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Information × Registration Number 0225U001684, (0124U001335) , R & D reports Title Development of an anthrax diagnostic tool by polymerase chain reaction with noncyclic amplification popup.stage_title Створення науково-методичних підходів для виявлення збудника сибірки за допомогою петлевої ізотермічної ампліфікації (LAMP) Head Tarasov Oleksandr A., Кандидат ветеринарних наук Registration Date 05-02-2025 Organization Institute of Veterinary Medicine of the National Academy of Agrarian Sciences of Ukraine popup.description1 To develop new approaches for fast and effective diagnosis of B.anthracis based on the method of non-cyclic amplification, namely loop isothermal amplification (LAMP), and to establish the sensitivity and reproducibility of the LAMP method. popup.description2 As a result of the study, three separate sets of primers were developed for LAMP aimed at identifying marker regions of the anthrax genome: BA5345 sequence from the chromosome (GenBank accession no. NC003997.3), lef sequence from the pXO1 plasmid (GenBank accession no. M30210.1) and capB sequence from the pXO2 plasmid (GenBank accession no. M24150.1). The results showed that optimal amplification occurs at an incubation temperature of 60 °C for 30-60 minutes. The optimal concentrations of primers for the assay were 1.6 ?M for FIP and BIP, 0.8 ?M for LF and LB, and 0.2 ?M for F3 and B3. The analytical sensitivity of the LAMP assays was 100 copies of genomic DNA per reaction. Colorimetric analysis of the endpoint of the LAMP reaction was determined using the intercalating dye SYTO16 and phenol red dye solution. Under visible light, the color in LAMP-positive reactions changed to yellowish-green, and in ultraviolet light showed fluorescence. In LAMP-negative reactions, the color of the dye remained unchanged and the dye did not fluoresce in UV light. The LAMP products on agarose gel electrophoresis showed characteristic tracks of the expected size. Sufficient sensitivity and specificity of the primers were confirmed. The test allows for specific differentiation of B. anthracis from other Bacillus species and other bacterial species, and allows for the direct detection of the pathogen in clinical samples without microbiological cultivation. Based on the results of the research, the "Guidelines for the detection of anthrax pathogen by looped isothermal amplification (LAMP)" (2024) were developed, and an application for the Patent of Ukraine for a utility model "Method for B. anthracis detecting using loop isothermal amplification (LAMP)" (2024) was applied Product Description popup.authors popup.nrat_date 2025-02-05 Close