Nucleotide sequence analysis of the 5·8S rDNA and adjacent ITS2 region of <i>Candida albicans</i> and related species

Lott, Timothy J.; Kuykendall, Randall J.; Reiss, Errol

doi:10.1002/yea.320091106

Cited by 98 publications

(70 citation statements)

References 41 publications

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“…The successful amplification of intact cells was probably due to factors associated with the sufficient content of template in the cell suspension and the high numbers of copies of the target region rRNA gene, which in fungi are present in hundreds of copies (41,64). The high copy number can act as a preamplification step, enabling an increase in amplicon yield (46,48).…”

Section: Discussionmentioning

confidence: 99%

Use of a Suspension Array for Rapid Identification of the Varieties and Genotypes of the Cryptococcus neoformans Species Complex

Diaz

Fell

2005

J Clin Microbiol

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Cryptococcus neoformans is an encapsulated fungal pathogen known to cause severe disease in immunocompromised patients. The disease, cryptococcosis, is mostly acquired by inhalation and can result in a chronic meningoencephalitis, which can be fatal. Here, we describe a molecular method to identify the varieties and genotypic groups within the C. neoformans species complex from culture-based assays. The method employs a novel flow cytometer with a dual laser system that allows the simultaneous detection of different target sequences in a multiplex and high-throughput format. The assay uses a liquid suspension hybridization format with specific oligonucleotide probes that are covalently bound to the surface of fluorescent color-coded microspheres. Biotinylated target amplicons, which hybridized to their complementary probe sequences, are quantified by the addition of the conjugate, streptavidin R-phycoerythrin. In this study we developed and validated eight probes derived from sequence analysis of the intergenic spacer region of the rRNA gene region. The assay proved to be specific and sensitive, allowed discrimination of a 1-bp mismatch with no apparent cross-reactivity, and detected 10 1 to 10 3 genome copies. The described protocol, which can be used directly with yeast cells or isolated DNA, can be undertaken in less than 1 h following PCR amplification and permits identification of species in a multiplex format. In addition to a multiplex capability, the assay allows the simultaneous detection of target sequences in a single reaction. The accuracy, speed, flexibility, and sensitivity of this technology are a few of the advantages that will make this assay useful for the diagnosis of human cryptococcal infections and other pathogenic diseases.

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Section: Discussionmentioning

confidence: 99%

Use of a Suspension Array for Rapid Identification of the Varieties and Genotypes of the Cryptococcus neoformans Species Complex

Diaz

Fell

2005

J Clin Microbiol

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“…Among the most important factors that limit the direct application of the all Candida genus-specific probe to blood samples is its design from the highly conserved 5.8S region of rDNA (16,20). Recently, human DNA-derived sequences were detected in blood specimens from healthy individuals by using real-time PCR with a probe targeting conserved regions of bacterial 16S rDNA (17).…”

Section: Discussionmentioning

confidence: 99%

Rapid Detection of Candida albicans in Clinical Blood Samples by Using a TaqMan-Based PCR Assay

et al. 2003

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We describe a rapid and reproducible PCR assay for quantitation of the Candida albicans ribosomal DNA (rDNA) in clinical blood samples based on the TaqMan principle (Applied Biosystems), in which a signal is generated by cleavage of a template-specific probe during amplification. We used two fluorogenic probes based on universal, fungus-specific primers, one for the detection of C. albicans species DNA and one for the detection of all Candida genus DNA. C. albicans blastoconidia mixed with whole blood in a titration experiment yielded a linear PCR signal over a range of 3 orders of magnitude. The TaqMan-based PCR assay for C. albicans exhibited a low limit of detection (5 CFU/ml of blood) and an excellent reproducibility (96 to 99%). While the C. albicans species-specific probe had 100% specificity for C. albicans, all Candida genus-specific probes cross-reacted with other organisms likely to coinfect patients with C. albicans infections. On the basis of these data, we determined the C. albicans loads with a species-specific probe from 122 blood samples from 61 hematology or oncology patients with clinically proven or suspected systemic Candida infections. Eleven positive samples exhibited a wide range of C. albicans loads, extending from 5 to 100,475 CFU/ml of blood. The sensitivity and specificity of the present assay were 100 and 97%, respectively, compared with the results of blood culture. These data indicate that the TaqMan-based PCR assay for quantitation of C. albicans with a species-specific probe provides an attractive alternative for the identification and quantitation of C. albicans rDNA in pure cultures and blood samples.Invasive candidiasis, a common cause of nosocomial infection, is a leading cause of infections among patients receiving bone marrow transplantations or those with leukemia or other cancers and is also associated with a high rate of morbidity and mortality (10,18,22). Given the rapidly fatal course of candidiasis, there is a need for improved methods for early diagnosis and subsequent initiation of antifungal therapy in order to have a significant impact on the death rate (1, 12). One such improvement is the application of rapid and sensitive methods based on PCR that enable the amplification and quantitation of a broad range of fungal pathogens directly from specimens and pure cultures.Two quantitative real-time PCR systems have been described for Candida detection. An assay based on the PCR LightCycler system (Roche Molecular Diagnostics Systems, Indianapolis, Ind.) was applied by Löeffler et al. (15) for quantification of Candida albicans DNA in blood to which known numbers of blastoconidia had been added. Those investigators also used the assay to determine the fungal burdens in a small number of blood samples taken from patients with hematological malignancies. More recently, Guiver et al. (7) and Bowman et al. (3) described the automated detection of fungal DNA by using the TaqMan assay (Applied Biosystems, Foster City, Calif.) with clinical isolates and murine tissues, respect...

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“…All primers and probes were synthesized by ␤-cyanoethyl phosphoramidite chemistry using a 394 or expedite automated DNA synthesizer (PE Applied Biosystems, Foster City, Calif.). ITS3, a universal fungal sequence located in the 5.8S region of the rRNA gene and contained within the region amplified by ITS1 and ITS4 primers (23,41), was biotinylated at the 5Ј end by incorporating dimethyoxytrityl-biotin-carbon-6-phosphoramidite during its synthesis. This biotinylated probe (ITS3-B) was then purified by reverse-phase liquid chromatography.…”

Section: Dna Isolationmentioning

confidence: 99%

Rapid Identification of Dimorphic and Yeast-Like Fungal Pathogens Using Specific DNA Probes

et al. 2001

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Specific oligonucleotide probes were developed to identify medically important fungi that display yeast-like morphology in vivo. Universal fungal primers ITS1 and ITS4, directed to the conserved regions of ribosomal DNA, were used to amplify DNA from Histoplasma capsulatum, Blastomyces dermatitidis, Coccidioides immitis, Paracoccidioides brasiliensis, Penicillium marneffei, Sporothrix schenckii, Cryptococcus neoformans, five Candida species, and Pneumocystis carinii. Specific oligonucleotide probes to identify these fungi, as well as a probe to detect all dimorphic, systemic pathogens, were developed. PCR amplicons were detected colorimetrically in an enzyme immunoassay format. The dimorphic probe hybridized with DNA from H. capsulatum, B. dermatitidis, C. immitis, P. brasiliensis, and P. marneffei but not with DNA from nondimorphic fungi. Specific probes for H. capsulatum, B. dermatitidis, C. immitis, P. brasiliensis, P. marneffei, S. schenckii, C. neoformans, and P. carinii hybridized with homologous but not heterologous DNA. Minor cross-reactivity was observed for the B. dermititidis probe used against C. immitis DNA and for the H. capsulatum probe used against Candida albicans DNA. However, the C. immitis probe did not cross-react with B. dermititidis DNA, nor did the dimorphic probe hybridize with C. albicans DNA. Therefore, these fungi could be differentiated by a process of elimination. In conclusion, probes developed to yeast-like pathogens were found to be highly specific and should prove to be useful in differentiating these organisms in the clinical setting.

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Nucleotide sequence analysis of the 5·8S rDNA and adjacent ITS2 region of Candida albicans and related species

Cited by 98 publications

References 41 publications

Use of a Suspension Array for Rapid Identification of the Varieties and Genotypes of the Cryptococcus neoformans Species Complex

Use of a Suspension Array for Rapid Identification of the Varieties and Genotypes of the Cryptococcus neoformans Species Complex

Rapid Detection of Candida albicans in Clinical Blood Samples by Using a TaqMan-Based PCR Assay

Rapid Identification of Dimorphic and Yeast-Like Fungal Pathogens Using Specific DNA Probes

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