Rapid Identification of Candida albicansand Other Human Pathogenic Yeasts by Using Short Oligonucleotides in a PCR

ABSTRACT A PCR system that can quickly and accurately identify 14 species of human pathogenic yeasts was developed. The procedure distinguished between nine species of a closely related clade, Lodderomyces elongisporus, Candida parapsilosis, a newCandida sp., C. sojae, C. tropicalis, C. maltosa, C. viswanathii,C. albicans, and C. dubliniensis and between another five more divergent species, Pichia guilliermondii,C. glabrata, C. zeylanoides, C. haemulonii, and C. haemulonii type II. A rapid DNA extraction procedure that yields purified DNA in about 1 h is also described. The system uses uniform conditions with four primers for each reaction, two 40- to 50-mer universal primers that serve as a positive control and two 23- to 30-mer species-specific primers. Species-specific primers were derived from a 600-nucleotide variable region (D1/D2) at the 5′ end of the large-subunit (26S) ribosomal DNA gene and were generally designed to use mismatches at the 3′ end. Universal primers were developed from conserved nucleotide sequences in the small-subunit (18S) rRNA gene. In this system, a control 1,200- to 1,300-base DNA fragment was produced in all reactions and a smaller 114- to 336-base DNA fragment was produced if the chromosomal DNA from the target species was present. The PCR procedure is rapid and easy to interpret and may be used with mixed cultures.

[1]  R. Gander,et al.  Evaluation of Gen-Probe's Histoplasma capsulatum and Cryptococcus neoformans AccuProbes , 1993, Journal of clinical microbiology.

[2]  D. Glenn,et al.  Evaluation of commercially available acridinium ester-labeled chemiluminescent DNA probes for culture identification of Blastomyces dermatitidis, Coccidioides immitis, Cryptococcus neoformans, and Histoplasma capsulatum , 1993, Journal of clinical microbiology.

[3]  T. C. White,et al.  Clinical, Cellular, and Molecular Factors That Contribute to Antifungal Drug Resistance , 1998, Clinical Microbiology Reviews.

[4]  J. Galgiani,et al.  Comparison of relative susceptibilities of Candida species to three antifungal agents as determined by unstandardized methods , 1987, Antimicrobial Agents and Chemotherapy.

[5]  T. Deák.  Molecular taxonomy of yeasts. , 1999, Acta microbiologica et immunologica Hungarica.

[6]  C. Kurtzman,et al.  Partial rRNA sequences in marine yeasts: a model for identification of marine eukaryotes. , 1992, Molecular marine biology and biotechnology.

[7]  M. Pfaller,et al.  Comparison of the Quantum II, API Yeast Ident, and AutoMicrobic systems for identification of clinical yeast isolates , 1988, Journal of clinical microbiology.

[8]  C. J. Robnett,et al.  MOLECULAR RELATIONSHIPS AMONG HYPHAL ASCOMYCETOUS YEASTS AND YEASTLIKE TAXA , 1995 .

[9]  N. Arnheim,et al.  Extension of base mispairs by Taq DNA polymerase: implications for single nucleotide discrimination in PCR. , 1992, Nucleic acids research.

[10]  F. Odds.  Resistance of yeasts to azole-derivative antifungals. , 1993, The Journal of antimicrobial chemotherapy.

[11]  G. Land,et al.  Evaluation of the Baxter-MicroScan 4-hour enzyme-based yeast identification system , 1991, Journal of clinical microbiology.

[12]  Henry D. Isenberg,et al.  Manual of Clinical Microbiology , 1991 .

[13]  G. Bodey,et al.  Azole antifungal agents. , 1992, Clinical infectious diseases : an official publication of the Infectious Diseases Society of America.

[14]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[15]  M. Henman,et al.  Antifungal drug susceptibilities of oral Candida dubliniensis isolates from human immunodeficiency virus (HIV)-infected and non-HIV-infected subjects and generation of stable fluconazole-resistant derivatives in vitro , 1997, Antimicrobial agents and chemotherapy.

[16]  G J Olsen,et al.  Phylogenetic group-specific oligodeoxynucleotide probes for identification of single microbial cells , 1988, Journal of bacteriology.

[17]  R. Matthews,et al.  Application of the polymerase chain reaction to the diagnosis of candidosis by amplification of an HSP 90 gene fragment. , 1993, Journal of medical microbiology.

[18]  Thomas J. White,et al.  PCR protocols: a guide to methods and applications. , 1990 .

[19]  R. Cannon,et al.  Detection of Candida albicans and other yeasts in blood by PCR , 1994, Journal of clinical microbiology.

[20]  G. St Germain,et al.  Evaluation of the MicroScan Rapid Yeast Identification panel , 1991, Journal of clinical microbiology.

[21]  P. Broda,et al.  Rapid preparation of DNA from filamentous fungi , 1985 .

[22]  T. White.  Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics , 1990 .

[23]  J. Rex,et al.  Resistance of Candida species to fluconazole , 1995, Antimicrobial agents and chemotherapy.

[24]  W. Rychlik,et al.  A computer program for choosing optimal oligonucleotides for filter hybridization, sequencing and in vitro amplification of DNA. , 1989, Nucleic acids research.

[25]  C. Kurtzman,et al.  Identification of clinically important ascomycetous yeasts based on nucleotide divergence in the 5' end of the large-subunit (26S) ribosomal DNA gene , 1997, Journal of clinical microbiology.

[26]  J. Fell.  Rapid identification of yeast species using three primers in a polymerase chain reaction. , 1993, Molecular marine biology and biotechnology.

[27]  L. E. Gray,et al.  Phylogenetic relationships of the soybean sudden death syndrome pathogen Fusarium solani f. sp. phaseoli inferred from rDNA sequence data and PCR primers for its identification. , 1995, Molecular plant-microbe interactions : MPMI.

[28]  R. Amann,et al.  Molecular and microscopic identification of sulfate-reducing bacteria in multispecies biofilms , 1992, Applied and environmental microbiology.

[29]  J. Lüthy,et al.  Polymerase chain reaction with additional primers allows identification of amplified DNA and recognition of specific alleles. , 1992, Molecular and cellular probes.