Real-time qPCR was utilized to measure bacterial 16S rRNA gene copies in feces in the StepOnePlus Real-Time PCR system (Thermo Fisher Scientific, Waltham, MA, USA) as in a previous study 29. Eight pairs of 16S rRNA gene primers specific to Firmicutes, Bacteroidetes, the C. leptum group, Bacteroides, Bifidobacterium, A. muciniphila, E. coli, and F. prausnitzii are listed in Supplementary Table 1. Standard curves were constructed with a 10-fold dilution series of the 16S rRNA gene fragment amplified from the reference strains that was cloned into a T&ATM Cloning Vector (Yeastern Biotech, Co., Ltd, Taipei, Taiwan). Each reaction mixture with a total volume of 10 μl was composed of 0.25 μl of each 10 μM primer, 5 μl of AceQ qPCR SYBR Green Master Mix (Vazyme Biotech Co., Piscataway, NJ, USA), 1 μl of sample DNA, and 3.5 μl sterilized ultra-pure water. Real-time PCR was carried out by the following cycle conditions: an initial holding at 95 °C for 30 s, followed by 40 cycles of denaturation at 95 °C for 3 s, then annealing/elongation at 60 °C for 40 s. Melting curve analysis was performed after amplification to determine the specificity. Quantitation of the eight taxonomic units was evaluated as the copy numbers of the 16S rRNA genes/gram of feces weight. All qPCR tests were performed in duplicate, and the presented data are the mean values of duplicate qPCR analysis.

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