title: Evaluation of growth conditions and DNA extraction techniques used in the molecular analysis of dermatophytes.
abstract: OBJECTIVE
  Recent molecular methods for diagnosis of superficial mycoses have determined the need for a rapid and easy method of extracting DNA. The aim of study was to determine growth conditions and techniques of DNA extraction for Microsporum canis, Trichophyton mentagrophytes and T. verrucosum.
  
  
  RESULTS
  Samples were prepared of each of the DNA extraction methods (phenol-chloroform, CTAB and four different kits) for all of the incubation periods (4, 7 and 10 days) of the cultures on the solid and in the liquid medium. The highest DNA concentrations were obtained using the phenol-chloroform method. The concentration of DNA extracted with the CTAB method accounted for 62·21%, for kits it corresponded from 35·53 to 15·41%. The analysis of the DNA weight yield revealed the highest isolation efficiency of the phenol-chloroform method, 1 mg of mycelium yielded 223·8 μg DNA. Lower DNA yield (by 39·32%) was obtained with the CTAB method; in the case of kits by 68·46-85·32%. In most of the techniques, the DNA yield on the solid medium was higher.
  
  
  CONCLUSIONS
  In summary, the highest DNA yield was noted in the 7-day cultures and extraction with the phenol-chloroform method. Importantly, the type of culture was not relevant for the diagnostic result.
  
  
  CONCLUSIONS
  Most mycoses are caused by fungi that reside in nature. The severity of the infection depends on the pathogenic attributes, socioeconomic factors and local environmental conditions. Recent diagnosis increasingly relies on not only the clinical features. Molecular identifications have determined the need for a rapid and easy method of extracting DNA. Usually two factors have to be considered: maximize the DNA yield and ensure that the extracted DNA is susceptible to enzymatic reactions. These data suggest that phenol-chloroform methods and a 7-day culture period may be useful for validation and constitute the first step of molecular diagnosis of dermatophytes.
authors.affiliation: Sub-Department of Veterinary Microbiology, Institute of Biological Bases of Animal Diseases, Faculty of Veterinary Medicine, University of Life Sciences, Lublin, Poland.
authors.forename: S
authors.initials: S
authors.lastname: Gnat
authors.affiliation: Sub-Department of Veterinary Microbiology, Institute of Biological Bases of Animal Diseases, Faculty of Veterinary Medicine, University of Life Sciences, Lublin, Poland.
authors.forename: A
authors.initials: A
authors.lastname: Nowakiewicz
authors.affiliation: Sub-Department of Veterinary Microbiology, Institute of Biological Bases of Animal Diseases, Faculty of Veterinary Medicine, University of Life Sciences, Lublin, Poland.
authors.forename: G
authors.initials: G
authors.lastname: Ziółkowska
authors.affiliation: Sub-Department of Veterinary Microbiology, Institute of Biological Bases of Animal Diseases, Faculty of Veterinary Medicine, University of Life Sciences, Lublin, Poland.
authors.forename: A
authors.initials: A
authors.lastname: Trościańczyk
authors.affiliation: Sub-Department of Veterinary Microbiology, Institute of Biological Bases of Animal Diseases, Faculty of Veterinary Medicine, University of Life Sciences, Lublin, Poland.
authors.forename: B
authors.initials: B
authors.lastname: Majer-Dziedzic
authors.affiliation: State Veterinary Laboratory, Lublin, Poland.
authors.forename: P
authors.initials: P
authors.lastname: Zięba
mesh_terms: D003883:Arthrodermataceae / Q000145:classification / Q000235:genetics* / Q000254:growth & development* / Q000302:isolation & purification; D005591:Chemical Fractionation / Q000379:methods*; D004271:DNA, Fungal / Q000235:genetics / Q000302:isolation & purification*; D003881:Dermatomycoses / Q000382:microbiology*
chemical_list: D004271:DNA, Fungal
keywords: DNA extraction techniques; Microsporum canis; Trichophyton mentagrophytes; Trichophyton verrucosum; culture method; dermatophytes; incubation time; molecular identification of dermatophytes