Bacterial 16S rRNA Gene Sequencing

Genomic DNA was extracted from cultures incubated at 37°C for 72 h using a bacterial DNA extraction kit (Sangon Biotech, Shanghai, China). The 16S rRNA gene was amplified by PCR using two universal primers, including primers 27F (5′- AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5’-TACGGTTACCTTGTTACGACTT-3′) (Liang et al., 2014 (link)). The 25 μL mixtures were composed of 1 μL template DNA, 12.5 μL of 2 × Taq PrimerSTAR HS (R040A), 1 μL of each primer (10μM), and 9.5 μL of double-distilled H₂O. The PCR program was 98°C for 1 min, followed by 30 cycles of 98°C for 30 s, 60°C for 30 s, and 72°C for 1.5 min, with a final 5 min extension at 72°C and completion at 4°C. The PCR product was analyzed by electrophoresis in 2% agarose gel and then sent to Sangon Company (Shanghai, China) for sanger sequencing using an ABI sequencer (3730xl DNA Analyzer). The phylogenetic tree was constructed by applying the neighbor-joining method using the MEGA 11 software package (Temple University, PA, United States) based on Maximum Composite Likelihood with 1,000 replicates of bootstrap values.