The Application of 26S rDNA PCR-RFLP in the Identification and Classification of Malassezia Yeast

Lee, Yang Won; Lim, Sang Hee; Ahn, Kyu Joong

Abstract

BACKGROUND: Malassezia yeast are lipophilic fungi that are found in 75~80% of healthy adults. The yeast are known to be associated with pityriasis versicolor, seborrheic dermatitis, Malassezia folliculitis, and recently its pathogenicity is being expanded to other various skin disorders, such as atopic dermatitis and acne vulgaris. Up to present, mycological studies on Malassezia yeast have been carried out mostly through morphological analysis and biochemical analysis. Recently however, various molecular biological techniques are being preferred over morphological analysis, which is not a suitable method for establishing taxonomic relationship between species, and more or less time-consuming.
OBJECTIVE
We sought to implement novel molecular biology technique, namely 26S rDNA PCRRFLP method in identifying and classifying Malassezia yeast, and assess its clinical applicability.
METHODS
Eleven standard strains and eight clinical isolates were thoroughly examined with special attention to the shape of the colonies, size and change in media. Subsequently, the colonies were classified according to Gueho classification. For molecular analysis, RFLP analysis was carried out after DNA was isolated from each organism and 26S rDNA was amplified through PCR. The results of identification were confirmed by 26S rDNA sequencing.
RESULTS
In PCR analysis to amplify the 26S rDNA, a 580bp PCR band was seen in all of eleven standard colonies. On analysis of PCR-RFLP of 26S rDNA using restriction enzymes Hha1 and BstF51, all of the database in the restriction pattern of each species was attained. On analyzing eight clinical isolates, a restriction pattern which was interspecifically distinguishable, was identified, and the result was in accord with the pattern obtained from 26S rDNA PCR-RFLP of standard colonies. Out of eight, seven clinical isolates colonies was in accord with the result of 26S rDNA PCR-RFLP. In order to assess the precision of 26S rDNA PCR-RFLP, 26S rDNA sequencing was performed, whose result was in accord with 26S rDNA PCR-RFLP analysis.
CONCLUSION
As evidenced above, 26S rDNA PCR-RFLP analysis could provide a sensitive and rapid identification system for Malassezia species, which may be applied to epidemiological surveys and clinical practice