[Basic methods for extraction and molecular analysis of micromycetic DNA]

The methods for extraction of nucleic acids from the fungi and clinical materials containing various microorganisms have currently modified and make it possible to extract DNA with its highest yield and to minimize its destruction. In particular, this concerns mycelial fungi that have a rigid cell wall. Addition of glass microbeads to the lysed sample, followed by mixture stirring, gives rise to undamaged DNA from fungi and their spores. Extracted DNA is suitable for the identification of micromycetes by qualitative or quantitative polymerase chain reaction (PCR), real-time PCR-based hybridization, and LightCycler PCR and for the polymorphism analysis of the spacer regions of ribosomal genes by the sequencing technique. A combination of quantitative PCR and sequencing permits co-determination of the number of gene target copies in 9 different micromycetes in the clinical material being examined, which is of great importance for the diagnosis and treatment of patients with higher immunosuppression and, of those after transplantation of organs and tissues in particular.