Rapid and Accurate Identification of Candida albicans and Candida dubliniensis by Real-Time PCR and Melting Curve Analysis

• Published in: Medical principles and practice Vol. 27; no. 6; pp. 543 - 548
• Main Authors: Asadzadeh, Mohammad, Ahmad, Suhail, Al-Sweih, Noura, Khan, Ziauddin
• Format: Journal Article
• Language: English
• Published: Basel, Switzerland S. Karger AG 01.03.2019
• Subjects: Antifungal agents; Deoxyribonucleic acid; DNA; Identification; Ions; Laboratories; Mass spectrometry; Methods; Original Paper; Polymerase chain reaction; Ribosomal DNA; Scientific imaging; Kuwait; Candida albicans; Melting curve analysis; Candida dubliniensis; Real-time polymerase chain reaction
• ISSN: 1011-7571; 1423-0151; 1423-0151
• DOI: 10.1159/000493426

Objective: Candida albicans and Candida dubliniensis are germ tube-positive pathogenic yeast species. Accurate identification of these two species is warranted since C. albicans is a highly pathogenic species while C. dubliniensis exhibits increased adherence to buccal epithelial cells, reduced susceptibility to azoles and resistance to flucytosine. We have developed a duplex real-time PCR assay for rapid detection and differentiation between clinical C. albicans and C. dubliniensis isolates. Materials and Methods: A duplex real-time PCR assay was developed by using two species-specific primer pairs and SYBR Green dye to differentiate C. albicans and C. dubliniensis isolates via melting curve analysis of real-time PCR amplicons. Amplification products were also analyzed by agarose gel electrophoresis to confirm real-time PCR results. Results: Melting temperatures (T m ) for reference strains of C. albicans and C. dubliniensis were 86.55 and 82.75°C, respectively. No amplicon was obtained with DNA from reference strains of 8 other common Candida spp. When real-time PCR was applied on 226 clinical isolates previously identified by the Vitek 2 system and/or PCR sequencing of rDNA, T m values for C. albicans (n = 113) and C. dubliniensis (n = 98) were 86.68 ± 0.529 and 82.616 ± 0.535°C, respectively. The results were confirmed by agarose gel electrophoresis. No amplicon was obtained from 15 isolates belonging to 9 other Candida spp. Conclusions: The real-time PCR assay described here does not require prior identification of clinical yeast isolates as C. albicans/C. dubliniensis by germ tube formation and accurately reports results within 2 h. Detection of amplicons by agarose gel electrophoresis is also suitable for resource-poor settings devoid of real-time PCR facilities.