Abstrakt

The broad application of the molecular techniques in mycoses diagnosis is related to increase of infections caused by fungi in many countries. The oldest typing methods relaying on fenotypic observation, physiological and biochemical examination have had very limited importance from decades. Novadays, the molecular biology methods took their place. Most of the genotyping methods have been devoleped to be applied for typing of bacteria and viruses and are applied for fungi typing. However, the proper choice of the method is possible only when differences between bacterial and fungal genotypes are considered. Moreover, such differences can be found even within one genus, e.g. diploid Candida albicans and haploid Candida glabrata. The next issue that should be considered is that the proper choice of the method depends on the research aim to be obtained. This issue is closely related to the discrimination potential of the particular methods e.g. DNA-DNA hybridisation methods allow to distinguish genera or species, but are not useful for distinguishing strains. Moreover, some of the methods, e.g. sequencing can be applied to distinguishing the isolates on almost all the taxonomic levels - order, family, genus, species or strains. However, the definition of typing of microorganisms is their distinguishing within species or subspecies level. The choice of the typing method also depends on the knowledge of the genomes of fungi to be typed. Some methods, such PCR fingerprinting (RAPD, AP-PCR, DAF) or techniques based on the adapters ligation (LM PCR), don’t require the sequences of the genomes or their fragments, the others are based on their comparison (Multilocus Sequence Typing - MLST). To understand the possibilities and limitations of genotyping methods, their principles should be carefully studied. Various sequencing strategies and PCR based techniques, which can be applied in genotyping, will be presented The techniques based on restriction fragment length polymorphism (RFLP) with or without hybridization to probes (Southern), although their use is less extensive and is limited mostly to research laboratories, will be also reviewed. Beside the choice of the technique, the influence of experimental errors on the obtained results should be also considered. Moreover, it is important to remember that small differences not always are meaningfull and do not always allow for recognising the different strains.

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