Rapid identification of yeast species using three primers in a polymerase chain reaction

Abstract

Classic methods for identification of yeasts rely on a variety of morphological and physiological tests that often take days to weeks to complete. We have been able to reduce the time to less than one day through the use of multiple segment-specific oligonucleotide priming of a region of the large subunit rDNA in a polymerase chain reaction. The "hot start" reaction was used with two universal external delimiting primers and one internal species-specific primer. Five specific primers were tested: a primer for a biologically similar group of Rhodotorula species, a generic (Cystofilobasidium) primer, and 3 species-specific primers (Leucosporidium scottii, Cryptococcus muscorum, and Rhodotorula mucilaginosa). In the absence of specific target DNA, the universal rDNA segment is amplified; in the presence of target DNA, the specific primer region is amplified. The technique is accurate within two base position differences when a 24 nucleotide-specific primer is used. The technique should be applicable to other marine eukaryotes.