Quantitative detection of Mucor species

Mucor species quantification was performed on a 7900HT Fast Real-Time PCR System using the primers and probes described previously34 (link), with slight modifications. Mucor (F) 5′ GTC TTT GAA CGC AAC TTG CG 3′, Mucor (R) 5′ CCG CCT GAT TTC AGA TCA AAT T 3′ and Mucor probe: 5′ TTCCAATGAGCACGCCTGTT-MGBNFQ 3′. DNA from Mucor circinelloides (CBS277.49), belonging to the mold collection of the Fungal Biodiversity Center (CBS) was used as a positive control. For Mucor detection, PCR was performed in a final volume of 20 μl containing 0.5 μM of each primer of the Mucormycetes species, 0.25 μM of the internal control primers and 0.2 μM of Mucor spp. probe. For 18S (reference gene) amplification, we used primers, probes and conditions described previously35 (link). PCR conditions for Mucor spp., 18S and internal control were: 2 min at 50 °C for Uracil-DNA Glycosylase treatment, 10 min at 95 °C for Taq activation, 15 s at 95 °C for denaturation and 1 min at 61.2 °C for annealing and extension (50 cycles). SDS software 2.3 and RQ Manager 1.2 (Applied Biosystems) were used to analyze the results with the comparative threshold cycle (Ct) method (2^−∆∆Ct). C_t values for each sample were normalized with the 18S reference gene. All data were expressed as an n-fold difference relative to a calibrator (a mix of DNA from 4 human faecal samples).