the qualitative parameters of sowing, amaranth was sown with ballast (blind seeds) with a VS-4.2 vegetable seeder. The study revealed that the evenness of seed distribution in a row during sowing with an experimental seeder was 22%, for plants – 24.6%, control – 24.6 and 72.1%, respectively. The use of an experimental seeder made it possible to sow in accordance with agrotechnical requirements and create the best conditions for plant nutrition, which led to an increase in the fresh yield of Scarlet amaranthof Kinelsky 254 variety by 2.6 times, grain – by 1.6 times