Differential Diagnosis of Candida Species With Real-Time Polymerase Chain Reaction and Melting Temperature Analyses (RTPCR-MTA).

Abstract

Genus Candida covers more than 50 species, half of which can cause infections in humans. Some of the Candida species exhibit drug resistance; therefore, there is an urgent need for rapid and accurate differentiation for rendering appropriate and effective management. Here, we report a new methodology employing real-time polymerase chain reaction (RTPCR) and melting temperature analyses (MTA) procedures. Fungal ribosomal internal transcribed spacer 2 (ITS2) has been confirmed with variable nucleotide sequences, which makes it possible to differentiate one species from another by checking their melting temperature following PCR amplification. The universal primers (panFg) covering entire ITS2 region, from 5.8S to 28S rRNA genes, were designed to differentially identify most Candida species with RTPCR-MTA procedure. Nucleic acids from five genomes of closely related Candida species, which were experimentally spiked into human blood, were extracted and amplified. PCR amplicons were called for melting temperature of Candida albicans (87.49°C), C. glabrata (86.85°C), C. krusei (90.24°C), C. parapsilosis (86.22°C), and C. tropicalis (86.08°C). The melting temperature of each amplicon was consistent and reproducible in three replicate experiments (SD ± 0.04-0.32). The new RTPCR-MTA methodology showed promise in differential diagnosis of closely related Candida species from environmental and clinical samples.