C-218. DNA Profiling For Pathogenic Candida Species Identification

W. Wei1, H-M. Pang1, G. W. Procop2;
1Advanced Analytical Technologies, Inc., Ames, IA, 2Cleveland Clin., Cleveland, OH.

Background: Traditional methods for yeast identification are often time consuming and laborious. For rapid identification of Candida from species to strain levels, a novel method of DNA profiling is used to generate a sequencing-like pattern for identification. Methods: A universal forward and reversed primer pair is used to amplify the D2 region of large rRNA subunit. The forward primer was labeled with a fluorescent dye while the reversed primer is not labeled. A proprietary reagent kits (SNAP71) was used to cleave the PCR products to many different sizes base-specific fragments. This reagent kits selectively cleaves the PCR products at A and G nucleotides positions to obtain base-specific fragmentation. A high throughput multiplexed capillary gel electrophoresis system with fluorescence detection is used to analyze the fragments with single base separation resolution. Results: A sequencing-liked fingerprint based on the location of adenine and guanine residues was generated. The fingerprint pattern was compared to a database for Candida species identification. All four clinical important Candida species (Candida Parapsilosis, Candida Albicans, Candida Tropicalis, and Candida Glabrata) can be identified easily by this DNA profiling method. The entire process takes ~2 1/2 hours from template to identification. Sub-strains can also be identified as long as they have genetic variants in the region of study. Conclusion: This method appears highly accurate for microorganism identification, likely because the information generated is based on the DNA sequence of regions previously shown to be of benefit for taxonomic categorization Candida species. In addition, high sample throughput can be obtained through the use of a multiplexed capillary gel electrophoresis system allowing multiple samples to be analyzed simultaneously.