Rapid and accurate identification of species belonging to the Candida parapsilosis complex by real-time PCR and melting curve analysis.

Candida parapsilosis is the second most frequent Candida species isolated from blood cultures. Since 2005, C. parapsilosis has been divided into three distinct species based on genetic traits: Candida parapsilosis, Candida metapsilosis and Candida orthopsilosis. The aim of this study was to develop a rapid real-time PCR assay able to distinguish these closely related species via a melting curve analysis. This identification method was optimized by using reference strains and well-characterized clinical isolates of Candida species. A single set of consensus primers was designed to amplify a 184 bp portion of the SADH gene in order to identify species based on the unique melt profile resulting from DNA sequence variations from each species of the complex. PCR products were detected with SYBR Green fluorescent dye and identification was established by melting curve analysis. For validation of the technique, a total of 116 clinical isolates, phenotypically identified as C. parapsilosis, were tested by real-time PCR and results were further compared with PCR-RFLP patterns of the SADH gene, used as the reference method. The melting curve analysis of amplified DNA could differentiate between C. parapsilosis (83.5 °C), C. metapsilosis (82.9 °C) and C. orthopsilosis (82.1 °C), with a sensitivity and specificity comparable to those of the reference method. One hundred and fourteen C. parapsilosis and two C. orthopsilosis isolates were identified among the clinical isolates. This method provides a simple, rapid and reliable identification of species belonging to the C. parapsilosis complex. This novel approach could be helpful for clinical and epidemiological investigations.