Specific identification of <i>Candida albicans</i> by hybridization with oligonucleotides derived from ribosomal DNA internal spacers

Botelho, Ana; Planta, Rudi J.

doi:10.1002/yea.320100603

Cited by 15 publications

(12 citation statements)

References 28 publications

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“…Moreover, between the highly conserved rDNA subunits are the internally transcribed spacers, which contain sequences unique to each Candida species, and thus the use of primers corresponding to these regions facilitates species identification. Although sev- eral PCR assays based on amplification of the rDNA have been recently reported, species identification usually involved further manipulation of the amplified products, i.e., restriction enzyme digestion and analysis (24,37), use of radioactive and enzyme-labeled probes (2,10,15,20,23,32,33,35), and DNA sequencing (25). Many of these procedures required prolonged hybridization times and the use of hazardous radioactive materials.…”

Section: Discussionmentioning

confidence: 99%

Seminested PCR for Diagnosis of Candidemia: Comparison with Culture, Antigen Detection, and Biochemical Methods for Species Identification

et al. 2002

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The rapid detection and identification of Candida species in clinical laboratories are extremely important for the management of patients with hematogenous candidiasis. The presently available culture and biochemical methods for detection and species identification of Candida are time-consuming and lack the required sensitivity and specificity. In this study, we have established a seminested PCR (snPCR) using universal and species-specific primers for detection of Candida species in serum specimens. The universal outer primers amplified the 3 end of 5.8S ribosomal DNA (rDNA) and the 5 end of 28S rDNA, including the internally transcribed spacer 2 (ITS2), generating 350-to 410-bp fragments from the four commonly encountered Candida species, viz., C. albicans, C. tropicalis, C. glabrata, and C. parapsilosis. The species-specific primers, complementary to unique sequences within the ITS2 of each test species, amplified species-specific DNA in the reamplification step of the snPCR. The sensitivity of Candida detection by snPCR in spiked serum specimens was close to 1 organism/ml. Evaluation of snPCR for specific identification of Candida species with 76 clinical Candida isolates showed 99% concordant results with the Vitek and/or ID32C yeast identification system. Further evaluation of snPCR for detection of Candida species in sera from culture-proven (n ‫؍‬ 12), suspected (n ‫؍‬ 16), and superficially colonized (n ‫؍‬ 10) patients and healthy subjects (n ‫؍‬ 12) showed that snPCR results were consistently negative with sera from healthy individuals and colonized patients. In culture-proven candidemia patients, the snPCR results were in full agreement with blood culture results with respect to both positivity and species identity. In addition, snPCR detected candidemia due to two Candida species in five patients, compared to three by blood culture. In the category of suspected candidemia with negative blood cultures for Candida, nine patients (56%) were positive by snPCR; two of them had dual infection with C. albicans and either C. tropicalis or C. glabrata. In conclusion, the snPCR developed in this study is specific and more sensitive than culture for the detection of Candida species in serum specimens. Moreover, the improved detection of cases of candidemia caused by more than one Candida species is an additional advantage.

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Section: Discussionmentioning

confidence: 99%

Seminested PCR for Diagnosis of Candidemia: Comparison with Culture, Antigen Detection, and Biochemical Methods for Species Identification

et al. 2002

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“…Different molecular techniques have been used to identify and characterize Candida species. However, except in the case of the clinical species (Botelho & Planta, 1994;Iwaguchi et al, 1990;Jordan, 1994), a standard method of classification has not been developed. In this study, we have analysed by RFLP of the 5.8s-ITS region 56 strains belonging to 30 species.…”

Section: Teleomorphic and Anamorphic Ascomycetous Yeastsmentioning

confidence: 99%

Identification of yeasts by RFLP analysis of the 5.8S rRNA gene and the two ribosomal internal transcribed spacers

Esteve-Zarzoso

Belloch

Uruburu

et al. 1999

International Journal of Systematic and Evolutionary Microbiology

817

588

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The identification and classification of yeasts have traditionally been based on morphological, physiological and biochemical traits. Various kits have been developed as rapid systems for yeast identification, but mostly for clinical diagnosis. In recent years, different molecular biology techniques have been developed for yeast identification, but there is no available database to identify a large number of species. In the present study, the restriction patterns generated from the region spanning the internal transcribed spacers (ITS1 and ITS2) and the 5.8s rRNA gene were used to identify a total of 132 yeast species belonging to 25 different genera, including teleomorphic and anamorphic ascomycetous and basidiomycetous yeasts. In many cases, the size of the PCR products and the restriction patterns obtained with endonucleases Cfol. Haelll and Hinfl yielded a unique profile for each species. Accordingly, the use of this molecular approach is proposed as a new rapid and easy method of routine yeast identification.

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“…This has been shown to be effective for C. albicans identification and would be equally applicable to non-albicans spp. [55]. A disadvantage of the technique for routine use is that the sequence of the internal spacer regions determined before a species-specific probe developed.…”

Section: Molecular Identification Of Candida Spp; Use Of Molecular Tmentioning

confidence: 99%

Molecular genetic approaches to identification, epidemiology and taxonomy of non-albicans Candida species

Sullivan

Henman²,

Moran³

et al. 1996

Journal of Medical Microbiology

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The reported incidence of fungal infections associated with non-albicans species from the Candida genus is increasing. Most of these infections occur in immunocompromised patients, particularly those infected with HW. The role of molecular genetic techniques alongside the existing techniques for the identification and typing of these organisms is discussed. Species-specific genomic DNA fragments cloned from C. tropicalis and C. krusei have been developed for identification and strain typing. Analysis of tRNA profiles has been shown to be effective for the identification of C. glabrats, C. guilliermondii, C. parapsilosis and C. tropicalis. A PCR method employing primers complimentary to large ribosomal subunit genes and the lanosterol-a-demethylase gene has been applied for several species, including C. glabrata, C. krusei and C. tropicalis.Strain typing by comparison of genomic DNA fingerprints has been demonstrated for C. tropicalis and C. krusei following hybridisation analysis with species-specific probes. Synthetic oligonucleotide probes-which do not have to be species-specific and which can detect minor polymorphisms-have also been used for strain typing of isolates of several non-albicans species. Random amplification of polymorphic DNA (RAPD) has also been used for analysis of C. glabrata, C. lusitaniae and C. tropicalis isolates. The potential for the application of these and other techniques to Candida spp. taxonomyand the example of a recently discovered novel species, C. dublidensis-is discussed.

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Specific identification of Candida albicans by hybridization with oligonucleotides derived from ribosomal DNA internal spacers

Cited by 15 publications

References 28 publications

Seminested PCR for Diagnosis of Candidemia: Comparison with Culture, Antigen Detection, and Biochemical Methods for Species Identification

Seminested PCR for Diagnosis of Candidemia: Comparison with Culture, Antigen Detection, and Biochemical Methods for Species Identification

Identification of yeasts by RFLP analysis of the 5.8S rRNA gene and the two ribosomal internal transcribed spacers

Molecular genetic approaches to identification, epidemiology and taxonomy of non-albicans Candida species

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