Quantifying Bacterial Biomass via qPCR

qPCR assays were conducted to determine total bacterial biomass using the SYBR Premix Ex TaqTM Kit (Takara, Beijing, China) and iQ5 Real-Time PCR System (Bio–Rad, Hercules, CA, USA). Universal primers Eub338/Eub518 [29 (link)] were used to identify the V4 regions in the bacterial 16S rRNA genes. The PCR mix contained ten microliters each of SYBR green I and ROX dye II (Applied Biosystems, Waltham, MA, USA), 0.4 microliters of each primer, 2.0 microliters of DNA, and sterile deionized water up to a total of twenty microliters. The qPCR conditions entailed initial denaturation at 95 °C for 5 min, followed by 37 cycles of denaturation at 95 °C for 45 s, annealing at 56 °C for 45 s, and a final extension at 72 °C for 2 min. The standard samples were diluted to conduct a set of 10-fold dilutions and then used to quantify gene expression with qPCR. The standard curves were generated according to a predetermined protocol [29 (link)], and the R²-value exceeded 0.99. All amplifications from each individual sample were measured in triplicate.