PCR-based identification of pathogenic Candida species using primer mixes specific to Candida DNA topoisomerase II genes.

For rapid identification of Candida to the species level, degenerated primers and specific primers based on the genomic sequences of DNA topoisomerase II of C. albicans, C. dubliniensis, C. tropicalis (genotypes I and II), C. parapsilosis (genotypes I and II), C. krusei, C. kefyr, C. guilliermondii, C. glabrata, C. lusitaniae and Y. lipolytica were designed and their specificities tested in PCR-based identifications. Each of the specific primers selectively and exclusively amplified its own DNA fragment, not only from the corresponding genomic DNA of the Candida sp. but also from DNA mixtures containing other DNAs from several fungal species. For a simpler PCR-based identification, the specific primers were divided into three groups (PsI, PsII and PsIII), each of which contained four specific primer pairs. PCR with the primer mixes yielded four different sizes of PCR product, corresponding to each Candida sp. in the sample DNA. To obtain higher sensitivity of PCR amplification, sample DNAs were preamplified by the degenerated primer pair (CDF28/CDR148), followed by the main amplification using the primer mixes. By including this nested PCR step, 40 fg yeast genomic DNA was detected in the sample. Furthermore, we applied this nested PCR to a clinical diagnosis, using splenic tissues from experimentally infected mice and several clinical materials from patients. In all cases, the nested PCR amplifications detected proper DNA fragments of Candida spp., which were also identified by the standard identification tests. These results suggest that nested PCR, using primer mixes of the Candida DNA topoisomerase II genes, is simple and feasible for the rapid detection/identification of Candida to species level in clinical materials.