2.3. DNA Extraction and Polymerase Chain Reaction-Denaturing Gradient Gel Electrophoresis (PCR-DGGE)

Mouse fecal samples were collected, and all samples were stored at −80 °C for backup, followed by the application of (E.Z.N.A.^® Stool DNAkit, Omega kit, Norcross, GA, USA) for the extraction of genomic DNA. For the extracted DNA samples, the V3 region of 16S rRNA was amplified using primers 338F-806R (338F, 5′-CGCCCGGGGCGCGCCCCGGGCGGGGCGGGCACGGGGGGCCTACGGGAGGCAGCAG-3′ and 518R, 5′-ATTACCGCGGCTGCTGG-3′). The PCR system contained 25 µL PCR Master Mix, 1 µL forward primer (10 Pmol/µL), 1 µL reverse primer (10 Pmol/µL), and 3 µL DNA, and the total system was adjusted to 50 µL using enzyme-free water. The concentration of DNA was determined using a NanoDrop2000 (Thermo Scientific, Waltham, MA, USA) and DNA was stored at −20 °C. The PCR-amplified products were transferred to a DGGE gel, placed in a 1× TAE solution, and electrophoresed at a constant temperature and pressure of 60 °C and 80 V, respectively, for 6 h. After electrophoresis, the gel was stained with EB for 30 min, and the pictures were developed with a UVI fully automated gel imaging system (Bio-Rad, Hercules, CA, USA). DNA fragments of different sequences were separated via PCR-DGGE; the bands at different positions represent different microorganisms.