Identification of yeasts of dairy origin by amplified ribosomal DNA restriction analysis (ARDRA)

Amplified ribosomal DNA restriction analysis was used for classification of 90 yeast strains of 32 species of dairy origin. Primers ITS1 and ITS4 yielded PCR products in the range 375 bp (Candida lipolytica) to 915 bp (Schizosaccharomyces pombe). Species-specific restriction products were liberated by the enzymes Hin6I, HinfI and BsuRI. Some species showed genetic variation, leading to PCR products of different sizes (Debaryomyces hansenii, C. lipolytica, Candida inconspicua, Candida sake). Restriction patterns of three Trichosporon species were similar; a species determination was possible by including Trichosporon reference strains on the gels. Candida krusei, an essential ripening culture of acid curd cheese, could be distinguished from Candida norvegensis and C. inconspicua. C. lipolytica, an important contaminant of smear cheeses, could be classified by the size of the PCR product and restriction patterns. The method provides a fast and reliable tool for identification of dairy yeasts. Size determination of the PCR products gives a narrow range of possible species, while subsequent restriction analysis allows accurate species classification of relevant dairy yeasts.