Abstract
We developed a LightCycler™ real-time polymerase chain reaction (RTPCR) and melting temperature (Tm) assay targeting the internal transcribed spacer 2 (ITS2) and D1/D2 regions of the fungal ribosomal DNA gene (rDNA), and applied it to differentiate and identify clinically isolated fungal strains, including 2 Scedosporium spp., 3 Aspergillus spp., and 8 yeast-like fungal species isolated in our laboratory between August 2008 and July 2016. PCR was carried out using the LightCycler™ 2.0 Instrument and Tm values of the two PCR products in each species were measured. The Tm values of the ITS2 and D1/D2 amplicons were (mean ± standard deviation [SD]) 89.6 ± 0.20 °C and 89.9 ± 0.07 °C for 3 strains of S. prolificans, and 91.0 °C and 91.7 °C for a single strain of S. apiospermum, effectively separating them. Furthermore, these Tm values were lower than those of Aspergillus spp., enabling differentiation between the two filamentous fungi. Of the 8 yeast-like fungi tested, 7 exhibited unique Tm profiles, whereas Candida krusei had a similar Tm profile to S. apiospermum. Taken together, the assay enabled the identification of 9 of 13 species studied. The Tm values of strains of C. albicans and A. fumigatus were determined with a sensitivity of 1 × 104 (ITS2 and D1/D2), and 1 × 105 (ITS2) and 1 × 102 (D1/D2) colony forming units per mL, respectively, and the assay consistently yielded Tm values for the ITS2 and D1/D2 products with SDs of ± 0.04–0.21 °C and ± 0.02–0.05 °C, respectively. The LightCycler™ RTPCR-Tm assay provides fast and reliable information for the differentiation and identification of clinically relevant fungal pathogens, and is of value to initiate effective antifungal therapy promptly.
