Identification of Medically Important Candida and Non-Candida Yeast Species by an Oligonucleotide Array

Yeast strains.A total of 419 target strains, representing 44 Candida species (275 strains) and 33 non-Candida species (144 strains), were used for identification by the array (Table 1). Among these strains, 309 were reference (or type) strains and 110 were clinical isolates. Reference strains were obtained from the American Type Culture Collection (ATCC, Manassas, VA), the Bioresource Collection and Research Center (BCRC, Hsinchu, Taiwan), and Centraalbureau voor Schimmelcultures (CBS, Utrecht, The Netherlands). Clinical isolates were obtained from the Mycology Reference Centre, Department of Microbiology, Leeds Teaching Hospitals Trust (Leeds, United Kingdom), the Laboratory of Parasitology and Mycology of Angers University Hospital (Angers, France), and the National Cheng Kung University Medical Center (Tainan, Taiwan). Isolates were identified to the species level based on traditional criteria (18) or with the API ID 32C system. In addition, a total of 33 nontarget strains (33 species) were used for specificity testing of the array (data not shown).

DNA extraction.Yeasts were subcultured on Sabouraud dextrose agar and incubated at 28°C for 24 to 48 h. Colonies of yeast were suspended in saline, and the genomic DNA was extracted by using the blood and tissue genomic DNA extraction Miniprep system (Viogene, Taipei, Taiwan), as described previously (24), except that the lyticase digestion step was omitted.

Amplification of the ITS regions for hybridization.Digoxigenin (DIG)-labeled amplicons for array hybridization were obtained by PCR using the fungus-specific universal primers ITS1 (5′-DIG-TCCGTAGGTGAACCTGCGG-3′) and ITS4 (5′-DIG-TCCTCCGCTTATTGATATGC-3′) (50). Each primer was labeled with a digoxigenin molecule at its 5′ end and was obtained from MdBio, Inc. (Taipei, Taiwan). The PCR products encompassed ITS 1, the 5.8S rRNA gene, ITS 2, and partial regions of the 18S and 28S rRNA genes. The reaction mixtures and thermocycling conditions used for PCR were described previously (24).

Design of oligonucleotide probes.The oligonucleotide probes (16- to 30-mers) used for the identification of 77 yeast species are listed in Table 2. Probes were designed from either the ITS 1 or the ITS 2 region. The corresponding ITS sequences used for probe design were available in the GenBank database or determined in our previous studies (2, 24). Sequences extracted from GenBank (Table 2) were confirmed by at least one sequence from another reference strain of the same species in public databases. The alignment of multiple ITS sequences was made by using Discovery Studio Gene software (DS Gene, version 1.5; Accelrys, Inc., San Diego, CA). Based on the aligned sequences, areas displaying high intraspecies similarities and low interspecies similarities were used for probe synthesis. The melting temperature, GC content, and possible secondary structure of a designed probe were examined by using Vector NTI software (Invitrogen Corporation, Carlsbad, CA). In addition, the designed probes were checked for partial sequence homology with other microorganisms in public databases using the basic local alignment search tool (BLAST) algorithm. A total of 79 species-specific probes and one group-specific probe were used for fabrication of the oligonucleotide array (Table 2). In addition, a probe designed from a conserved sequence of the fungal 5.8S rRNA gene was used as a positive control probe (probe code, PC) (Table 2). Five to 17 bases of thymine were added to the 3′ or 5′ ends of probes that displayed weak hybridization signals after preliminary testing (1). Moreover, a digoxigenin-labeled bacterial universal primer 6R (5-DIG-GGGTTYCCCCRTTCRGAAAT-3, where Y is C or T and R is A or G) (probe code, M) was spotted on the array and used as a position marker (Fig. 1 and 2) (48). All probes were synthesized by MdBio, Inc. (Taipei, Taiwan).

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FIG. 1.

Layout of oligonucleotide probes on the array (0.75 by 0.9 cm). The positive control probe PC (located at D12 and I1) was designed from a conserved region of the fungal 5.8S rRNA gene. Probe NC (located at E7, I2 to I5, I11, and I12) was a negative control (tracking dye only). Probe M (located at E1 to E12 and at A7 to I7, except E7) was a DIG-labeled bacterial universal primer and was used as a position marker. The group-specific probe TAQAS1 (located at H10) is underlined. The corresponding sequences of all probes are listed in Table 2.

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FIG. 2.

Hybridization results for 77 yeast species. Chips 1 to 45 (A) and 46 to 78 (B) are the hybridization patterns of Candida spp. (44 species) and non-Candida spp. (33 species), respectively. The chips are alphabetically arranged according to the species names. The corresponding probes hybridized on the arrays are indicated in Fig. 1, and the corresponding sequences of the hybridized probes are shown in Table 2. The positive control probe was located at D12 and I1 on each array.

Fabrication of oligonucleotide arrays.The oligonucleotide probes were diluted 1:1 (final concentration, 10 μM) with a tracking dye solution composed of glycerol, dimethyl sulfoxide, sodium EDTA, and bromophenol blue (3). The probes were spotted onto a positively charged nylon membrane (Roche, Mannheim, Germany) by an Ezspot arrayer (model no. SR-A300; EZlife Technology, Taipei, Taiwan) using a 400-μm-diameter solid pin as described previously (48). The array (0.75 by 0.9 cm) contained 108 dots, including 80 dots for the identification of 77 yeast species (16 genera), 2 for positive controls (probe code, PC; final concentration, 5 μM), 7 for negative controls (probe code, NC; tracking dye only), and 19 for position markers (probe code, M; final concentration, 0.4 μM) (Fig. 1). The position markers formed a cross on the array after hybridization and helped to locate the hybridized probes (Fig. 1 and 2). Once all probes had been applied, the membrane was exposed to shortwave UV (Stratalinker 1800; Stratagene, La Jolla, CA) for 30 s.

Hybridization procedures.Except where otherwise indicated, the hybridization procedures were carried out at room temperature (approximately 27°C) with a shaking speed of 60 rpm. Most reagents except buffers were obtained from the DIG nucleic acid detection kit (Roche). The hybridization procedures were the same as those described previously (20), except that the hybridization step was conducted at 50°C for 90 min. Unbound oligonucleotides on the array were removed by three washes (3 min each) in 0.5× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate)-0.1% sodium dodecyl sulfate (SDS). Each array was prehybridized at room temperature for 2 h with hybridization solution (5′ SSC, 1% [wt/vol] blocking reagent, 0.1% N-laurylsarcosine, and 0.02% SDS) in an individual well of a 24-well cell culture plate. The DIG-labeled PCR product amplified from an isolate was denatured at 95°C and immediately cooled on an ice bath. Ten microliters of denatured PCR products were diluted with 0.3 ml of hybridization solution and added to each well. Hybridization was carried out at 50°C for 90 min. After removal of the nonhybridized PCR products, the array was washed four times (5 min each) with 0.25× SSC-0.1% SDS, followed by incubation for 1 h with blocking solution (1% [wt/vol] blocking reagent dissolved in maleic acid buffer [0.1 M maleic acid, 0.15 M NaCl, pH 7.5]). After removal of the blocking solution, 0.3 ml of alkaline phosphatase-conjugated anti-DIG antibodies (diluted 1:2,500) was added to each well and incubated for 1 h. The array was washed three times (each 15 min) with washing solution (0.3% [vol/vol] Tween 20 in maleic acid buffer), followed by washing with detection buffer (0.1 M Tris-HCl, 0.15 M NaCl, pH 9.5) for 5 min. Finally, 0.3 ml of alkaline phosphatase substrates (nitroblue tetrazolium chloride-5-bromo-4-chloro-3-indolylphosphate) was added to each array and incubated at 37°C for 30 to 60 min without shaking. The hybridized array was washed three times with distilled water and air dried. The image of the hybridization pattern was captured and processed by a scanner (PowerLook 3000; UMAX, Taipei, Taiwan).

Definition of sensitivity and specificity.A yeast strain was identified as one of the 77 target yeasts when the probe designed for the species and the positive control probes (Fig. 2) were hybridized. Sensitivity was defined as the number of target strains correctly identified (true positives) divided by the total number of target strains tested (30). Specificity was defined as the number of nontarget strains producing negative hybridization reactions (true negatives) divided by the total number of nontarget strains tested (30).

Analysis of discrepant strains.For strains producing discrepant identification between the methods based on phenotypic characteristics and array hybridization, the D1-D2 region of the large-subunit RNA gene, ITS 1, and ITS 2 were amplified by PCR, sequenced, and then compared with sequences in public databases using the BLAST algorithm for species clarification. The fungus-specific, universal primers ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS2 (5′-GCTGCGTTCTTCATCGATGC-3′) were used to amplify the ITS 1 region, while the primer pair ITS3 (5′-GCATCGATGAAGAACGCAGC-3′) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′) was used to amplify the ITS 2 region (50). The D1-D2 region was amplified by primers NL1 (5′-GCATATCAATAAGCGGAGGAAAAG-3′) and NL4 (5′-GGTCCGTGTTTCAAGACGG-3′) (23). The conditions used for amplification of the ITS 1, ITS 2, and D1-D2 regions were the same as those for amplifying the whole ITS segment as described previously (24). PCR products were purified with a PCR-M cleanup kit (Viogene, Taipei, Taiwan) and sequenced on a model 377 sequencing system (Applied Biosystems, Taipei, Taiwan).

Limit of detection of the array.Two strains (C. albicans BCRC 20512 and Kloeckera apis BCRC 22112) were used to determine the limit of detection of the oligonucleotide array. The DNAs of both strains were serially diluted 10-fold (10 ng/μl to 1 fg/μl) with a carrier DNA (1 ng/μl) extracted from a bacterium (Xanthobacter flavus BCRC 12271) by the boiling method (32). After PCR amplification of the diluted DNA, the amplicon was hybridized to the oligonucleotide array.

Oligonucleotide probe development.Initially, one to five probes were designed for each of the 77 target species. Through a preliminary hybridization test, probes cross-reacting with heterologous species or producing weak hybridization signals with their homologous species were discarded. Finally, 80 probes (Table 2) were selected for the fabrication of the array. Under most conditions, a single probe was used to identify a species (Table 2). Among these probes, a group-specific probe (probe code, TAQAS1) was designed; the probe could hybridize with species of Trichosporon aquatile, Trichosporon asahii, and Trichosporon inkin. However, each of the three Trichosporon species had its own specific probe (Table 2).

Due to the presence of three genotypes among isolates of Candida parapsilosis (27, 42, 46), three probes (probe codes, CP6, CP8, and CP10) were designed to identify this frequently isolated species. Probe CP6 was shared by all three genotypes of C. parapsilosis; however, strains of genotypes II or III were able to hybridize with, in addition to probe CP6, an additional probe (CP8 or CP10). For example, C. parapsilosis BCRC 20515 (genotype I) hybridized only to probe CP6 (Fig. 2A). However C. parapsilosis BCRC 20865 (genotype III) hybridized to probes CP6 and CP10 (Fig. 2A), while clinical isolate C. parapsilosis 770 (genotype II) hybridized to probes CP6 and CP8 (data not shown). It was observed that the hybridization signals of probes CP8 and CP10 were relatively weaker compared to that of probe CP6.

Twelve probes were intentionally designed to incorporate one single base mismatch with their respective complementary target sequences (Table 2) to eliminate weak cross-hybridizations produced by nonhomologous species. These 12 probes were used to identify Candida colliculosa, Candida dattila, Candida globosa, Candida haemulonii, Candida lambica, Candida lipolytica, Candida tropicalis, Candida viswanathii, Candida zeylanoides, Debaryomyces maramus, Lachancea fermentati, and Trichosporon pullulans (Table 2). The 12 modified probes had no cross-hybridization with other species, but still displayed good hybridization signals with their respective target yeasts.

Identification of reference strains by the array.A total of 342 reference strains, including 309 target strains (Table 1) and 33 nontarget strains (Table 3), were tested. Figure 2 shows the hybridization results of reference strains of different target species. All 309 target strains were correctly identified by the oligonucleotide array, producing a test sensitivity of 100%. Of the 33 nontarget reference strains (33 species), only one strain, Trichosporon ovoides CBS 7556, cross-hybridized to the group-specific probe (probe code, TAQAS1) shared by Trichosporon aquatile, Trichosporon asahii, and Trichosporon inkin. The remaining 32 strains did not produce any hybridization signals with probes on the array except the positive control probe. Therefore, the test specificity of the array was 97% (32/33).

Identification of clinical isolates by the array.A total of 110 clinical isolates, including 14 Candida species (96 strains) and 3 non-Candida species (15 strains), were analyzed by the array (Table 1). Of the 110 isolates, 98 were correctly identified and 12 produced discrepant identifications by phenotypic characteristics and array hybridization. Among the 12 isolates, 5 (Candida pelliculosa LMA 892971 and Trichosporon cutaneum LMA 94117, 94256, 931422, and 931440) were not identified by the oligonucleotide array. By sequencing the ITS 1, ITS 2, and D1-D2 regions of the five isolates, our previous study demonstrated that Candida pelliculosa LMA 892971 (Pichia anomala, teleomorph) was a misidentification of Pichia fabianii and that the four isolates of Trichosporon cutaneum were misidentifications of Trichosporon dermatis (24). Both ITS 2 and D1-D2 regions of Candida pelliculosa LMA 892971 had a sequence similarity of 100% with reference sequences (accession numbers AF335967 and AF335971) of Pichia fabianii in GenBank. Likewise, the four discrepant isolates of Trichosporon cutaneum (LMA 94117, 94256, 931422, and 931440) displayed a sequence similarity of 100% with reference sequences (accession number AF143557 in the ITS 1 and ITS 2 regions and accession number AF143555 in the D1-D2 region) of Trichosporon dermatis in GenBank (24). Since Pichia fabianii and Trichosporon dermatis were nontarget species in this study, their PCR products did not hybridize with any probes on the array except the positive control probe.

Of the remaining seven discrepant isolates, Candida dubliniensis RB 1168, Candida guilliermondii RB 1055, Candida inconspicua LMA 90289, Candida inconspicua RB 1226, Candida krusei RB 1237, Candida rugosa RB 1158, and Cryptococcus albidus LMA 935479 were identified as Candida albicans, Candida parapsilosis, Candida krusei, Candida glabrata, Pichia norvegensis, Saccharomyces cerevisiae, and Cryptococcus neoformans, respectively, by the array. The accuracy of identification of the seven discrepant isolates by hybridization was confirmed by sequence analysis of the ITS 1, ITS 2, and D1-D2 regions in our previous study (24). As the 12 discrepant clinical isolates (5 unidentified isolates and 7 misidentified isolates) were proved to be misidentified by phenotypic methods, the test sensitivity of clinical isolates by the array was 100% (105/105). If reference strains and clinical isolates were taken together, the overall test sensitivity of the array was also 100% (414/414).

Limit of detection of the array.Serial 10-fold dilutions of DNA extracted from Candida albicans BCRC 20512 and Kloeckera apis BCRC 22112 were used to determine the limit of detection of the array. The present method was able to detect genomic DNA at a level of 10 pg per assay of both strains (data not shown).