Species-identification of dermatophytes Trichophyton, Microsporum and Epidermophyton by PCR and PCR-RFLP targeting of the DNA topoisomerase II genes.
BACKGROUND
We have focused on the DNA topoisomerase II genes of pathogenic fungi and have previously applied polymerase chain reaction (PCR)-based identification of several species including the some of the major dermatophyte species.


OBJECTIVE
To identify the dermatophytes (18 species) to a species level by PCR and PCR-restriction fragment length polymorphism (RFLP) techniques, without determining the nucleotide sequence.


METHODS
The genomic DNAs of the dermatophytes (ten species of Trichophyton, seven species of Microsporum, and Epidermaphyton floccosum) were amplified by PCR using a common primer set (dPsD1) for the dermatophytes, followed by nested PCR using other primer sets (dPsD2, PsT and PsME) that contained primers specific for the DNA topoisomerase II genes of the dermatophytes. PCRs using PsT and PsME were used for the species-identification of Trichophyton, Microsporum and E. floccosum. The PCR products generated by dPsD2 were digested with restriction enzymes (Hinc II, Hinf, Afl II and PflM I), and the restriction profiles were analyzed.


RESULTS
Of the eighteen species of dermatophytes, five species (T. rubrum, T. violaceum, M. canis, M. gypseum and E. floccosum) were specifically identified by the PCR using PsT and PsME to the species level, and the remaining species were identified by the unique restriction profiles for each species in the PCR-RFLP analysis, except that the restriction profile of T. mentagrophytes var. interdigitale was identical to that of T. mentagrophytes var. quinckeanum.


CONCLUSIONS
PCR and PCR-RFLP techniques targeting the DNA topoisomerase II gene are simple and rapid, and quite useful as tools for the identification of dermatophytes to the species level.
Kanbe
Toshio
T
Division of Molecular Mycology and Medicine, Department of Advanced Medical Science, Center for Neural Disease and Cancer, Nagoya University Graduate School of Medicine, 65 Tsurumai, Showa-ku, Nagoya 466-8550, Japan.
Suzuki
Yasuhiro
Y
Kamiya
Atsushi
A
Mochizuki
Takashi
T
Kawasaki
Masako
M
Fujihiro
Machiko
M
Kikuchi
Akihiko
A
D004250:DNA Topoisomerases, Type II / Q000235:genetics*; D004271:DNA, Fungal; D004821:Epidermophyton / Q000201:enzymology* / Q000302:isolation & purification*; D008864:Microsporum / Q000201:enzymology* / Q000302:isolation & purification*; D015335:Molecular Probes; D016133:Polymerase Chain Reaction; D012150:Polymorphism, Restriction Fragment Length; D012680:Sensitivity and Specificity; D014249:Trichophyton / Q000201:enzymology* / Q000302:isolation & purification*
D004271:DNA, Fungal; D015335:Molecular Probes; D004250:DNA Topoisomerases, Type II