16S rRNA gene sequence

Under sterile conditions, 1 gram of fecal sample was weighed and 9 mL of sterile normal saline (with a mass fraction of 0.85%) was added. The samples were thoroughly mixed by vortexing. During this process, each sample was handled separately to avoid contamination between samples. The homogenate was then diluted 10-fold with 9 mL of sterile normal saline until reaching 10−6. 0.2 mL of the 10−3, 10−4, and 10−5 dilutions of the bacterial solution were taken and spread on different culture media for anaerobic cultivation at 37 ℃ for 48-72 hours. The morphology of the colonies was carefully observed, and single colonies with different morphological characteristics were selected from the plates and inoculated onto BHI agar medium using the plate streaking method. The cultures were incubated for 48 hours. The above process was repeated to purify the colonies. Single colonies with diverse morphological characteristics were selected and inoculated into BHI liquid medium. The cultures were stained with Gram staining, and the stained slides were observed under a BX50 optical microscope to record the cell morphology and arrangement. The isolates with uniform distribution, single morphology, and different colors were selected and stored at −80 ℃. The bacterial genomic DNA of the above bacteria was extracted using the Tiangen bacterial genomic DNA extraction kit. The DNA of the qualified samples was amplified using polymerase chain reaction (PCR) for DNA fragments. The amplification and sequencing were performed using the universal primers 27F (5′-AGAGTTTGATCCTCGCTCAG-3′) and 1492R (5′-GGTTACCTTGTTACGACTT-3′) for amplification and sequencing. The PCR amplification system and conditions were referenced. The PCR products were detected using 0.8% agarose gel electrophoresis technology. There was a bright and clear band at 1500 bp without a trailing phenomenon, indicating that the PCR products could meet the sequencing requirements. The PCR amplification products of the strains were sent to Shanghai Saiheng Biotechnology Co., Ltd. for sequencing under a low-temperature condition.