Quantitative Real-Time PCR Analysis of Gut Microbiome

Extracted gDNA samples were used as templates for quantitative real-time PCR (RT-qPCR) using the 2× TSINGKE Master qPCR Mix (SYBR Green I) (TSE201, Tsingke, Beijing, China) in an ABI7500 (Thermo Fisher Scientific, Waltham, MA, USA) sequence detector. Three replicates per sample in each pair of primers were examined. PCR mixtures included 10 μL 2 × Mix, 0.4 μL forward primer (10 μM), 0.4 μL reverse primer (10 μM), 1 μL template gDNA (80 ng/μL), and 8.2 μL distilled H₂O. The PCR cycling conditions were as follows: 95 °C for 30 s, 40 cycles of 95 °C for 10 s, and 60 °C for 30 s, ultimately tested at 95 °C for 15 s, 60 °C for 60 s, and 95 °C for 15 s. The standard curve method and QuantStudio™ 7 Flex Real-Time PCR Software (Applied Biosystems, Foster, CA, USA) were used for data analysis. Relative abundance of gut microbes was determined using the 2^−ΔΔCt method [60 (link)], where the 16S rRNA gene amplified by the total bacterial primer set was used as an internal reference [61 (link),62 (link)]. Thus, the standard (NLD) group was considered as a control, and Fold Changes in abundance were expressed relative to the standard (NLD) group. Primer sets for quantification of the Bacteroidetes phylum, Firmicutes phylum, Clostridiales order, Lachnospiraceae family, and Ruminococcaceae family were designed and synthesized by Sangon Biotech Co., Ltd., Shanghai, China, (Table 1).