Use of PCR-denaturing gradient gel electrophoresis for the discrimination of Candida species isolated from natural habitats.

Candida species are opportunistic microbes that cause chronic infections for a human being. Therefore, the exact identification of Candida species is extremely important for improved therapeutic strategy against these species. Identification based on conventional methods cannot differentiate between some of yeasts species, hence PCR based molecular techniques and sequencing could be an alternative tool for the yeasts identification. A quick molecular method based on the polymerase chain reaction (PCR) and Denaturing Gradient Gel Electrophoresis (DGGE) was applied for distinguishing strains belonging to the Candida species. Six different species designated as AH-20, AH-21, AH-22, AH-23, AH-24 and AH-25 were isolated from soil samples, and their exact identification was detected based on the D1/D2 domain of the 26S rRNA gene amplification and sequence determination. Alignment results and the comparison of 26S rRNA gene sequences of the isolates to 26S rRNA gene sequences available in the GenBank database, as well as the phylogenetic analysis, confirmed the accurate position of the isolates as Candida intermedia strain AH-20, Candida boidinii strain AH-21, Candida tropicalis strain AH-22, Candida mengyuniae strain AH-23, Candida maltosa strain AH-24 and Candida maltosa strain AH-25. Fragments of the D1/D2 domain of 26S rRNA gene were amplified using NL1-GC/LS2 primers and separated by the DGGE. Results showed that all Candida species reported in this study were well discriminated by a distinct band in the DGGE profile. Our results demonstrated that DGGE technique using NL1-GC/LS2 primers could use for the rapid discrimination of yeast strains belonging to the same genera.