Development of Candida-Specific Real-Time PCR Assays for the Detection and Identification of Eight Medically Important Candida Species

Jing Zhang; Guo-Chiuan Hung; Kenjiro Nagamine; Bingjie Li; Shien Tsai; Shyh-Ching Lo

doi:10.4137/MBI.S38517

Development of Candida-Specific Real-Time PCR Assays for the Detection and Identification of Eight Medically Important Candida Species

Microbiol Insights. 2016 Apr 18:9:21-8. doi: 10.4137/MBI.S38517. eCollection 2016.

Authors

Jing Zhang¹, Guo-Chiuan Hung¹, Kenjiro Nagamine², Bingjie Li¹, Shien Tsai¹, Shyh-Ching Lo³

Affiliations

¹ Tissue Microbiology Laboratory, Division of Cellular and Gene Therapies, Office of Cellular, Tissue and Gene Therapies, Center for Biologics Evaluation and Research, US Food and Drug Administration, Silver Spring, MD, USA.
² Tissue Microbiology Laboratory, Division of Cellular and Gene Therapies, Office of Cellular, Tissue and Gene Therapies, Center for Biologics Evaluation and Research, US Food and Drug Administration, Silver Spring, MD, USA.; Department of Oral and Maxillofacial Surgery, Nagoya University Graduate School of Medicine, Nagoya, Aichi, Japan.
³ Tissue Microbiology Laboratory, Division of Cellular and Gene Therapies, Office of Cellular, Tissue and Gene Therapies, Center for Biologics Evaluation and Research, US Food and Drug Administration, Silver Spring, MD, USA.; Senior Investigator, Medical Officer, Tissue Microbiology Laboratory, Division of Cellular and Gene Therapies, Center for Biologics Evaluation and Research, US Food and Drug Administration, Silver Spring, MD, USA.

Abstract

Culture-based identification methods have been the gold standard for the diagnosis of fungal infection. Currently, molecular technologies such as real-time PCR assays with short turnaround time can provide desirable alternatives for the rapid detection of Candida microbes. However, most of the published PCR primer sets are not Candida specific and likely to amplify DNA from common environmental contaminants, such as Aspergillus microbes. In this study, we designed pan-Candida primer sets based on the ribosomal DNA-coding regions conserved within Candida but distinct from those of Aspergillus and Penicillium. We demonstrate that the final two selected pan-Candida primer sets would not amplify Aspergillus DNA and could be used to differentiate eight medically important Candida pathogens in real-time PCR assays based on their melting profiles, with a sensitivity of detection as low as 10 fg of Candida genomic DNA. Moreover, we further evaluated and selected species-specific primer sets covering Candida albicans, Candida glabrata, Candida tropicalis, and Candida dubliniensis and show that they had high sensitivity and specificity. These real-time PCR primer sets could potentially be assembled into a single PCR array for the rapid detection of Candida species in various clinical settings, such as corneal transplantation.

Keywords: melting profiles; primer design; ribosomal DNA; species differentiation; specific PCR.