rapid identiﬁcation of candida dubliniensis using a …of allelic differences in the human...

JOURNAL OF CLINICAL MICROBIOLOGY,0095-1137/00/$04.0010

Aug. 2000, p. 2829–2836 Vol. 38, No. 8

Copyright © 2000, American Society for Microbiology. All Rights Reserved.

Rapid Identification of Candida dubliniensis Using aSpecies-Specific Molecular Beacon

STEVEN PARK,1 MAY WONG,2 SALVATORE A. E. MARRAS,1 EMILY W. CROSS,1

TIMOTHY E. KIEHN,2 VISHNU CHATURVEDI,3 SANJAY TYAGI,1 AND DAVID S. PERLIN1*

Public Health Research Institute,1 and Memorial Sloan-Kettering Cancer Center,2 New York, andWadsworth Center, New York State Health Department, Albany,3 New York

Received 6 March 2000/Returned for modification 1 May 2000/Accepted 14 May 2000

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

Candida dubliniensis is a newly recognized opportunisticpathogen that has been linked to oral candidiasis in humanimmunodeficiency virus (HIV)-infected patients (16, 25, 45,47), although it has also been observed in blood isolates frombone marrow transplant patients and oral and vaginal isolatesfrom non-HIV-infected patients (26, 29, 35). C. dubliniensiswas initially difficult to distinguish from other Candida speciesin standard clinical laboratory tests because of its closelyshared phenotypic and genotypic characteristics with C. albi-cans (45, 46). However, more recently, phenotypic character-istics, including carbon assimilation (7, 13, 33, 39), growthtemperature (34), immunofluorescence (3), and DNA-basedmolecular approaches (7–9, 11, 15, 18, 48) have been used todistinguish C. dubliniensis from other Candida species. C. dub-liniensis is largely susceptible to existing antifungal agents (30),although it can rapidly develop in vitro fluconazole resistance(29). A small percentage of isolates with fluconazole resistancehave been reported (29, 30), with some isolates expressingcommon classes of multidrug transporters (28). Ultimately,this propensity may present a problem in the oral cavity, whereother resistant Candida species from HIV-infected patients arefrequently encountered (1, 19, 29, 52). Given the growing rec-ognition of C. dubliniensis as an opportunistic pathogen ofimmunosuppressed patients, a rapid and reliable method forthe identification of this non-C. albicans species is an impor-tant clinical goal for proper disease management.

The internal transcribed spacer 2 (ITS2) is a spacer regionflanked by the 5.8S and 28S rRNA genes and has been used toidentify other clinically important fungi such as Pneumocystis,Aspergillus, and Cryptococcus spp. (11, 20–22, 36, 49). The ITS2region can be amplified with universal fungal primers ITS3 andITS4 specific for conserved sequences in the ends of the 5.8

and 28S rRNA genes (22). Use of the ITS2 region for speciesidentification requires either direct sequence analysis, which ishighly accurate but time-consuming, or detection with se-quence-specific hybridization probes. However, the use of lin-ear probes for detection, whether amplified or not, can poseproblems of sensitivity and false-positive results, depending onthe probe sequence and hybridization conditions (14). Re-cently, molecular beacons were introduced to overcome theselimitations (51).

Molecular beacons are small, single-stranded nucleic acidhairpin probes that brightly fluoresce when they are bound totheir targets (50, 51). They possess a loop-and-stem structurein which the loop contains the complementary target sequenceand the stem forms by the annealing of short complementarynucleotide sequence arms adjacent to the target sequence (Fig.1A). A fluorophore is covalently linked to the end of the stemsequence, and a quencher is covalently linked to the other end.In free solution, molecular beacons do not fluoresce becausethe stem structure keeps the fluorophore close to the quencherand the fluorescence energy is absorbed and released as heat.However, in the presence of target DNA, the loop sequenceanneals to the target, a probe-target hybrid is formed, forcingthe stems that contain the fluorophore and quencher to disas-sociate, and fluorescence occurs. Molecular beacons have asignificant advantage over conventional nucleic acid probesbecause of their fidelity and ability for allele discrimination(24, 50). This property has recently been exploited for thedetection of single-nucleotide base pair changes within therpoB gene of Mycobacterium tuberculosis, which confers resis-tance to the antibiotic rifampin (31, 32), and for the detectionof allelic differences in the human b-chemokine receptor 5(CCR5) gene (17). In addition, molecular beacons have beenused to rapidly detect and quantitate four retroviruses respon-sible for AIDS and T-cell lymphoma/leukemia (53).

In this report, we describe a method for the rapid identifi-cation of C. dubliniensis in which a species-specific molecularbeacon probe that recognizes a 22-nucleotide target region in

* Corresponding author. Mailing address: Public Health ResearchInstitute, 455 First Ave., New York, NY 10016. Phone: (212) 578-0820.Fax: (212) 578-0804. E-mail: [email protected].

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the ITS2 region of this organism is used. The results of appli-cation of this probe were compared with those of more con-ventional molecular biology-based approaches that involverandom amplified polymorphic DNA (RAPD) analysis andrestriction endonuclease analysis (REA).

MATERIALS AND METHODSStrains and growth conditions. Candida reference strains C. albicans ATCC

90028 C. glabrata ATCC 90030, and C. krusei ATCC 6258, were obtained fromAmerican Type Culture Collection (Manassas, Va.). C. dubliniensis referencestrain NCPF3949 was obtained from the National Collection of Yeast Cultures(Norwich, England). Clinical isolates of C. albicans and C. dubliniensis (isolatesM1-23 and CST 23, respectively) were obtained from the Microbiology Labora-

tory at Memorial Sloan-Kettering Cancer Center. The isolates were obtainedfrom 21 patients over a period of 2 months (6 July 1998 to 13 September 1998)and were not epidemiologically related. Two strains, strains M1 (ATCC 18804)and M3, were laboratory test strains included in the panel of test strains. Theyeasts were presumably identified by tests for detection of the formation of germtubes at 37°C in horse serum (Life Technologies, Grand Island, N.Y.), produc-tion of chlamydospores on cornmeal agar with polysorbate 80 (Becton DickinsonMicrobiology Systems, Cockeysville, Md.), substrate assimilation with the API20C AUX and ID 32C systems (bioMerieux Inc., Hazelwood, Mo.), colorimetricgrowth on CHROMagar Candida plates (DRG International, Mountainside,N.J.), and growth at 45°C. Growth at 45°C was assessed by removing a singlecolony and streaking it over the surface of a Sabouraud dextrose agar plate(Becton Dickinson), which was incubated at 45°C for 48 h. Colony formation onthe last three quadrants of the plate was considered good growth, while growth

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 probesare 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 rigiddouble helix, a conformational reorganization occurs that separates the quencher from the fluorophore, restoring fluorescence. The figure is adapted from Tyagi andKramer (51). (B) Nucleotide sequence of the Candida species-specific molecular beacons. The 22-nucleotide target sequence is complementary to the ITS2 region ofeach Candida species. The fluorophore tetrachloro-6-fluorescein (TET) was attached to the sulfhydryl group on the 59 arm sequence and 4-(49-dimethylaminophe-nylazo)benzoic acid (DABCYL), a quencher, was attached to an amino group on the 39 arm sequence to form the stem region of the molecular beacon.

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on the first quadrant was considered poor growth (13, 30). All strains weremaintained on Sabouraud dextrose agar (4% [wt/vol] dextrose, 1% [wt/vol]peptone, 1.5% [wt/vol] agar [pH 5.6]) and were grown with shaking (;250 rpm)at 30°C in YPD medium (1% [wt/vol] yeast extract, 2% [wt/vol] peptone, 2%[wt/vol] dextrose [pH 5.7]).

RAPD analysis of genomic DNA. Yeast cells were incubated at 37°C for 1 h ina suspension that contained 1 M sorbitol, 0.1 M EDTA, 0.1% (wt/vol) Zymol-yase-100T (Seikaguku Corp., Tokyo, Japan), and 1% (vol/vol) 2-mercaptoetha-nol and that was adjusted to pH 7.5. Chromosomal DNA was purified with theWizard genomic DNA purification kit (Promega, Madison, Wis.). Standard PCRamplifications were performed in a 50-ml reaction mixture that consisted of 33.5ml of nuclease-free water (Promega), 5 ml of 103 Buffer A (Promega), 3 ml of 25mM MgCl2, 4 ml of 2.5 mM deoxynucleoside triphosphates (PE Applied Biosys-tems, Foster City, Calif.), 2 ml of primer 1 (25 mM; 59-AACGCGCAAC-39), 2 mlof primer 2 (25 mM; 59-GAGGGTGGNGGNTCT-39) (IDTDNA, Coralville,Iowa), and 2 ml of chromosomal DNA (approximately 100 ng). The reaction wasinitiated by the addition of 0.5 ml of 5 U of Taq polymerase (Promega) per ml.PCR was performed in a PTC-150 Minicycler (MJ Research, Waltham, Mass.)with 45 cycles of a three-step program that consisted of 94°C for 1 min (step 1),37°C for 1 min (step 2), and 72°C for 3 min (step 3). The PCR products (4 ml)were separated on 20% polyacrylamide gels in an X-Cell gel apparatus (Novex,Carlsbad, Calif.) for ;3 h at 175 V.

Statistical analysis. Similarity coefficients based on the DNA fingerprintingpatterns of prominent bands of #1,000 bp among all isolates were calculated asthe ratio of matches over the total number of bands scored. Similarities werecalculated as the arithmetic mean of all pairwise distances between strain fin-gerprint patterns. Student’s t test was used to compare genetic similarities be-tween different groups of isolates. Banding patterns and similarity coefficientswere determined with the Molecular Analyst/Fingerprinting Plus v.1.12 (Bio-Rad Laboratories, Hercules, Calif.) and the statistical software GB-STAT 6.5(Scolari, London, England). P values of ,0.05 were considered significant.

ITS2 amplification and REA. PCR amplification of the ITS2 region wasperformed with fungus-specific universal primers ITS3 (59-GCATCGATGAAGAACGCAGC-39) and ITS4 (59-TCCTCCGCTTATTGATATGC-39) to amplifya conserved portion of the 5.8S ribosomal DNA (rDNA) region, the adjacentITS2 region, and a small portion of the 28S rDNA region, yielding products of0.338 kb for C. albicans and 0.343 for C. dubliniensis. Full DNA sequence analysisof the PCR products obtained with universal fungal primers ITS3 and ITS4specific for rDNA genes was used to confirm the species identification, as de-scribed previously (11). The reaction mixture (total volume, 100 ml) consisted of2 ml of genomic DNA (;200 ng), 1 ml of 25 mM dNTP (Promega), 1 ml of 5 Uof Amplitaq Gold Taq polymerase (PE Applied Biosystems) per ml, 10 ml of 103buffer (100 mM Tris-HCl [pH 8.3], 500 mM KCl, 15 mM MgCl2, 0.01% [wt/vol]gelatin), 1 ml of universal fungal primers ITS3 and ITS4 (25 mM) (IDTDNA)(14), and 84 ml of nuclease-free water (Promega). The PCR mixture was sub-jected to PCR with the following cycling conditions: 95°C for 10 min, followed by40 cycles of 95°C for 30 s, 58°C for 1 min, and 72°C for 1 min and a final step of72°C for 5 min. PCR-amplified products were purified with the Qiaquick DNAPurification Kit (Qiagen, Valencia, Calif.). The PCR amplification products(;500 ng) were digested with 1 ml of restriction enzymes NspBII (C. albicansITS2 specific) (AP Biotech, Piscataway, N.J.) and BsmAI (C. dubliniensis ITS2

specific) (New England Biolabs, Beverly, Mass.) for 1 h and were analyzed by gelelectrophoresis in a 1.5% agarose gel.

Molecular beacon design and analysis. Molecular beacons specific for theITS2 region of C. albicans and C. dubliniensis were designed on the basis ofpublished probe sequences (11) that were independently confirmed by DNAsequence analysis at the New York University DNA sequencing facility. Targetsequence selection, beacon design, and synthesis were optimized by standardprotocols available on the Public Health Research Institute’s website for molec-ular beacons (http//www.molecular-beacons.com), as described by Tyagi andcolleagues (50, 51). Each molecular beacon possessed a 6-nucleotide arm se-quence and a 22-nucleotide probe target recognition sequence, as follows: 59-GCTAAGGCGGTCTCTGGCGTCG (C. dubliniensis) and 59-TAGGTCTAACCAAAACATTGC (C. albicans). The arm sequences 59-GCGAGG and 39-CCTCGC were designed to form a stable stem hybrid at the annealing temperatureof the PCR to ensure that nonhybridized probes remained in a hairpin confor-mation (no fluorescence). The fluorophore tetrachloro-6-carboxyfluorescein andthe quencher 4-(49-dimethylaminophenylazo)benzoic acid (Molecular ProbesInc., Eugene, Oreg.) were covalently attached to the 59 and 39 ends of the armsequences, respectively. Real-time PCR amplification was performed with 2 ml ofspecies-specific molecular beacons (100 ng) in a 50-ml reaction volume thatcontained 2 ml of genomic DNA (200 ng), 0.5 ml of 25 mM nucleotide mix(Promega), 0.5 ml of 5 U of Amplitaq Gold Taq polymerase (PE AppliedBiosystems) per ml, 5 ml of 103 buffer (100 mM Tris-HCl [pH 8.3], 500 mM KCl,15 mM MgCl2, 0.01% [wt/vol] gelatin), 5 ml of 25 mM MgCl2, 1 ml of universalfungal primers ITS3 and ITS4 (1 mg/ml), and 33 ml of nuclease-free water(Promega). The PCR mixture was subjected to PCR with the following cyclingconditions: 95°C for 10 min, followed by 40 cycles of 95°C for 30 s, 58°C for 1 min,and 72°C for 1 min in a Prism 7700 96-well spectrofluorometric thermal cycler(PE Applied Biosystems).

RESULTSRAPD analysis. A collection of 23 Candida isolates that

mostly contained C. albicans and a smaller number of C. dub-liniensis strains was assembled by the Microbiology Laboratoryat Memorial Sloan-Kettering Cancer Center from stock cul-tures. The collection was provided for a blind evaluation of C.dubliniensis. Initially, all 23 strains were subjected to randomprimed RAPD genotyping analysis to assess the relativegenomic relatedness of the strains. The RAPD fragments forall 23 isolates displayed a profile of more than 15 prominentbands, with sizes ranging from 0.1 to 1 kb (Fig. 2), with nearlyidentical profiles produced in three separate trials. Two dis-tinct banding profiles were identified in which band similaritiesof greater than 0.8 (band similarity coefficient, 0 to 3 banddifferences) were found when the band profiles for commonstrains in the set were compared (P , 0.05), but similarities of

FIG. 2. RAPD analysis of 23 Candida isolates. Genomic DNA was extracted and purified from each isolate, and PCR amplification was performed with randomprimers, 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). p, suspected C. dubliniensis strain.

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less than 0.5 were found when the band profiles betweenstrains of the two sets were compared (P , 0.05). The majorgroup included strains M1, M2, M4, M6 to M9, M12 to M14,M17, M18, and M20 to M23, whose patterns matched that ofa reference C. albicans strain, while the minor group consistedof M3, M5, M10, M11, M15, M16, and M19, whose patternsmatched the pattern observed from a C. dubliniensis referencestrain (not shown).

REA. PCR amplification of the ITS2 region with universalfungal primers ITS3 and ITS4 (22) was used to generate;0.3-kb fragments from all 23 isolates. The fragments wereanalyzed with restriction enzyme BsmAI, which has a specificsequence recognition sequence in this region from C. dublini-ensis. Figure 3A shows that 7 of the 23 amplified products werecut by this restriction enzyme, yielding fragments of 0.25 and0.9 kb. These restricted amplicons were derived from the samesubset of seven strains identified by RAPD analysis (Fig. 2).The remaining PCR-amplified products could be cut with re-striction enzyme NspBII, which specifically recognizes a site inthe ITS2 region from C. albicans to yield fragments of ;0.18and 0.16 kb (Fig. 3B).

Specificity and sensitivity of molecular beacon assay. Spe-cies-specific molecular beacons were designed to target se-quences within the ITS2 regions of C. albicans and C. dublini-ensis (Fig. 1B). Real-time PCR was performed with each of themolecular beacons with DNA from reference strains of C.albicans, C. dubliniensis, C. glabrata, and C. krusei. Fluores-cence was detected for each of the molecular beacons only inthe prescence of its proper DNA target (Fig. 4). There was noeffect of excess nontarget amplicons on the signal of the mo-lecular beacon. The sensitivity of the C. dubliniensis-specificmolecular beacon was evaluated by serially diluting genomicDNA 105-fold from a starting amount of 100 ng. The molecularbeacon was able to detect the target when ;100 pg of theinitial genomic DNA was present (Fig. 5). This result furtherillustrates that molecular beacons have the ability to quantifytarget DNA in a real-time PCR assay (6).

Detection of C. dubliniensis. The molecular beacon specificfor C. dubliniensis was used to probe all 23 Candida isolates,along with reference strains of C. albicans, C. dubliniensis, C.glabrata, and C. krusei. Figure 6 shows that cycle-dependent

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).p, C. dubliniensis strains.

FIG. 4. Selectivities of species-specific molecular beacons for reference Can-dida strains. In this experiment, species-specific molecular beacons, as indicatedin the panels, were used to probe individual DNAs from the four Candidaspecies. Real-time PCR amplification of the ITS2 regions of four referenceCandida strains was performed with 100 ng of beacons and ;200 ng of genomicDNA for 40 cycles in a Prism 7700 96-well spectrofluorometric thermal cycler(PE Applied Biosystems).



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amplification of the signal from the C. dubliniensis-specificmolecular beacon was obtained only for the seven suspected C.dubliniensis strains, strains M3, M5, M10, M11, M15, M16, andM19. All 23 isolates were also probed with the C. albicans-specific molecular beacon, and a fluorescence signal was ob-tained only for the 16 isolates which the C. dubliniensis probedid not detect (data not shown). Table 1 summarizes the re-sults obtained by the three different DNA-based species iden-tification methods (analysis with molecular beacons, RAPDanalysis, and REA) and demonstrates that the results obtainedby analysis with molecular beacons showed a 100% correlationwith the results obtained by the other approaches.

DISCUSSION

The increasing prevalence of fungal infections caused bynon-C. albicans species that display clinical resistance or re-duced susceptibility to common azole-based antifungal drugshas created a need to rapidly differentiate between Candidaspecies early in infection to assist in proper therapeutic man-agement. Conventional morphology and carbon assimilationtests require several days or more for identification (54) and

may misidentify some species. C. dubliniensis is an example ofsuch a misidentified organism that is now readily turning up inthe stock culture collections of C. albicans from numerousclinical laboratories (45, 47). In the last few years, several newmethods have been developed to distinguish the phenotypicproperties of this organism from those of other Candida spe-cies, including chlamydospore formation (44), carbon assimi-lation (39), temperature-dependent growth (34), colony col-oration on CHROMagar (16), and immunofluorescence (3), ashave commercially available systems (API20C AUX, RapIDYeast Plus, VITEK YBC, and VITEK 2 ID-YST) (13, 33).Despite advances in phenotypic detection, species identifica-tion by these routes is relatively slow. Genotyping with hybrid-ization probes such as the C. albicans mid-repeat-sequenceprobes 27A and Ca3 (2, 8, 40) and the C. dubliniensis-specificcomplex probe Cd25-1 (15) can be used to provide species andsubspecies information. These techniques, however, are typi-cally too labor intensive for a clinical laboratory setting and aremore suited for epidemiological investigations.

Nucleic acid amplification of species-specific target se-quences by PCR or ligase chain reaction, or with RNA-depen-dent Qb replicase provides the most rapid alternative to stan-dard testing (14) since few fungal cells are required. This highlevel of sensitivity is particularly attractive for the early detec-tion of fungemias, which are difficult to detect by conventionalprocedures like blood culture or those that depend upon acompetent immune system (27). The detection of 1 to 2 CFUper ml of blood can generally be achieved, provided the targetgene is present in numerous (.100) copies (10). While thesensitivity associated with PCR amplification can be maxi-mized to detect low levels of pathogenic organisms, PCR as-says applied in clinical diagnostics have drawbacks due topotential contamination with environmental organisms, coam-plification of human DNA, false priming, and/or the necessityfor additional hybridization steps (43). Furthermore, PCRproducts are rarely validated other than by size, and detectionschemes that involve linear probes may have limited fidelityand limited overall sensitivity.

Recently, a promising method for rapid identification ofCandida spp., including C. dubliniensis, with sequence-specificdigoxigenin-labeled hybridization probes in a PCR-enzyme im-munoassay format was described (11, 12, 41, 42). This methodis robust but requires a posthybridization step to remove thecontaminating unhybridized probe that can lead to false-posi-tive results. Typically, any time that linear probes are used fordetection, there is a risk that false annealing may occur. Probe-target hybridization is highly temperature dependent, and de-pending on the nucleotide composition of the probe, randomannealing can pose a problem, especially when one is dealingwith sequences with high G1C contents, since the temperatureprofile for annealing is shifted downward (5, 38).

To overcome a number of the inherent problems associatedwith nucleic acid amplification and detection, we have appliedmolecular beacon technology to detect PCR-amplified targetsequences in the ITS2 regions of C. dubliniensis and C. albi-cans. Using this technology, we readily detected all seven C.dubliniensis strains from a panel of 23 unknown Candida iso-lates (Fig. 6). The molecular beacon analysis data were inde-pendently validated by less robust molecular approaches in-volving RAPD analysis-based genotyping and REA of targetrecognition sequences in the ITS2 regions of C. dubliniensisand C. albicans. The ITS2 region of fungi is a suitable target forspecies identification because of its variability among species(11, 21, 22). In addition, only a single primer set, ITS3 andITS4, is required to amplify this region from fungi because ofthe highly conserved domains of the 5.8S and 28S rDNA genes

FIG. 5. Real-time PCR amplification for determination of relative sensitivitywas performed for 40 cycles with C. dubliniensis genomic DNA (;100 ng) thatwas serially diluted 105-fold, as indicated, in the presence of a fixed amount (100ng) of the molecular beacon. Relative fluorescence was monitored in a PEApplied Biosystems 7700 Prism 96-well spectrofluorometric thermal cycler.

FIG. 6. Real-time detection of C. dubliniensis from a blinded panel of 23Candida isolates was accomplished by PCR amplification of the ITS2 region inthe presence of a molecular beacon specific for C. dubliniensis. Relative fluores-cence was monitored in a PE Applied Biosystems 7700 Prism 96-well spectroflu-orometric thermal cycler. Control DNAs from reference strains of C. albicans, C.krusei, and C. glabrata were also evaluated.



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that flank it. The targeting of high-copy-number RNA genes isalso an advantage because it increases the sensitivity of detec-tion of amplified DNA without the need for nested PCR tech-niques (12). The total time required for sample preparationand analysis of target sequences is typically less than 6 h start-ing from pregrown colonies of ;1 mm.

Molecular beacons have a distinct advantage over linearfluorescent probes because of their stem-and-loop structures(4, 6). In this conformation, nonhybridized beacons remaindark because the fluorophore is maintained close to thequencher. When bound to its target, the beacon opens andfluoresces brightly. There is no requirement for isolation ofprobe-target hybrids to measure fluorescence, which elimi-nates any possible posthybridization contamination. Thus, mo-lecular beacons can be added prior to amplification and real-time fluorescence can be measured in a single-step assay. Real-time monitoring of the PCR amplification allows a quantitativemeasure of the starting template based on the fluorescencesignal as a function of PCR cycle (23), a feature unavailable toendpoint assays such as PCR-enzyme immunoassay.

The use of hairpin-shaped molecular beacons in PCR assaysprovides several advantages over the use of linear probes be-cause the stem-loop structure imparts an increased ability todiscriminate single-base-pair mismatches compared to thatfrom the use of linear probes such as TaqMan (4, 50). Thehairpin shape makes mismatched probe-target hybrids lessthermally stable than hybrids between corresponding linearprobes. Thus, molecular beacons are more useful for allelediscrimination, which adds to their fidelity in monitoring of

authentic products in PCR amplifications. To successfullymonitor a PCR assay, the molecular beacon should be de-signed to hybridize to its target at the PCR annealing temper-ature, whereas the free molecular beacon should stay closedand nonfluorescent at higher temperatures. A probe sequenceshould be chosen such that the molecular beacon dissociatesfrom its target at a temperature 7 to 10°C higher than theannealing temperature of the PCR amplification (6) (see thePublic Health Research Institute website for molecular bea-cons support). Finally, unlike linear hydrolysis probes, thequenching of molecular beacons has been shown to occurthrough a collisional mechanism that involves a direct transferof energy from the fluorophore to the quencher (4). Thisproperty enables a common quencher molecule to be used withbeacons, which increases the number of possible fluorophoresthat can be used as reporters. This is especially important formultiplexing of molecular beacons in a PCR assay, which havebeen reported for the detection of viruses (53) and drug resis-tance-conferring mutations in M. tuberculosis (31, 37).

In summary, our results indicate that species-specific molec-ular beacons are a highly reliable tool for molecular biology-based identification of C. dubliniensis that overcomes many ofthe inherent problems associated with nucleic acid amplifica-tion and detection. The results in this study extend previousapplications of molecular beacons and demonstrate that theyprovide a rapid and highly reliable method for detection ofsequence-specific amplified DNA. Molecular beacons are idealtools for clinical diagnostics because of their stability, highsignal-noise property, real-time monitoring capability, and

TABLE 1. DNA-based species identification

Strain Isolation date(mo/day/yr)

Isolatesource

Candida detection by:

RAPD analysis REA Analysis with molecularbeacons

C. albicans C. dubliniensis C. albicans C. dubliniensis C. albicans C. dubliniensis

Test strainsM1 a a ● ● ●M2 09/02/98 Mouth ● ● ●M3 b b ● ● ●M4 09/13/98 Mouth ● ● ●M5 09/08/98 Sputum ● ● ●M6 07/06/98 Facial wound ● ● ●M7 07/08/98 Throat ● ● ●M8 07/13/98 Mouth ● ● ●M9 09/02/98 Sputum ● ● ●M10 07/23/98 Tongue ● ● ●M11 08/02/98 Blood ● ● ●M12 07/24/98 Mouth ● ● ●M13 08/08/98 Bronchial wash ● ● ●M14 08/10/98 Sputum ● ● ●M15 08/12/98 Bronchial wash ● ● ●M16 08/14/98 Tracheal aspirate ●M17 08/25/98 Pleural fluid ● ● ●M18 08/11/98 Sputum ● ● ●M19 08/26/98 Stool ●M20 08/14/98 Mouth ● ● ●M21 08/19/98 Pleural fluid ● ● ●M22 08/15/98 Sputum ● ● ●M23 08/18/98 Nasopharnyx ● ● ●

Control strainsATCC 90028 ● ● ●NCPF 3949 ● ● ●

a ATCC 18804.b New York State Health Department proficiency test strain.



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high-throughput potential. We are developing a panel of mo-lecular beacons for molecular identification of numerous fungi.Furthermore, since molecular beacons can be used with severaldifferent fluorophores, they are amenable for use for detectionof multiplex sequences in a single reaction tube (53).

ACKNOWLEDGMENTS

This work was supported by a sole-source contract from the NewState Department of Health, Albany, N.Y. (to D.S.P.).

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identification of up to three Candida species in a single reaction tube by a 59exonuclease assay using fluorescent DNA probes. J. Clin. Microbiol. 37:165–170.

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