Rapid identification of Candida dubliniensis using a species-specific molecular beacon

Abstract

Candida dubliniensis is an opportunistic fungal pathogen that has been linked to oral candidiasis in AIDS patients, although it has recently been isolated from other body sites. DNA sequence analysis of the internal transcribed spacer 2 (ITS2) region of rRNA genes from reference Candida strains was used to develop molecular beacon probes for rapid, high-fidelity identification of C. dubliniensis as well as C. albicans. Molecular beacons are small nucleic acid hairpin probes that brightly fluoresce when they are bound to their targets and have a significant advantage over conventional nucleic acid probes because they exhibit a higher degree of specificity with better signal-to-noise ratios. When applied to an unknown collection of 23 strains that largely contained C. albicans and a smaller amount of C. dubliniensis, the species-specific probes were 100% accurate in identifying both species following PCR amplification of the ITS2 region. The results obtained with the molecular beacons were independently verified by random amplified polymorphic DNA analysis-based genotyping and by restriction enzyme analysis with enzymes BsmAI and NspBII, which cleave recognition sequences within the ITS2 regions of C. dubliniensis and C. albicans, respectively. Molecular beacons are promising new probes for the rapid detection of Candida species.

FIG. 1

(A) Molecular beacon consists of a stem-loop structure with a fluorophore and a quencher bound to the ends of the probe. In free solution, these probes are nonfluorescent because the stem hybrid keeps the fluorophore close to the quencher. When the probe sequence in the loop hybridizes to its target, forming a rigid double helix, a conformational reorganization occurs that separates the quencher from the fluorophore, restoring fluorescence. The figure is adapted from Tyagi and Kramer (51). (B) Nucleotide sequence of the Candida species-specific molecular beacons. The 22-nucleotide target sequence is complementary to the ITS2 region of each Candida species. The fluorophore tetrachloro-6-fluorescein (TET) was attached to the sulfhydryl group on the 5′ arm sequence and 4-(4′-dimethylaminophenylazo)benzoic acid (DABCYL), a quencher, was attached to an amino group on the 3′ arm sequence to form the stem region of the molecular beacon.

FIG. 2

RAPD analysis of 23 Candida isolates. Genomic DNA was extracted and purified from each isolate, and PCR amplification was performed with random primers, as described in Materials and Methods. The PCR-amplified products were run on a 20% Tris-borate-EDTA–polyacrylamide gel and stained with GelStar (FMC Bioproducts). ∗, suspected C. dubliniensis strain.

FIG. 3

The ITS2 region was PCR amplified with universal fungal primers ITS3 and ITS4, and the products were digested with restriction enzymes BsmAI (C. dubliniensis specific) (A) and NspBII (C. albicans specific) (B). The restriction fragments were run on a 1.2% agarose gel and stained with GelStar (FMC Bioproducts). ∗, C. dubliniensis strains.

FIG. 4

Selectivities of species-specific molecular beacons for reference Candida strains. In this experiment, species-specific molecular beacons, as indicated in the panels, were used to probe individual DNAs from the four Candida species. Real-time PCR amplification of the ITS2 regions of four reference Candida strains was performed with 100 ng of beacons and ∼200 ng of genomic DNA for 40 cycles in a Prism 7700 96-well spectrofluorometric thermal cycler (PE Applied Biosystems).

FIG. 5

Real-time PCR amplification for determination of relative sensitivity was performed for 40 cycles with C. dubliniensis genomic DNA (∼100 ng) that was serially diluted 10⁵-fold, as indicated, in the presence of a fixed amount (100 ng) of the molecular beacon. Relative fluorescence was monitored in a PE Applied Biosystems 7700 Prism 96-well spectrofluorometric thermal cycler.

FIG. 6

Real-time detection of C. dubliniensis from a blinded panel of 23 Candida isolates was accomplished by PCR amplification of the ITS2 region in the presence of a molecular beacon specific for C. dubliniensis. Relative fluorescence was monitored in a PE Applied Biosystems 7700 Prism 96-well spectrofluorometric thermal cycler. Control DNAs from reference strains of C. albicans, C. krusei, and C. glabrata were also evaluated.