The Application of Pyrosequencing Method in the Identification and Classification of Malassezia Yeasts

Song, Young Chan; Lim, Sang Hee; Jung, Bo Ra; Lee, Yang Won; Choe, Yong Beom; Ahn, Kyu Joong

Abstract

BACKGROUND: Malassezia yeasts are lipophilic fungi that are found in 75~80% of healthy adults. Recently, various molecular biological techniques are being preferred to identify and classify the Malassezia yeasts. Pyrosequencing is a real-time DNA sequencing technique. This technology has the potential advantage of accuracy, ease-of-use, high flexibility and is now emerging as a popular platform for microbial typing.
OBJECTIVE
We sought to implement novel molecular biology technique, namely pyrosequencing method in identifying and classifying Malassezia yeasts, and assess its clinical applicability.
METHODS
We obtained ribosomal RNA sequences of 11 Malassezia standard strains from NCBI database. Primers for the initial PCR amplification of the target region (ITS2) and sequencing primers within the regions amplified by the PCR primers were designed using Pyrosequencing Assay Design Software (Biotage AB, Uppsala, Sweden). We obtained PCR amplifying fragments of genomic DNA isolated from the Malassezia yeasts. And pyrosequence reactions were performed using reagents provided with the PSQ 96 Sample Preparation kit.
RESULTS
In the PCR analysis, all of 11 standard strains are shown at the 130 bp levels. In the pyrosequencing analysis, M. obtusa and M. furfur sequences were corresponded among 11 Malassezia standard strains. But, in 4 cases, Malassezia strains mismatched with expected Malassezia strain and in rest of 5 Malassezia strains, pyrosequencing was failed.
CONCLUSION
As evidenced above, pyrosequencing analysis could provide a sensitive and rapid identification system for Malassezia species. But it still has many limitation to be applied to epidemiological surveys and clinical practice.