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J Med Microbiol 53 (2004), 1207-1214; DOI: 10.1099/jmm.0.45779-0
© 2004 Society for General Microbiology
ISSN 0022-2615

Rapid identification and differentiation of fungal DNA in dermatological specimens by LightCycler PCR

Ralf Gutzmer, Susanne Mommert, Uta Küttler, Thomas Werfel and Alexander Kapp

Department of Dermatology and Allergology, Hannover Medical University, Ricklinger Str. 5, D-30449 Hannover, Germany

Correspondence Ralf Gutzmer rgutzmer{at}gmx.de

Received June 16, 2004
Accepted August 12, 2004

The aim was to develop a LightCycler PCR method for the rapid detection and differentiation of fungal DNA in dermatological specimens such as skin scales and skin swabs. LightCycler PCR assays were established for seven primer sets specific for fungal DNA. For each primer set LightCycler melting points were defined by amplification of DNA from 21 fungi and sensitivity was determined by amplification of serial dilutions of fungal DNA. A protocol was established that allows detection and differentiation of mould and yeast DNA with one highly sensitive PCR reaction by assessment of LightCycler melting points. Two subsequent LightCycler PCR reactions and one RFLP reaction allowed the differentiation of dermatophytes and non-dermatophyte moulds and the subclassification of yeasts. Analysis of clinical samples from 38 patients with fungal skin diseases provided conclusive new diagnostic information in 9/38 cases (23.7 %) by this PCR protocol that was not equally provided by direct microscopy and mycological culture. Thus the LightCycler PCR protocol established here represents a rapid diagnostic tool that aids in the diagnosis of fungal skin disease in a substantial number of patients.

Abbreviations: ITS, internal transcribed spacer; PBMCs, peripheral blood mononuclear cells.




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