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.