2.6. PCR amplification of 18S rRNA and ITS1

Both PCR assays were performed in a 20μL final reaction volume, which consisted of 10 μL 2xEasyTaq PCR superMix (TransGene Biotech; contained EasyTaq DNA polymerase, dNTPs, and optimised buffer), 3 μL of DNA template, 0.8 μL of each forward (HMT-F) and reverse (HMT-R) primer for amplification of a partial sequence of 18S rRNA, and of LITSR and L5.8S primers for ITS1. The volume of the reaction was completed with the addition of nuclease free water. PCR mixtures were spun down briefly (5–10 s), then placed in a thermal cycler (TCY, Crealcon, NL) and subjected to the following cycling conditions: initial denaturation at 94 °C for 4 min, 35 cycles of denaturation at 94 °C for 30 s, annealing at 56 °C for 30 s, extension at 72 °C for 40 s, and a final extension step at 72 °C for 8 min.

The amplified DNA fragments were visualised via 1.5% agarose gel electrophoresis, using prime save dye (GeneAid) in TBE buffer at 100 V for 60 min at room temperature. Gels were photographed after electrophoresis and amplicon size was determined by comparison with a 100-bp DNA ladder (TransGene Biotech).