Quantitative PCR for Fungal 18S rRNA

Quantitative PCR (Q-PCR) assessed the total abundance of the fungal 18S rRNA genes. Primers FR1 (5′-AICCATTCAATCGGTAIT-3′) and FF390 (3′-CGATAACGAACGAGA CCT-5′) (24 (link), 49 (link)) were used with the SYBR Premix Ex Taq (TaKaRa, Japan). The 10 μl reaction volume contained 1× SYBR Premix Ex Taq, 0.25 μM each primer, and ∼10 ng of DNA template. The Q-PCR was performed on a DNAEngine Peltier Thermal Cycler with a Chromo4 Real-Time PCR Detector (Bio-Rad, USA). The reactions were amplified with an initial denaturation at 95°C for 2 min, followed by 40 cycles of 95°C for 5s, annealing at 46°C for 30s and elongation at 72°C for 30s. Standard curves were constructed using known amounts of standard linearized plasmid, a combination of the pTOPO-TA vector (Gene-better, Beijing, China) and the target gene derived from genomic DNA of Rhodosporidium diobovatum.