Bulletin "Veterinary biotechnology"

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Veterynarna biotehnologija – Veterinary biotechnology, 2022, 40, 43-52 [in Ukrainian]. https://doi.org/10.31073/vet_biotech40-04

ZAKHAROVA O., PhD, e-mail: This email address is being protected from spambots. You need JavaScript enabled to view it. TARASOV O., PhD,e-mail: This email address is being protected from spambots. You need JavaScript enabled to view it., YANGOL YU.A., junior researcher, e-mail: This email address is being protected from spambots. You need JavaScript enabled to view it.

Institute of Veterinary Medicine NAAS

KOLYCH N.B., PhD, Associate Professor, e-mail: This email address is being protected from spambots. You need JavaScript enabled to view it.

National University of Life and Environmental Sciences of Ukraine

 

 DETECTION OF TOXIN-PRODUSING FUNGI OF FUSARIUM GENUS USING REAL-TIME POLYMERASE CHAIN REACTION

 Introduction. The problem of toxinogenic fungi impact to grain and animal feed is actual, due to the numerous publications confirm the high level of grain damage by toxin-producing fungi, including the genus Fusarium. According to the published data, more than half of grain products are affected by toxins, including Fusarium one. Almost 12% of the affected grains contained fusariotoxin in the maximum permissible contamination level.

The goal of the work was to validate the method of detection of DNA of toxin-producing fungi of Fusarium genus using a polymerase chain reaction in real time in accordance with international standards.

Materials and methods. It was used 5 toxin-producing isolates of Fusarium genus fungi from museum of the Institute of Veterinary Medicine of theNAAS. Studies of morphological and cultural properties were performed using conventional microbiological methods.

The fusariotoxin producing isolates were detected by PCR-RT test (real-time method) using set of primers recommended by Yli-Mattila et al. (2008). The assay parameters were optimized by us. The results of experimental studies are processed by conventional methods of statistics.

Results of research and discussion. According to the analysis of the results of microbiological studies, it was found that all the studied isolates had the main typical properties characteristic of microscopic fungi of the genus Fusarium spp.

The number of genome equivalents (GЕ/cm3) was determined in the experimental samples, which was 1×105 GE/cm3, which was diluted to 1×101 GE/cm3. Five repeated tests were performed for each concentration.

The limit of analytical sensitivity of the method at concentrations of 1×105 GE/cm3 and 1×104 GE/cm3 was 100%. Using 1×103 GE/cm3 and 1×102 GE/cm3 concentrations, the confidence intervals were 60% and 20%, respectively. Thus, the detection limit (LOD) for the test was determined as 1×104 GE/cm3.

Conclusions and prospects for further research. As a result of research, it was found that PCR in real-time allows to detect toxin-producing microscopic fungi of Fusarium genus.

The results of the evaluation of the sensitivity of the method allowed to detect the target DNA by the concentration at least 104 GE/cm3. This concentration is much lower than those reported for contaminated substrate by Fusarium genus fungi.

The use of PCR in real-time to detect toxin-producing microscopic fungi of the genus Fusarium in Ukraine is important for improving control over mycotoxins poisoning of animals and support the high quality of agricultural products.

Keywords: Fusarium,fumonisine, PCR, isolate, toxigenic strain.

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