Mastercycler ep realplex 2s device

Real-time PCR was conducted to estimate the abundance of bacterial and fungal species. The reactions were based on 2× TaKaRa SYBR^® Premix Taq™ (Bio technology Dalian Co. Ltd., Dalian, China) using a Mastercycler ep realplex 2S device (Eppendorf, Germany). The primer pairs were Eub338/Eub518 (bacteria) and ITS1-F/ITS2 (fungi). Each 25 μL reaction contained 10 µL 2× SYBR^® Premix ExTaq™, 0.5 µL of each primer, 0.5 μL 50× ROX Reference Dye II and 2 µL DNA. For the bacterial assay, the PCR comprised an initial denaturation of 95 °C/10 min, followed by 40 cycles of 95 °C/10 s, 54 °C/10 s, 72 °C/30 s, while for the fungal amplicons, the initial denaturation step was 95 °C/5 min and the cycling regime was 45 cycles of 95 °C/15 s, 53 °C/30 s, 72 °C/45 s. Standard curves were generated using a 10-fold serial dilution (from 10⁹ to 10⁴ copies) of a plasmid containing a full length copy of Saccharomyces cerevisiae 18S rRNA or Escherichia coli 16S rRNA [3 (link),14 (link)]. Plasmid was extracted by Axyprep™ Plasmid Miniprep Kit (Axygen, Hangzhou, China). Each biological sample was subjected to three technical replicates.

qPCR was performed using a Mastercycler ep realplex 2S device (Eppendorf, Germany) in conjunction with SYBR Premix Ex Taq II (TaKaRa, Dalian, China). Reactions were performed in a total volume of 20 μL containing 5 μL of diluted cDNA, 0.6 μL of each of forward and reverse primer (10 μM), 10 μL of 2 × SYBR Premix and 3.8 μL of ddH₂O. The amplification program comprised an initial denaturation step (95℃ for 2 min), followed by 40 cycles of 95℃ for 5 s, 60℃ for 30 s, and 72 ℃ for 30 s, and a melting curve protocol (60–95 ℃ with a temperature increment of 0.5 ℃ s⁻¹). Each RT-qPCR was performed for three biological and three technical replicates, and negative controls were included for each primer pair. Amplification efficiency (E) and correlation coefficient (R²) values were obtained from standard curves generated using a tenfold diluted cDNA series, the starting quantity of cDNA was 500 ng/μL^{50 (link)}.