RAPID IDENTIFICATION OF CANDIDA-ALBICANS AND OTHER HUMAN PATHOGENIC YEASTS BY USING SHORT OLIGONUCLEOTIDES IN A PCR

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