Identification of Malassezia species from patient skin scales by PCR-RFLP G. Gaitanis, A. Velegraki, E. Frangoulis, A. Mitroussia, A. Tsigonia, A. Tzimogianni,

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Isolation and Expression of a Malassezia globosa Lipase Gene, LIP1

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Identification of Malassezia species from patient skin scales by PCR-RFLP G. Gaitanis, A. Velegraki, E. Frangoulis, A. Mitroussia, A. Tsigonia, A. Tzimogianni, A. Katsambas, N.J. Legakis Clinical Microbiology and Infection Volume 8, Issue 3, Pages 162-173 (March 2002) DOI: 10.1046/j.1469-0691.2002.00383.x Copyright © 2002 European Society of Clinical Infectious Diseases Terms and Conditions

Figure 1 Sequences of ITS 3/4 amplification products of Malassezia Type, Neotype and Reference CBS strains. HinfI restriction sites are marked in bold type and the AluI sites are marked by arrows. Clinical Microbiology and Infection 2002 8, 162-173DOI: (10.1046/j.1469-0691.2002.00383.x) Copyright © 2002 European Society of Clinical Infectious Diseases Terms and Conditions

Figure 2 ITS 3/4 amplification products and corresponding restriction fragments of Malassezia species from pure cultures in 2.5–3% standard agarose gels. (a) Lane 1, M. furfur (Mf, CBS 6001); lane 3, M. globosa (Mg, GM 56); lane 6, M. obtusa (Mo, CBS 7876); lane 8, M. restricta (Mr, CBS 7877); lane 10, M. sympodialis (Msy, CBS 7222); lane 13, M. pachydermatis (Mp, CBS 1879); lane 15, M. slooffiae (Ms, CBS 7956). Each Malassezia species tested is shown above the corresponding lanes. ITS 3/4 HinfI digests of each Malassezia species tested (panels a,b), lane 2, Mf (no restriction); lane 4, Mg; lane 7, Mo; lane 9, Mr; lane 11, Msy; lane 14, Mp; lane 17, Ms. Lanes 5, 12 and 16, molecular size marker MspI digest of pBR322 DNA. Clinical Microbiology and Infection 2002 8, 162-173DOI: (10.1046/j.1469-0691.2002.00383.x) Copyright © 2002 European Society of Clinical Infectious Diseases Terms and Conditions

Figure 3 ITS 3/4 AluI digests of Malassezia spp. Lane 1, M. furfur (Mf) clinical strain; lane 2, M. obtusa (Mo) CBS 7876; lane 3, M. slooffiae (Ms) CBS 7956; lane 4, M. pachydermatis (Mp) (dog otitis); lane 5, M. restricta (Mr) (clinical strain); lane 6, M. globosa (Mg) (clinical strain); lane 7, M. sympodialis (Msy) CBS 7222. Lane 8, molecular size marker MspI digest of pBR322 DNA. M. obtusa, M. restricta and M. sympodialis do not possess a restriction site for AluI, while one site is recognized on M. furfur, M. globosa, M. pachydermatis and M. slooffiae differentiating all four of them. Clinical Microbiology and Infection 2002 8, 162-173DOI: (10.1046/j.1469-0691.2002.00383.x) Copyright © 2002 European Society of Clinical Infectious Diseases Terms and Conditions

Figure 4 ITS 3/4 direct Malassezia and other yeast species detection from seborrheic dermatitis (SD) and pityriasis versicolor (PV) skin scales (Ssc) run in 2.5% standard agarose gels. (a) Lane 1, M. restricta from pure culture; lane 2, M. restricta (Mr) amplicon incubated with AluI (no restriction site recognized); lane 3, amplified target DNA from SD interscapular region skin scales identical to that of M. restricta (Ssc) incubated with AluI (no restriction site recognized). This result was later confirmed by culture and conventional identification. The approximately 330 and 242 bp smaller amplification products conform to those of C. albicans and Y. (Candida) lipolytica, respectively, the latter also isolated in culture. No AluI sites are recognized on either of these secondary products. (b) Lane 6, amplified target DNA of approximately 450 bp from trunk PV skin scales identical to that of M. globosa (Ssc); lane 7, AluI digest of the amplification product identical to that obtained from Type and clinical pure cultures of M. globosa (Ssc × AluI). Smearing, evident in lanes 3 and 6, was always obtained when target Ssc DNA was electrophoresed. (c) Y. lipolytica amplification products, HinfI and AluI digests from pure cultures of interscapular region SD skin scales run in 3.5% MetaPhor agarose (FMC, Rockland, ME, USA). Lanes 9 and 10, amplification products of Y. lipolytica (Yl) DNA from pure cultures; lane 11, HinfI digest (Yl × HinfI). The approximately 42 bp fragment was only visible in MetaPhore agarose. Lane 12, product incubated with AluI (Yl × AluI) (no AluI restriction site recognized). Lanes 4, 5 and 8, molecular size marker MspI digest of pBR322 DNA. Clinical Microbiology and Infection 2002 8, 162-173DOI: (10.1046/j.1469-0691.2002.00383.x) Copyright © 2002 European Society of Clinical Infectious Diseases Terms and Conditions