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Published on: December 28, 2017
Development of Melting-Curve-Based Real-Time PCR for Differentiating Medically Important Candida Species
Eliandro Reis Tavares1,2, Virginia Prezzi Santos3, Isabela Madeira de Castro3
1Departamento de Microbiologia, Centro de Ciências Biológicas, Universidade Estadual de Londrina, Londrina CEP 86057-970, PR, Brazil.
Abstract:
Candida species are the primary fungal pathogens of invasive infections associated with high morbidity and mortality. The identification of these microorganisms is critical for therapeutic management and control of hospital infection. Herein, assays targeting the Intergenic Spacer 2 (IGS2) and Internal Transcribed Spacer 1 (ITS1) from the rDNA locus were developed to differentiate Candida species. Based on consensus nucleotide sequences, specific primers and positive controls were designed, and standard PCR and real-time PCR (qPCR) assays were performed. All primers resulted in specific amplification of the molecular targets of each species with no amplifications of the negative template control. Furthermore, the primers were highly specific when tested with a range of fungal DNAs and no cross-reactivity was observed among Candida species. The assays presented a limit of detection (LoD) of 10 copies of positive control per reaction for all specific primers designed. Overall, our results showed that qPCR assays employing primers targeting the regions IGS2 and ITS1 completely differentiated between Candida albicans, Candida auris, Candida parapsilosis, Candida tropicalis, and Nakazeomyces glabratus, with great accuracy and no amplification of DNA from other fungal species.
Insights
Accurate identification of Candida species is crucial for treating invasive fungal infections. New real-time PCR assays targeting IGS2 and ITS1 regions offer specific and sensitive differentiation of key Candida species, improving patient care.
Area of Science:
- Medical Mycology
- Molecular Diagnostics
- Infectious Diseases
Background:
- Invasive fungal infections caused by Candida species lead to significant morbidity and mortality.
- Accurate and rapid identification of Candida species is essential for effective therapeutic management and hospital infection control.
Purpose of the Study:
- To develop and validate specific molecular assays for the accurate differentiation of clinically relevant Candida species.
- To evaluate the performance of real-time PCR (qPCR) assays targeting the rDNA locus for Candida species identification.
Main Methods:
- Design and synthesis of specific primers and positive controls targeting the Intergenic Spacer 2 (IGS2) and Internal Transcribed Spacer 1 (ITS1) regions.
- Performance evaluation using standard PCR and quantitative real-time PCR (qPCR) assays.
- Specificity testing against a panel of fungal DNAs and assessment of the limit of detection (LoD).
Main Results:
- Developed specific primers and positive controls for IGS2 and ITS1 regions, showing no amplification in negative controls.
- Achieved high specificity, with no cross-reactivity observed among tested Candida species or other fungal DNAs.
- Established a limit of detection of 10 copies per reaction for all designed primers, demonstrating high sensitivity.
Conclusions:
- qPCR assays targeting IGS2 and ITS1 regions accurately differentiate between Candida albicans, Candida auris, Candida parapsilosis, Candida tropicalis, and Nakazeomyces glabratus.
- The developed molecular assays provide a sensitive and specific tool for identifying key Candida species, aiding in clinical management.
- These assays contribute to improved diagnostics for invasive fungal infections, supporting better patient outcomes and infection control strategies.
