Use of denaturing gradient gel electrophoresis for the identification of mixed oral yeasts in human saliva

Abstract

A PCR-denaturing gradient gel electrophoresis (DGGE) method was established for the simultaneous presumptive identification of multiple yeast species commonly present in the oral cavity. Published primer sets targeting different regions of the Saccharomyces cerevisiae 26-28S rRNA gene (denoted primer sets N and U) and the 18S rRNA gene (primer set E) were evaluated with ten Candida and four non-Candida yeast species, and twenty Candida albicans isolates. Optimized PCR-DGGE conditions using primer set N were applied to presumptively identify, by band matching, yeasts in the saliva of 25 individuals. Identities were confirmed by DNA sequencing and compared with those using CHROMagar Candida culture. All primer sets yielded detectable DGGE bands for all species tested. Primer set N yielded mainly single bands and could distinguish all species examined, including differentiating Candida dubliniensis from C. albicans. Primer set U was less discriminatory among species but yielded multiple bands that distinguished subspecies groups within C. albicans. Primer set E gave poor yeast discrimination. DGGE analysis identified yeasts in 17 of the 25 saliva samples. Six saliva samples contained two yeast species: three contained C. albicans and three C. dubliniensis. C. dubliniensis was present alone in one saliva sample (total prevalence 16 %). CHROMagar culture detected yeasts in 16 of the yeast-containing saliva samples and did not enable identification of 7 yeast species identified by DGGE. In conclusion, DGGE identification of oral yeast species with primer set N is a relatively fast and reliable method for the simultaneous presumptive identification of mixed yeasts in oral saliva samples.