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Information × Registration Number 0225U005358, (0124U002158) , R & D reports Title Development of a tool for diagnosing Fusarium wilt in grain by the polymerase chain reaction (PCR) method popup.stage_title Створення технологічного регламенту на тест-набір з діагностики фузаріозів зерна методом ПЛР Head Zakharova Olga , Кандидат біологічних наук Registration Date 30-12-2025 Organization Institute of Veterinary Medicine of the National Academy of Agrarian Sciences of Ukraine popup.description1 To develop a tool for the diagnosis of grain fusarium wilt using the PCR method, which will contribute to more effective and early diagnosis of grain fusarium wilt popup.description2 Optimization of primers and PCR conditions. Based on the sequences of CAL and TEF genes, 7 primer systems were designed, including a universal pair for genus determination and 6 species-specific pairs. The optimal temperature regime (94–58–72°C), the composition of the reaction mixture and the concentration of PairMix were worked out. The size of amplicons (340–600 bp) was checked by electrophoresis. Development of the concept of completing the test kit. The structure of the kit was formed: 2×Hot-Start Master Mix, 7 primer mixtures, positive and negative controls. Study of test samples of corn grain. PCR analysis was performed and it was found that 55 out of 60 isolates (91.7%) were positive for Fusarium DNA. Species typing of isolates. For positive isolates, amplification was performed with 6 species-specific primers. Checking the toxinogenicity of isolates. TLC was performed for qualitative screening and ELISA (R-Biopharm) for quantitative determination of T-2 toxin, zearalenone and fumonisin B1. Of the 55 PCR-positive isolates, 27 (49.1%) showed toxin production. Correlation matrices between species and toxin were constructed. Reagent reproducibility and stability studies were conducted. Batch control, testing of positive and negative controls, analysis of discrepancies between repeated reactions (<10%). The stability of MasterMix and primer mixtures in the storage cycle (−20°C) was assessed. Improvement of the composition and preparation of technological regulations. The packaging volumes of primer mixtures have been adjusted, the requirements for quality control and storage of reagents have been updated, and the final version of the technological regulations for the diagnostic kit has been formed. Technological regulations for the manufacture and control of the diagnostic PCR kit for the detection of DNA of fungi of the genus Fusarium have been developed in accordance with the requirements of DSTU ISO/IEC 17025:2019. Product Description popup.authors Olga Zakharova Yana P. Krytsa Tetiana V. Sydorenko Янголь Юлія Анатоліївна Моложанова Альона Василівна popup.nrat_date 2025-12-30 Close