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Mycology

Rapid Identification of Fungi by Using the ITS2 Genetic Region and an Automated Fluorescent Capillary Electrophoresis System

Christine Y. Turenne, Steven E. Sanche, Daryl J. Hoban, James A. Karlowsky, Amin M. Kabani
Christine Y. Turenne
Department of Medical Microbiology, Faculty of Medicine, and
Departments of Clinical Microbiologyand
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Steven E. Sanche
Division of Infectious Diseases, Royal University Hospital, Saskatoon, Saskatchewan, Canada
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Daryl J. Hoban
Department of Medical Microbiology, Faculty of Medicine, and
Faculty of Pharmacy, University of Manitoba, and
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James A. Karlowsky
Department of Medical Microbiology, Faculty of Medicine, and
Departments of Clinical Microbiologyand
Faculty of Pharmacy, University of Manitoba, and
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Amin M. Kabani
Department of Medical Microbiology, Faculty of Medicine, and
Departments of Clinical Microbiologyand
Medicine, Health Sciences Centre, Winnipeg, Manitoba R3A 1R9, Canada, and
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DOI: 10.1128/JCM.37.6.1846-1851.1999
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  • Fig. 1.
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    Fig. 1.

    Schematic representation of the fungal ribosomal genes containing the primer target areas used in the amplification of the ITS2 region.

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    Fig. 2.

    Specificity of universal ITS2 primers against bacteria and human genomic DNA. PCR amplification using the ITS4 and ITS86 primer pair was performed as described in Materials and Methods. The following DNA templates were used for PCR (by lane): 1,Staphylococcus epidermidis ATCC 12228; 2, Escherichia coli ATCC 25922; 3, Staphylococcus aureus ATCC 25923; 4, Pseudomonas aeruginosa ATCC 27853; 5, Clostridium perfringens ATCC 13124; 6, human whole blood; 7, human leukocytes; 8, human liver; 9, Candida albicans ATCC 10231; and 10, H2O contamination control.

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    Fig. 3.

    Detection of fungal species by agarose gel electrophoresis. PCR amplification of variable Candidaspecies from culture colonies using the ITS4 and ITS86 universal fungal primers was performed as described in Materials and Methods. Lanes: L, 123-bp ladder; 1, Candida albicans; 2, Candida kefyr; 3, Candida zeylanoides; 4, Candida tropicalis; 5, Candida krusei; 6, Candida glabrata; 7, Candida guilliermondii; 8, Candida lusitaniae; 9, Candida parapsilosis.

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    Fig. 4.

    Electropherograms of five Candida species as analyzed by the ABI PRISM 310 genetic analyzer. Control strains (graphs 1 to 5) were amplified by using ITS4 and fluorescently labeled ITS86 and dUTP as described in Materials and Methods. Each was run separately on the capillary electrophoresis system along with an internal size standard (GeneScan ROX-500). Standard peaks are shown as a separate electropherogram (graph 6) for clarity of illustration. The standard peak sizes are 139, 150, 150, 200, 240, 300, 340, 350, and 400 bp. Graphs: 1, Candida albicans ATCC 10231; 2, Candida tropicalis ATCC 66029; 3, Candida glabrata ATCC 90030; 4, Candida lusitaniae ATCC 42720; 5, Candida krusei ATCC 6258.

Tables

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  • Table 1.

    Base pair determination and associated standard deviation of the ITS2-containing PCR fragments of various species during multiple fragment analysis runs

    OrganismStrain (n)aITS2 fragment length (SD)b
    Candida albicansATCC 10231 (42)279.15 (0.4)
    Candida tropicalisATCC 66029 (8)268.61 (0.5)
    Candida glabrataATCC 90030 (7)359.47 (0.4)
    Candida parapsilosisATCC 22019 (7)251.33 (0.4)
    Candida kruseiATCC 6258 (6)282.21 (0.2)
    Candida lusitaniaeATCC 42720 (8)198.67 (0.5)
    Candida guilliermondiiCAP F1-95 (9)320.87 (0.5)
    Candida kefyrATCC 4135 (6)372.35 (0.2)
    Saccharomyces cerevisiaeATCC 9763 (8)363.94 (0.3)
    • ↵a n, number of runs (including PCR amplification and detection by capillary electrophoresis).

    • ↵b Lengths are in base pairs. SD, standard deviation.

  • Table 2.

    Length determination (in base pairs) of the PCR fragment containing the ITS2 of known fungal isolates

    OrganismStrain(s)aConventional IDPCR fragment length (bp)
    Yeasts
     Candida albicansATCC 10231, SCC, HSC1159, HSC67442, HSC67630, HSC68392, HSC81363API 20C-AUX279
     Candida kefyrATCC 4135, SCCAPI 20C-AUX372
     Candida guilliermondiiCAP F1-95, SCCAPI 20C-AUX321
     Candida tropicalisATCC 66029, SCC, HSC75317, HSC75418, HSC75422, HSC78308, HSC78408, HSC81682, HSC81701, HSC71273, HSC71278, HSC29977, HSC71277API 20C-AUX269
     Candida kruseiATCC 6258, SCC, CT1, K1865, CA37, E175API 20C-AUX282
     Candida glabrataATCC 90030, SCC, HSC17253, HSC20818, HSC20819API 20C-AUX360
     Candida zeylanoidesCAP F18-93API 20C-AUX316
     Candida lusitaniaeATCC 42720API 20C-AUX199
     Candida parapsilosisATCC 22019, SCC, HSC5966, HSC21193API 20C-AUX251
     Candida pelliculosaATCC 8168API 20C-AUX317
     Candida pseudotropicalisATCC 66028API 20C-AUX372
     Candida stellatoideaATCC 36232API 20C-AUX279
     Trichosporon beigeliiATCC 28592API 20C-AUX295
    HSC6088API20C-AUX298
     Geotrichum candidumATCC 34614API 20C-AUX, mannitol192
     Cryptococcus neoformansATCC 90112API 20C-AUX, urease, phenoloxidase315
     Cryptococcus albidusATCC 10666API 20C-AUX350
     Cryptococcus laurentiiATCC 18803API 20C-AUX306
     Rhodotorula rubraATCC 9449, SCC HSC20116, HSC37777API 20C-AUX348
     Blastoschizomyces capitatusATCC 10663API 20C-AUX248
     Saccharomyces cerevisiaeATCC 9763API 20C-AUX363
     Malassezia furfurATCC 14521API 20C-AUX494
    Other opportunistic fungi
     Aspergillus fumigatusCAP F6-91Morphology284
     Aspergillus flavusATCC 10124Morphology288
     Aspergillus nigerATCC 16404Morphology290
     Aspergillus terreusATCC 28301Morphology292
     Alternaria alternataCAP F13-93Morphology285
     Penicilliumsp.SCCMorphology283
     Acremonium strictumCAP F4-96Morphology294
     Fusarium solaniCAP F15-95Morphology286
     Paecilomyces sp.SCCMorphology287
     Rhizopus sp.SCCMorphology319
     Absidia corymbiferaATCC 66271Morphology410
     Mucor sp.SCCMorphology312
     Cunninghamella bertholletiaeATCC 42115Morphology347
     Pseudallescheria boydiiATCC 58085Morphology321
     Scedosporium prolificansSCCMorphology303
     Phialophora verrucosaATCC 28181Morphology308
     Phialophora richardsiaeATCC 26465Morphology276
     Fonsecaea pedrosoiATCC 28174Morphology319
     Cladosporium carrioniiATCC 32279Morphology303
     Wangiella dermatitidisSCCMorphology334
     Exophiala jeanselmeiATCC 10224Morphology311
     Stemphylium ilicisATCC 18160Morphology280
    Dimorphic fungi
     Penicillium marneffeiATCC 18224Morphology280
     Sporothrix schenckiiATCC 10212Morphology, conversion to yeast phase310
     Blastomyces dermatitidisHSC patient isolateNucleic acid probe315
     Histoplasma capsulatumHSC patient isolateNucleic acid probe299
    Dermatophytes
     Microsporum canisCAP F4-90Morphology321
     Microsporum gypseumCAP F20-90Morphology321
     Microsporum audouiniiATCC 42558Morphology318
     Microsporum nanumSCCMorphology324
     Microsporum gallinaeSCCMorphology325
     Microsporum cookeiSCCMorphology314
     Trichophyton schoenleiniiSCCMorphology, nutritional and temp (°C) requirements306
     Trichophyton terrestreSCCMorphology, nutritional and temp (°C) requirements298
     Trichophyton violaceumSCCMorphology315
     Trichophyton mentagrophytesATCC 28185Morphology, urease, hair performation303
     Trichophyton rubrumATCC 28188Morphology307
     Trichophyton tonsuransATCC 28942Morphology and nutritional requirements302
     Trichophyton verrucosumCAP F15-1994, SSCMorphology, nutritional and temp (°C) requirements307
     Epidermophyton floccosumATCC 52066Morphology366
    • ↵a SCC and HSC, stock culture collection and patient isolates, respectively, obtained from the Health Sciences Centre, Winnipeg, Manitoba, Canada.

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Rapid Identification of Fungi by Using the ITS2 Genetic Region and an Automated Fluorescent Capillary Electrophoresis System
Christine Y. Turenne, Steven E. Sanche, Daryl J. Hoban, James A. Karlowsky, Amin M. Kabani
Journal of Clinical Microbiology Jun 1999, 37 (6) 1846-1851; DOI: 10.1128/JCM.37.6.1846-1851.1999

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Rapid Identification of Fungi by Using the ITS2 Genetic Region and an Automated Fluorescent Capillary Electrophoresis System
Christine Y. Turenne, Steven E. Sanche, Daryl J. Hoban, James A. Karlowsky, Amin M. Kabani
Journal of Clinical Microbiology Jun 1999, 37 (6) 1846-1851; DOI: 10.1128/JCM.37.6.1846-1851.1999
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KEYWORDS

DNA, Ribosomal
fungi
Genes, Fungal

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