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Title: RAPID IDENTIFICATION OF CANDIDA ALBICANS AND OTHER HUMAN PATHOGENIC YEASTS USING SHORT OLIGONUCLEOTIDES IN A POLYMERASE CHAIN REACTION (PCR)

Author
item Mannarelli, Bruno
item Kurtzman, Cletus

Submitted to: Journal of Clinical Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/16/1998
Publication Date: N/A
Citation: N/A

Interpretive Summary: A quick method to identify yeasts pathogenic for humans and animals is described. The genetic method is based on short DNA fragments in a polymerase chain reaction (PCR), and, to our knowledge, is the first study that has shown that the PCR method can distinguish between closely related yeasts. Pathogenic yeasts cause millions of human and animal infections a year. At present, most yeast infections are treated as a general fungal infection with antibiotics intended to control a broad array of yeasts. The specific yeast causing the infection is seldom identified and the antibiotic treatment is usually continued for a long period of time. This practice is not always successful since the widespread use of generalized antibiotics has resulted in a rapid rise in yeasts resistant to antibiotics. Drug resistance is now a major problem in the treatment of human and animal infections. Research is presently geared toward developing new antibiotics, but just as logical an approach is to develop quick and accurate identification techniques for microorganisms. The procedure described can yield definitive results in less than a day and would allow clinicians to administer the most effective drug and correct dose.

Technical Abstract: A PCR system was developed that can quickly and accurately identify 14 species of human pathogenic yeasts. The procedure distinguished between nine species of a closely related clade, Lodderomyces elongisporus, Candida parapsilosis, Candida sp., C. sojae, C. tropicalis, C. maltosa, C. viswanathii, C. albicans, and C. dubliniensis; and another five more divergent species, Pichia guilliermondii, Candida glabrata, C. zeylanoides, C. haemulonii, and C. haemulonii Type II. A rapid DNA extraction procedure that yields purified DNA in about one hour is also described. The system used uniform conditions with 4 primers in each reaction, two 40-50-mer universal primers and two 23-30-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) rDNA 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 1,200-1,300 base DNA fragment was produced in all reactions, and a smaller 114-336 base DNA fragment was produced if the chromosomal DNA from the target species was present. The PCR procedure is rapid, easy to interpret, and can be used with mixed cultures.