Study On The Identification And Analyzation Of Malassezia Species Using Micro-Fluidic Chips

Part Ⅰ:Classification of Genus Malassezia and their distribution in common Malassezia-related DiseasesObjective:To study the classification of Genus Malassezia and their distribution in two common Malassezia-related skin diseases.Methods:To analyze the association between Malassezia species and pityriasis versicolor as well as Malassezia folliculitis by isolating pathogen from patients with the diseases, and standard strains were as control. The clinical isolates were identified with DNA sequencing, using ITS2region PCR.Results:A total of83Malassezia isolates were obtained from72patients with pityriasis versicolor and11patients with Malassezia folliculitis, including41strains of Malassezia sympodialis (49.40%),26strains of Malassezia furfur (31.33%),10strains of Malassezia globosa (12.05%),2strains of Malassezia japonica (2.41%),1strain of Malassezia restricta (1.20%),1strain of Malassezia yamatoensis (1.20%),1strain of Malassezia dermatis (1.20%) and1strain of mix infection of Malassezia furfur and Malassezia sympodialis (1.20%). The construction of species in the two diseases was possibly associated with type of the diseases. Malassezia sympodialis were isolated from a half of the patients with pityriasis versicolor.Conclusion:The construction of Malassezia species varied in different diseases and Malassezia sympodialis is associated with pityriasis versicolor closely; PCR and sequencing of ITS2region in rDNA can be applied to the classification and identification of Malassezia strains.Part Ⅱ:Establishment of identification-database of Common Malassezia using Micro-Fluidic ChipsObjective:To invesgate the application advantage of micro-fluidic chips in identification of Malassezia speciesMethods:Clinical strains were isolated from patients with pityriasis versicolor and Malassezia folliculitis. And standard isolates were also collected. The clinical isolates were identified with DNA sequencing, random amplified polymorphic DNA (RAPD-PCR) cluster analysis tree and genotype quantitative analysis of micro-fluidic chip.Results:A total of83Malassezia isolates were obtained from72patients with pityriasis versicolor and11patients with Malassezia folliculitis. Genomic DNA of most strains was successfully amplified with two primers(S22、S24), while the DNA bands from primer S22were rather stable and clear. By micro-fluidic chip, the amplified strain-specific DNA fragments were different in size in different species. And interspecies and intraspecies polymorphisms can be observed. On the basis of DNA sequencing, combined with micro-fluidic chips and RAPD method,10different Malassezia species were genotyped. The following eight species can be distinguished: M.furfur, M.sympodialis, M.globosa, M. pachydermatis, M. slooffiae, M.Japonica, M.yamatoensis and M. dermatis.Conclusion:Combination of micro-fluidic chip analysis and RAPD method is a rapid, high-throughput, high-sensitivity detection analysis technology. And there are some advantages for distinguishing of strains between species and identification of new Malassezia species. The micro-fluidic chip analysis is suitable not only for genetic diversity of interspecies strains and phylogenetic analysis of genetic relationship, but also for epidemiological analysis of the diseases caused by this species.