Stool DNA was extracted as described previously (20). The copy numbers of the genes attributable to FL1-BP and FL2-BP and 16S ribosomal RNA (rRNA) of total bacteria and genus Bifidobacterium in fecal DNA were quantified using an SYBR Green system (TB Green Premix Ex Taq II, TaKaRa Bio). Relative abundance (%) was calculated by dividing the copy numbers of the transporter genes or genus Bifidobacterium-specific 16S rRNA gene by the total bacterial 16S rRNA gene copies. The average values of technical duplicates (<6% difference of threshold cycle) were reported. The primers used for qPCR analysis were listed in table S3 [nos. 25 to 29; note that two different primer pairs were used for quantifying FL2-BP orthologs (clusters I and II and cluster III)] (Fig. 3D and fig. S6C). Standard curves were created using B. infantis JCM 1222T and B. kashiwanohense JCM 15439T genomic DNAs as templates. The detection limits of our qPCR system for FL1-BP and FL2-BP genes were 1.1 × 104 and 6.3 × 104 copies/g feces, respectively, while that for genus Bifidobacterium was 2.1 × 105 copies/g feces. The specificity of each primer set was confirmed before the analysis.

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