Development of Melting-Curve-Based Real-Time PCR for Differentiating Medically Important Candida Species

TL;DR

This paper develops a real-time PCR method to accurately distinguish between important Candida species using specific DNA regions.

Contribution

The study introduces novel qPCR assays targeting IGS2 and ITS1 regions for precise differentiation of medically significant Candida species.

Findings

qPCR assays using IGS2 and ITS1 primers accurately differentiated Candida species without cross-reactivity.

The assays achieved a detection limit of 10 DNA copies per reaction for each species tested.

No amplification occurred with non-target fungal DNA, ensuring high specificity.

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.

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