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

Abstract

The incidence of yeast infections has increased in the recent decades, with Candida albicans still being the most common cause of infections. However, infections caused by less common yeasts have been widely reported in recent years. Based on the internal transcribed spacer 1 (ITS 1) and ITS 2 sequences of the rRNA genes, an oligonucleotide array was developed to identify 77 species of clinically relevant yeasts belonging to 16 genera. The ITS regions were amplified by PCR with a pair of fungus-specific primers, followed by hybridization of the digoxigenin-labeled PCR product to a panel of oligonucleotide probes immobilized on a nylon membrane for species identification. A collection of 452 yeast strains (419 target and 33 nontarget strains) was tested, and a sensitivity of 100% and a specificity of 97% were obtained by the array. The detection limit of the array was 10 pg of yeast genomic DNA per assay. In conclusion, yeast identification by the present method is highly reliable and can be used as an alternative to the conventional identification methods. The whole procedure can be finished within 24 h, starting from isolated colonies.

Development of an Oligonucleotide Array for Direct Detection of Fungi in Sputum Samples from Patients with Cystic Fibrosis

“…In addition, two probes (Taeme4 and Taeme6) were used to identify Talaromyces emersonii due to intraspecies sequence divergence in the ITS regions (Table 2). Similarly, the C. parapsilosis complex was identified by a combination of three probes (33). One base mismatch was intentionally introduced into the probe used to identify C. tropicalis to eliminate weak crosshybridization caused by a few nontarget Candida species, but FIG.…”

“…C. krusei was not included in the list of the target species (Table 2) since the yeast was rarely found in sputum samples from CF patients (22). In our previous study, the probe (code CGL1, Table 2) used to identify C. glabrata was found to have no cross-hybridization with C. krusei (33). There- 3540 (patient 24), A. flavus, A. fumigatus, A. terreus, C. glabrata, and C. parapsilosis.…”

“…The designed probes were checked for melting temperature, internal repeat, secondary structure, and GC content by using the software Vector NTI Advance 9 (Invitrogen, Carlsbad, CA) and checked for potential cross-reactivities with other fungal species by using the BLASTN program (http: //blast.ncbi.nlm.nih.gov/). A total of 24 probes (final concentration, 10 M), including some previously described probes (30,33), were used to fabricate the arrays by using nylon membranes ( Table 2). In addition, a positive-control probe was designed from a conserved region in the 5.8S rRNA gene.…”

“…In addition, a total of 141 nontarget strains corresponding to 135 species (see Table S1 in the supplemental material) were used to test the specificity of the array. Fungal DNA was extracted as described previously (30,33). Clinical specimens and DNA extraction.…”

“…Oligonucleotide probes were synthesized by MDBio Inc. (Taipei, Taiwan). Arrays were prepared with an automatic arrayer (Ezspot, Taipei, Taiwan) by using a 400-m-diameter solid pin as described previously (33). The array (0.5 by 0.5 cm) contained 36 dots (6 ϫ 6 dots); among them, 23 dots were used for identification of the fungal species listed in Table 2, one dot was a positive control (code PC), two dots were negative controls (code NC, tracking dye only), and 10 dots were position markers (code M, a digoxigenin-labeled ITS4 primer).…”