Ann Dermatol.  2009 Nov;21(4):352-357. 10.5021/ad.2009.21.4.352.

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

• Oh BH
• Song YC
• Lee YW
• Choe YB
• Ahn KJ

• KMID: 2156496
• DOI: http://doi.org/10.5021/ad.2009.21.4.352

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.

Keyword

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

MeSH Terms

Base Sequence
Dermatitis, Seborrheic
DNA, Ribosomal
Folliculitis
Humans
Malassezia
Polymerase Chain Reaction
Polymorphism, Restriction Fragment Length
Skin
Skin Diseases
Yeasts
DNA, Ribosomal

Figure

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  Fig. 1 Flow chart of the nested PCR and RFLP methods.

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  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).

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  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.

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