Qiaamp dna stool mini kit

The genomic DNA of feces was extracted using a QIAamp DNA Stool Mini Kit (Tiangen, Beijing, China) according to the manufacturer’s instructions. The integrity and size of the DNA were verified through 1% agarose gel electrophoresis, and DNA concentrations were determined. The 16S rRNA-based amplification process was executed by utilizing the primers 341F (5′-CCTAYGGGRBGCASCAG-3′) and 806R (5′-GGACTACNNGGGTATCTAAT-3′), which are directionally targeting the V3-V4 hypervariable regions of the 16S rRNA gene. The amplicons underwent a purification, quantification, and normalization process before being combined in equal molar concentrations. The sequencing of the pooled amplicons was then conducted using an Illumina Novaseq 6000 sequencing instrument (Illumina, Inc., San Diego, CA, USA).
Effective sequencing data were then clustered into operational taxonomic units (OTUs) with 97% similarity cut-off using USEARCH (version 11). The RDP Classifier (version 2.13) algorithm was used to annotate taxonomic information for each representative sequence using the Silva Database (SSU138). The Mothur program (version 1.30.2) was employed to analyze rarefaction curves and calculate richness estimators and diversity indices. Linear discriminant analysis (LDA) coupled with effect size (LEfSe) was applied to evaluate the differentially abundant taxon.

Genomic DNA was extracted from mouse feces using the QIAamp DNA Stool Mini Kit (Tiangen, China). The quantity and quality of DNA were assessed using a NanoDrop2000 (Thermo Fisher Scientific, USA). Qing110-specific primers were designed based on the 16S rRNA gene sequences of the Qing110 strain, which were obtained from whole-genome sequencing. The designed primers for Qing110 were as follows:
Qing110 Forward Primer: 5′-CATGCAAGTCGAACGAGGGTC-3′
Qing110 Reverse Primer: 5′-CGATGCCGTCTCTGTCCCTA-3′. In addition, primers for total bacteria were used for comparison:
Total Bacteria Forward Primer: 5′-ACTCCTACGGGAGGCAGCAGT-3′
Total Bacteria Reverse Primer: 5′-ATTACCGCGGCTGCTGGC-3′.
The qPCRs were performed with the designed primers. The reaction conditions included an initial denaturation step at 95°C for 30 seconds, followed by 40 cycles of denaturation at 95°C for 5 seconds and annealing/extension at 60°C for 34 seconds. To determine the relative abundance of Qing110, the obtained qPCR data were normalized to the total bacterial load. This normalization step allowed for the quantification of Qing110 relative to the overall bacterial population in the fecal samples.

Lincomycin hydrochloride (LH) was purchased from Anhui Shuanghe Pharmaceutical Co., Ltd. (Anhui, China). ELISA kits were purchased from Shanghai MLBIO Biotechnology Co., Ltd. QIAamp DNA stool mini kit was manufactured by TIANGEN (Germany). Other reagents were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China).
According to our previous methods, CSP-1 was isolated from Cereus sinensis (19 (link), 21 (link)). After being soaked in NaCl solution (2%) to remove impurities, fresh Cereus sinensis was crushed and mixed with an equal volume of acetone for 12 h. The solution was filtered and freeze-dried to obtain degreased Cereus sinensis powder. The powder was mixed with distilled water at 72°C for 3 h and then precipitated with 3 times the volume of ethanol for 2 days. The above solution was centrifuged to obtain the precipitate, which was the crude polysaccharides. 50 mg/mL crude polysaccharide solution was mixed with 3% trichloroacetic acid for 12 h to remove protein. The solution was centrifuged, concentrated, dialyzed (3500D MWCO), and lyophilized. 5 mL of 20 mg/mL polysaccharide solution were further purified by column chromatography, involving DEAE-52 ion-exchange column and Sephadex G-100 column. 0.3 mol/L NaCl solution was used as eluate. The solution containing polysaccharides was collected, concentrated, dialyzed, and lyophilized to obtain CSP-1.