The bacterial communities in the fecal samples were investigated by Illumina MiSeq high-throughput sequencing. The V3 and V4 regions of the 16S rDNA gene were selected for PCR. The primers were barcoded as 338F (5′-ACTCCTACGGGAGGCAGCAG-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′), where the barcode was an eight-base sequence unique to each sample. The 20-μl PCR reaction mixture was composed of 4 μl of 5 × FastPfu buffer, 2 μl of 2.5 mM dNTPs, 5 μM each of forward and reverse primer, 0.4 μl TransStart Fastpfu DNA Polymerase (TransGen Biotech, Beijing, China), and 10 ng DNA template. The following cycling parameters were used: maintenance at 95°C for 2 min, 27 cycles of 95°C for 30 s, 55°C for 30 s, and 72°C for 30 s, and a final extension at 72°C for 5 min. Triplicate reaction mixtures were pooled for each sample, purified using an AxyPrep DNA gel extraction kit (Axygen, Union City, CA, United States), and quantified using a QuantiFluor-ST fluorescence quantitative system (Promega, Madison, WI, United States). Amplicons from different samples were sent out for sequencing on an Illumina MiSeq platform at Shanghai Majorbio Bio-Pharm Technology Co., Ltd. (Shanghai, China).

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