Quantifying Ruminococcus in Fecal Microbiome

Bacterial genomic DNA was isolated from faecal pellets using a QIAamp Stool Mini Kit (Qiagen). DNA encoding 16S rRNA was quantified by SYBR Green dye incorporation (Takara) analysed using an ABI Prism 7700 thermal cycler and detector system (Thermo Fisher Scientific)^{41 (link)}. qPCR was carried out according to the manufacturers’ instructions. The PCR primer sequences used to universally amplify 16S rRNA of all bacteria were 5′-GTGCCAGCMGCCGCGGTAA-3′ and 5′-GACTACCAGGGTATCTAAT-3′. The sequences used to specifically amplify 16S rRNA of Ruminococcus were 5′-CTAGGTGAAGATACTGACGGTAACCTG-3′ and 5′-GTATTACCGCGGCTGCTGGCAC-3′^{42 (link)}. The relative amount of Ruminococcus to whole bacteria was calculated based on the difference in the threshold cycle between universal and specific PCR products.