Development of Candida-Specific Real-Time PCR Assays for the Detection and Identification of Eight Medically Important Candida Species.
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.
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.
Jing
J
Zhang
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.
Guo-Chiuan
GC
Hung
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.
Kenjiro
K
Nagamine
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.
Bingjie
B
Li
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.
Shien
S
Tsai
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.
Shyh-Ching
SC
Lo
melting profiles; primer design; ribosomal DNA; species differentiation; specific PCR