Quantifying Bacterial Diversity in Colonic Digesta

Bacterial DNA in colonic digesta was extracted by using the Stool DNA Kit (Omega Bio-Tek, Doraville, CA, USA). All primers and probes were listed in Table 4 and designed following the previous report [25 (link)]. Microbial real-time quantitative PCR was performed in a QuanStudio™ 6 Flex Real-Time PCR System (Applied Biosystems, Foster, CA, USA). Briefly, the total bacteria was detected using SYBR^® Premix Ex Taq™ II reagent (TaKaRa, Dalian, China), and the Bacillus, Lactobacillus, E. coli and Bifidobacterium were detected using PrimerScriptTM PCR kit (TaKaRa, Dalian, China) following the previous methods [26 (link)]. Furthermore, for the quantification of bacteria, specific standard curves were generated by constructing standard plasmids as presented by Chen et al. (2013) [26 (link)].