Article Figures & Data
Figures
- Fig. 1.
Schematic representation of the fungal ribosomal genes containing the primer target areas used in the amplification of the ITS2 region.
- 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.
- 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.
- 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
- Table 1.
Base pair determination and associated standard deviation of the ITS2-containing PCR fragments of various species during multiple fragment analysis runs
Organism Strain (n)a ITS2 fragment length (SD)b Candida albicans ATCC 10231 (42) 279.15 (0.4) Candida tropicalis ATCC 66029 (8) 268.61 (0.5) Candida glabrata ATCC 90030 (7) 359.47 (0.4) Candida parapsilosis ATCC 22019 (7) 251.33 (0.4) Candida krusei ATCC 6258 (6) 282.21 (0.2) Candida lusitaniae ATCC 42720 (8) 198.67 (0.5) Candida guilliermondii CAP F1-95 (9) 320.87 (0.5) Candida kefyr ATCC 4135 (6) 372.35 (0.2) Saccharomyces cerevisiae ATCC 9763 (8) 363.94 (0.3) - Table 2.
Length determination (in base pairs) of the PCR fragment containing the ITS2 of known fungal isolates
Organism Strain(s)a Conventional ID PCR fragment length (bp) Yeasts Candida albicans ATCC 10231, SCC, HSC1159, HSC67442, HSC67630, HSC68392, HSC81363 API 20C-AUX 279 Candida kefyr ATCC 4135, SCC API 20C-AUX 372 Candida guilliermondii CAP F1-95, SCC API 20C-AUX 321 Candida tropicalis ATCC 66029, SCC, HSC75317, HSC75418, HSC75422, HSC78308, HSC78408, HSC81682, HSC81701, HSC71273, HSC71278, HSC29977, HSC71277 API 20C-AUX 269 Candida krusei ATCC 6258, SCC, CT1, K1865, CA37, E175 API 20C-AUX 282 Candida glabrata ATCC 90030, SCC, HSC17253, HSC20818, HSC20819 API 20C-AUX 360 Candida zeylanoides CAP F18-93 API 20C-AUX 316 Candida lusitaniae ATCC 42720 API 20C-AUX 199 Candida parapsilosis ATCC 22019, SCC, HSC5966, HSC21193 API 20C-AUX 251 Candida pelliculosa ATCC 8168 API 20C-AUX 317 Candida pseudotropicalis ATCC 66028 API 20C-AUX 372 Candida stellatoidea ATCC 36232 API 20C-AUX 279 Trichosporon beigelii ATCC 28592 API 20C-AUX 295 HSC6088 API20C-AUX 298 Geotrichum candidum ATCC 34614 API 20C-AUX, mannitol 192 Cryptococcus neoformans ATCC 90112 API 20C-AUX, urease, phenoloxidase 315 Cryptococcus albidus ATCC 10666 API 20C-AUX 350 Cryptococcus laurentii ATCC 18803 API 20C-AUX 306 Rhodotorula rubra ATCC 9449, SCC HSC20116, HSC37777 API 20C-AUX 348 Blastoschizomyces capitatus ATCC 10663 API 20C-AUX 248 Saccharomyces cerevisiae ATCC 9763 API 20C-AUX 363 Malassezia furfur ATCC 14521 API 20C-AUX 494 Other opportunistic fungi Aspergillus fumigatus CAP F6-91 Morphology 284 Aspergillus flavus ATCC 10124 Morphology 288 Aspergillus niger ATCC 16404 Morphology 290 Aspergillus terreus ATCC 28301 Morphology 292 Alternaria alternata CAP F13-93 Morphology 285 Penicilliumsp. SCC Morphology 283 Acremonium strictum CAP F4-96 Morphology 294 Fusarium solani CAP F15-95 Morphology 286 Paecilomyces sp. SCC Morphology 287 Rhizopus sp. SCC Morphology 319 Absidia corymbifera ATCC 66271 Morphology 410 Mucor sp. SCC Morphology 312 Cunninghamella bertholletiae ATCC 42115 Morphology 347 Pseudallescheria boydii ATCC 58085 Morphology 321 Scedosporium prolificans SCC Morphology 303 Phialophora verrucosa ATCC 28181 Morphology 308 Phialophora richardsiae ATCC 26465 Morphology 276 Fonsecaea pedrosoi ATCC 28174 Morphology 319 Cladosporium carrionii ATCC 32279 Morphology 303 Wangiella dermatitidis SCC Morphology 334 Exophiala jeanselmei ATCC 10224 Morphology 311 Stemphylium ilicis ATCC 18160 Morphology 280 Dimorphic fungi Penicillium marneffei ATCC 18224 Morphology 280 Sporothrix schenckii ATCC 10212 Morphology, conversion to yeast phase 310 Blastomyces dermatitidis HSC patient isolate Nucleic acid probe 315 Histoplasma capsulatum HSC patient isolate Nucleic acid probe 299 Dermatophytes Microsporum canis CAP F4-90 Morphology 321 Microsporum gypseum CAP F20-90 Morphology 321 Microsporum audouinii ATCC 42558 Morphology 318 Microsporum nanum SCC Morphology 324 Microsporum gallinae SCC Morphology 325 Microsporum cookei SCC Morphology 314 Trichophyton schoenleinii SCC Morphology, nutritional and temp (°C) requirements 306 Trichophyton terrestre SCC Morphology, nutritional and temp (°C) requirements 298 Trichophyton violaceum SCC Morphology 315 Trichophyton mentagrophytes ATCC 28185 Morphology, urease, hair performation 303 Trichophyton rubrum ATCC 28188 Morphology 307 Trichophyton tonsurans ATCC 28942 Morphology and nutritional requirements 302 Trichophyton verrucosum CAP F15-1994, SSC Morphology, nutritional and temp (°C) requirements 307 Epidermophyton floccosum ATCC 52066 Morphology 366 ↵a SCC and HSC, stock culture collection and patient isolates, respectively, obtained from the Health Sciences Centre, Winnipeg, Manitoba, Canada.
