Quantification of Bacterial and Fungal DNA

Quantification of the bacterial 16S rRNA gene and fungal 18S rRNA gene was accomplished using real-time PCR. Bacterial DNA was quantified using the primers 1055f and 1392r and the TaqMan probe 16Staq1115 (Harms et al., 2003 (link)). Fungal DNA quantification was conducted using the primers FungiQuant-F and FungiQuant-R and the TaqMan probe FungiQuant-Prb (Liu et al., 2012 (link)). For fungi, universal fungal primers, and TaqMan probes covered the 1,199–1,549 S. cerevisiae numbering region of the 18S rRNA-encoding gene. Each reaction mixture was prepared in a total volume of 25 μL with 12.5 μL Premix Ex Taq (Probe qPCR, Takara Bio), 0.2 μM of each primer, 0.25 μM TaqMan probe, and 2 μL of standard or extracted DNA. For the assay, PCR amplification was performed in a Thermal Cycler Dice Real Time System (TP-850, Takara Bio, Otsu, Japan) under the conditions of initial denaturation for 30 s at 95°C followed by 40 cycles of 5 s at 95°C and 30 s at 60°C. DNA standards for bacteria and fungi were prepared from serial dilutions of the pGEM-T Easy Vector (Promega) containing the 16S rRNA gene from Escherichia coli and the 18S rRNA gene from Cladosporium sp., respectively. Duplicate aliquots of the standards and the samples were included in each PCR run and all assays included a negative control without template DNA.