Interlaboratory aprobation of primers for molecular genetic identification of Fusarium link fungus
DOI:
https://doi.org/10.31548/dopovidi2019.06.017Keywords:
molecular genetic research methods, polymerase chain reaction, fusariumAbstract
There are given results of innerlaboratory approbation of methodology of molecular genetic identification of Fusarium fungus by using PCR method. The researches were carried out by using polymerase chain reaction method with detection of amplification products in agarose gel. For determination specificity of the methodology there were used different strains of fungus: Fusarium, Alternaria Nees, Aspergillus P. Micheli ex Haller, Cladosporium Link, Drechslera S. Ito, Mucor Fresen., Rhizopus Ehrenb., Trichoderma Pers.
The extraction of DNA was carried out by using guanidine-thiocyanate method with sorption on silicon oxide which was described by R. Boom with co-authors. For researches there were used primers that flank part of ITC region of ribosomal DNA Fusarium spp, 431 n.p. After amplification in thermocycler 2720 (Applied Biosystems) with appropriate temperature mode its products were analyzed by separation in 1,5 % agarose gel.
During the research of specificity of the methodology all the species of Fusarium fungus were identified as positive by using given primers. Cross-reactions with micromycetes of other species were not observed. Sensitivity of PCR-analysis using given primers is 0,12 ng/mcl. The results of detection of Fusarium fungus directly from corn grains and its recycling products show that most corn plants on fields are infected by micromycetes of given genus.
The results of innerlaboratory evaluation of primers for Fusarium spp identification with researching such parameters as specificity and sensitivity show that tested methodology gives ability to detect Fusarium fungus in grain products quite fast and reliably and also it can be used for confirmation of dubious results of mycological analysis.
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