Advanced Receptions in the Methodology of Extraction Total and Viral Nucleic Acids From Basidiomycetes

Abstract

Existing governmental programs aimed to increase mushrooms production and significant market demand for mushrooms contributed to the expansion of such production. Meanwhile, until now practically don`t exist highly precised molecular diagnostics for the detection and identification of mycoviruses that strike the Basidiomycetes in Ukraine. The purpose and objectives are to study modern methods of extraction of total RNA from Basidiomycetes and their modifications and improvements as well as the molecular biological systems test for the identification of Basidiomycetes mycoviruses. This research was carried out by way of with follow techniques: molecular biological (the method of extraction of total RNA, dsDNA, precipitation, amplification); biotechnological (obtaining and subcultivation of samples of mycelium in vitro using electrophoresis in agarose and polyacrylamide gels (PAGE); microbiological (obtaining pure mushroom culture, determining the hydrogen index (pH) of the nutrient medium), mycological (a measurement of growth), virology, microscopy. Interpretation statistics data was carried out with the help of computer software. Screening of mushroom`s samples has been performed in the Kyiv region. The virus infection and prevalence of viral diseases of mushrooms have been researched. The modification of method of extraction total and viral nucleic acids from Basidiomycetes has been proposed. It is based on the acceleration of the stage of cellulose-chromatographic purification of nucleic acids and fractionation of dsRNA. It is proposed to propagate the first stage of low-speed centrifugation at high speeds from 6000g to 8000g. Such centrifugation is performed according to the method after the first phenol isolation of total nucleic acids. In the future it is proposed to perform only one instead of two stages of the cellulose gradient for double-stranded ribonucleic acids of the test sample. As a result of the modification a larger number of nucleic acids has been received. An alternative step was introduced for reducing the loss of RNA from natural abrasives by increase the weight of samples to 10 g, 30 ml STE buffer for initial washing, 50 % of the phenol`s volume, 17 ml of chloroform, and 2 ml of isoamyl alcohol.