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.

于无菌操作条件下，称取1克粪便样本，加入9 mL质量分数为0.85%的无菌生理盐水，通过涡旋振荡使样本充分混匀。操作过程中每份样本单独处理，避免样本间发生交叉污染。随后将均质后的菌液以无菌生理盐水进行10倍梯度稀释，直至稀释至10⁻⁶。分别吸取10⁻³、10⁻⁴、10⁻⁵稀释度的菌液各0.2 mL，涂布于不同培养基上，置于37℃下厌氧培养48~72小时。仔细观察菌落形态，从平板上挑取形态特征各异的单菌落，采用平板划线法接种至脑心浸液琼脂培养基（BHI agar medium）中，于37℃培养48小时，重复上述操作以完成菌落纯化。挑取形态多样的单菌落接种至脑心浸液液体培养基中，进行革兰氏染色（Gram staining），将染色后的玻片置于BX50型光学显微镜（BX50 optical microscope）下观察，记录细胞形态与排列方式。选取分布均匀、形态单一且色泽各异的菌株，于-80℃条件下低温保存。采用天根（Tiangen）细菌基因组DNA提取试剂盒提取上述菌株的细菌基因组DNA。对合格样本的DNA，通过聚合酶链式反应（PCR）扩增目的DNA片段，使用通用引物27F（5′-AGAGTTTGATCCTCGCTCAG-3′）和1492R（5′-GGTTACCTTGTTACGACTT-3′）完成扩增与测序。PCR扩增体系及条件参照相关规范执行。采用0.8%琼脂糖凝胶电泳技术检测PCR扩增产物，在1500 bp处可见明亮清晰的条带且无拖尾现象，表明扩增产物符合测序要求。将菌株的PCR扩增产物置于低温条件下，送至上海赛恒生物技术有限公司（Shanghai Saiheng Biotechnology Co., Ltd.）进行测序。