Evaluating Primer Performance in Environmental DNA Analyses

To evaluate the performance of the primer pairs amplifying target DNA from a heterogeneous pool of DNA in environmental samples, all primer pairs were tested in a qPCR set-up. A 2-fold dilution series (1∶1 to 1∶64) was made from twelve DNA samples (ranging from 5 ng µl⁻¹ to 78 pg µl⁻¹, including one no-template control (NTC) for each sample). Amplification was performed in optical 96-well plates using a 7500 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) and SYBR Green chemistry. PCR conditions were as follows: initial denaturation at 95°C for two minutes, followed by 40 cycles of 95°C (30 s), 55°C (30 s) and 72°C (60 s) and a final extension phase at 72°C for 10 minutes followed by the generation of a dissociation curve to verify amplification specificity. These qPCR conditions were chosen to mimic the PCR conditions used during the PCR step prior to emPCR and amplicon pyrosequencing. Reactions contained 2.5 µL template DNA, 5 µL 2× Fast SYBR Green Master Mix (Applied Biosystems, Foster City, CA, USA), 0.3 µl forward and reverse primers (3.3 µM each) and 1.9 µL nuclease-free H₂O in a total volume of 10 µL. PCR efficiencies (E) were calculated as E = (10^−1/slope−1)×100.
To assess a potential PCR-bias at the phylum level, DNA was extracted from 15 pure cultures provided by the Mycothèque de l'Université Catholique de Louvain (BCCM/MUCL) including 5 basidiomycetes (Lentinula edodes (MUCL 44827), Agrocybe praecox (MUCL 46727), Coniophora marmorata (MUCL 39471), Suillus luteus (UH-Slu-LM8-n1) and Antrodia vaillantii (MUCL 54533)), 5 ascomycetes (Cladosporium cladosporioides (MUCL 53652), Cryptosporiopsis radicicola (MUCL 53485), Monilinia laxa (MUCL 30841), Arthroderma otae (MUCL 39756) and Galactomyces geotrichum (MUCL 52377)), 2 glomeromycetes (Rhizophagus clareus (MUCL 46238) and Rhizophagus sp. (MUCL 41833)) and 3 zygomycetes (Mortierella verticillata (MUCL 9658), Absidia corymbifera (MUCL 38907) and Mucor hiemalis (MUCL 15439), also see Table S4). DNA was extracted from cultures using the DNeasy Plant Mini Kit according to the manufacturer's instructions (Qiagen, Venlo, Netherlands). DNA concentrations extracted from pure cultures used for qPCR ranged from 5 ng µl⁻¹ to 20 ng µl⁻¹. PCR bias at the phylum level was tested according to the qPCR protocol described above.