qPCR assay design
Extracted DNA from fungal cultures was checked with PCR using ITS gene fragment primers forward 5’-GCATCGATGAAGAACGCAGCGA-3’ and reverse 5’-GGYCAGCKCCGGCCGGGTC-3’ used in a previous study (Peterson et al., 2020) to confirm that purified DNA from the reference strains could be successfully amplified. Primers and probe were selected by manually examining nucleotide alignments of each coding sequence for regions of dissimilarity. The intronic region of the N. barbatae NADH dehydrogenase subunit I (nad1 ) gene was selected due to a larger number of polymorphisms compared with other genes (FIGURE 1). A custom primer and probe set was developed using the PrimerQuest™ Tool (Integrated DNA Technologies, Inc) and produced with the 6-FAM five-prime reporter dye and the ZEN/Iowa Black FQ double-quenched probe system (Integrated DNA Technologies, Inc). The qPCR reactions consisted of 10 µl SensiFAST Probe No-ROX Kit (Meridian Bioscience), 0.4 µM each of forward primer 5’-TGATCATGTTTAGTCTCTGAAGGT-3’ and reverse primer 5’-AGGCTAAGCTGATTTGGTCTC-3’, 0.1 µM of the probe 5’-6-FAM/TCCCTGCTG/ZEN/ATTGCCATATATTAGGT/FQ/-3’, and 2 µl DNA template made up to a volume of 20 µl using molecular grade water. The cycling parameters included an initial denaturation step of 95oC for 5 mins followed by 35-40 cycles of 95oC for 20 seconds and 63oC for 30 seconds using a Rotor-Gene Q HRM 5plex Platform (Qiagen). The resulting 114 bp amplicon was confirmed as the correct region using Sanger sequencing. A positive result was called for any sample crossing the cycle threshold (C t) as determined by the Rotor-Gene Q software.