Comparison of Nested PCR and RFLP for Identification and Classification of Malassezia Yeasts from Healthy Human Skin

Abstract

Background: Malassezia yeasts are normal flora of the skin found in 75~98% of healthy subjects. The accurate identification of the Malassezia species is important for determining the pathogenesis of the Malassezia yeasts with regard to various skin diseases such as Malassezia folliculitis, seborrheic dermatitis, and atopic dermatitis.

Objective: This research was conducted to determine a more accurate and rapid molecular test for the identification and classification of Malassezia yeasts.

Methods: We compared the accuracy and efficacy of restriction fragment length polymorphism (RFLP) and the nested polymerase chain reaction (PCR) for the identification of Malassezia yeasts.

Results: Although both methods demonstrated rapid and reliable results with regard to identification, the nested PCR method was faster. However, 7 different Malassezia species (1.2%) were identified by the nested PCR compared to the RFLP method.

Conclusion: Our results show that RFLP method was relatively more accurate and reliable for the detection of various Malassezia species compared to the nested PCR. But, in the aspect of simplicity and time saving, the latter method has its own advantages. In addition, the 26S rDNA, which was targeted in this study, contains highly conserved base sequences and enough sequence variation for inter-species identification of Malassezia yeasts.

Keywords: 26S rDNA PCR-RFLP; Malassezia yeasts; Molecular biological classification; Nested PCR.

Fig. 1

Flow chart of the nested PCR and RFLP methods.

Fig. 2

PCR-RFLP patterns of 26S rDNA PCR digested with Hha I (A), BtsC I (B) of 11 Malassezia standard strains. Lanes: M. molecular marker; 1. M. furfur (KCTC 7743); 2. M. sympodialis (KCTC 7985); 3. M. globosa (CBS 7966); 4. M. restricta (KCTC 7848); 5. M. slooffiae (KCTC 17431); 6. M. pachydermatis (KCTC 17008); 7. M. japonica (CBS 9432); 8. M. nana (JCM 12085); 9. M. dermatis (JCM 11348); 10. M. obtusa (KCTC 7847); 11. M. yamatoensis (CBS 9725).

Fig. 3

Nested PCR products of standard Malassezia species. Lanes: M. molecular marker; 1. M. dermatis (JCM 11348); 2. M. furfur (KCTC 7743); 3. M. globosa (CBS 7966); 4. M. japonica (CBS 9432); 5. M. nana (JCM 12085); 6. M. obtuse (KCTC 7847); 7. M. pachydermatis (KCTC 17008); 8. M. restricta (KCTC 7848); 9. M. slooffiae (KCTC 17431); 10. M. sympodialis (KCTC 7985); 11. M. yamatoensis (CBS 9725); C. negative control.