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Identification of medically important yeasts using PCR-based detection of DNA sequence polymorphisms in the internal
Y C Chen1, J D Eisner, M M Kattar
1Departments of Laboratory Medicine, Division of Bioinformatics, University of Washington, Seattle, Washington, USA.
Abstract:
Identification of medically relevant yeasts can be time-consuming and inaccurate with current methods. We evaluated PCR-based detection of sequence polymorphisms in the internal transcribed spacer 2 (ITS2) region of the rRNA genes as a means of fungal identification. Clinical isolates (401), reference strains (6), and type strains (27), representing 34 species of yeasts were examined. The length of PCR-amplified ITS2 region DNA was determined with single-base precision in less than 30 min by using automated capillary electrophoresis. Unique, species-specific PCR products ranging from 237 to 429 bp were obtained from 92% of the clinical isolates. The remaining 8%, divided into groups with ITS2 regions which differed by /=99%. Seven clinical isolates contained ITS2 sequences that did not agree with their phenotypic identification, and ITS2-based phylogenetic analyses indicate the possibility of new or clinically unusual species in the Rhodotorula and Candida genera. This work establishes an initial database, validated with over 400 clinical isolates, of ITS2 length and sequence polymorphisms for 34 species of yeasts. We conclude that size and restriction analysis of PCR-amplified ITS2 region DNA is a rapid and reliable method to identify clinically significant yeasts, including potentially new or emerging pathogenic species.
Insights
Accurate identification of medically important yeasts is crucial. This study introduces a rapid PCR method targeting the internal transcribed spacer 2 (ITS2) region for reliable yeast identification, including new species.
Area of Science:
- Medical Mycology
- Molecular Biology
- Genetics
Background:
- Accurate identification of medically significant yeasts is essential for effective patient treatment.
- Current identification methods can be slow and prone to inaccuracies.
- Novel molecular techniques are needed to improve yeast identification efficiency and reliability.
Purpose of the Study:
- To evaluate a PCR-based method for identifying yeasts by analyzing sequence polymorphisms in the internal transcribed spacer 2 (ITS2) region.
- To establish a database of ITS2 length and sequence polymorphisms for 34 yeast species.
- To assess the method's accuracy and speed compared to traditional identification techniques.
Main Methods:
- Utilized PCR to amplify the ITS2 region of rRNA genes from 401 clinical yeast isolates, 6 reference strains, and 27 type strains.
- Determined the length of PCR products with single-base precision using automated capillary electrophoresis (under 30 min).
- Employed restriction enzyme analysis and DNA sequencing for species with closely related ITS2 lengths.
Main Results:
- Achieved unique, species-specific PCR products for 92% of clinical isolates, with lengths ranging from 237 to 429 bp.
- Identified the remaining 8% using restriction enzyme analysis due to minor length differences (< =2 bp).
- Demonstrated high concordance (99% sequence identity) between phenotypic and ITS2-based identification for 427 of 434 isolates, with potential new species identified in Candida and Rhodotorula.
Conclusions:
- PCR-based analysis of ITS2 region DNA length and sequence polymorphisms is a rapid, reliable method for identifying clinically significant yeasts.
- This technique can distinguish between closely related species and identify potentially new or emerging pathogenic yeast species.
- The established ITS2 database provides a valuable resource for molecular yeast identification.