Method for enriching and screening Fusatienibacter in intestinal bacteria

Abstract

The invention provides a method for enriching and screening Fusatienibacter intestinal bacteria, which is suitable for being operated under conventional conditions in a common laboratory. According to the method, a basic anaerobic culture medium and a special enrichment culture are prepared, and molecular biology and a traditional culture technology are combined, so that the efficient separation of the Fusicaribacter is realized. The method comprises the specific steps of culture medium preparation, enrichment culture, nucleic acid extraction, high-throughput sequencing, strain separation and molecular identification. According to the method disclosed by the invention, the unique culture medium formula and culture conditions are utilized, so that the problems of harsh growth conditions, low separation efficiency and the like of the Fusicaribacter bacteria in the traditional method are solved. By optimizing the culture process and adding specific agents, target strains can be efficiently enriched under the aerobic condition, and the screening workload is reduced. The method provides technical support for functional research and application of the Fusicatinibacter, and has the advantages of simplicity and convenience in operation, low cost and high efficiency.

Description

Technical Field

[0001] The present invention relates to the enrichment and isolation of a specific type of microorganism, and particularly to the isolation of strains with research and application value from human intestinal bacteria. Combining a specially designed in vitro culture device and operation method, it is suitable for ordinary laboratories to carry out work under conventional experimental conditions. Background Art

[0002] The gut microbiome is one of the most complex microecosystems in the human body, consisting of trillions of microorganisms, including bacteria, fungi, viruses, and archaea. These microorganisms form complex symbiotic relationships with the host and have a profound impact on the host's health. In recent years, with the development of high-throughput sequencing technology and bioinformatics, significant progress has been made in the study of the gut microbiota, revealing its key roles in metabolism, immune regulation, neurodevelopment, and disease occurrence. The genus Fusicatenibacter is an emerging genus in the gut microbiota and has received extensive attention in recent years due to its potential probiotic functions. The genus Fusicatenibacter belongs to the phylum Firmicutes and was first described and named by Sakamoto et al. in 2018. Studies have shown that the abundance of Fusicatenibacter strains is associated with various disease states, such as inflammatory bowel disease (IBD), irritable bowel syndrome (IBS), and metabolic syndrome. The metabolic characteristics of Fusicatenibacter strains are one of the key directions for functional research. Strains of this genus can ferment a variety of carbohydrates to produce short-chain fatty acids (SCFAs), such as acetic acid, propionic acid, and butyric acid. SCFAs are important products of gut microbiota metabolism, which not only provide energy for the host but also have anti-inflammatory, immune regulatory, and gut barrier function-maintaining effects. In particular, butyric acid has been shown to promote the proliferation and differentiation of intestinal epithelial cells, enhance gut barrier function, and inhibit inflammatory responses. Therefore, Fusicatenibacter strains may play an important role in regulating the gut microenvironment and host health by producing SCFAs. In addition, Fusicatenibacter strains also have potential immune regulatory functions. Studies have shown that strains of this genus can inhibit excessive inflammatory responses and promote immune tolerance by regulating the host immune system response. For example, Fusicatenibacter saccharivorans can induce the differentiation of regulatory T cells (Tregs), thereby inhibiting inflammatory responses and maintaining immune homeostasis. This property makes it potentially useful in the treatment of inflammatory bowel disease, allergic diseases, and autoimmune diseases. Although the functional potential of Fusicatenibacter strains is great, their abundance in the gut microbiota is usually low, and they are difficult to isolate and culture. Traditional microbial isolation methods rely on selective media and anaerobic culture techniques, but due to the complexity of the gut microbiota and the special growth requirements of Fusicatenibacter strains, the isolation efficiency is often low. Therefore, the development of an efficient and specific enrichment and screening method is of great significance for in-depth study of the functions and applications of Fusicatenibacter strains. In recent years, based on the progress of molecular biology and high-throughput screening technology, researchers have developed a variety of novel strain enrichment and screening methods.For example, metagenomic analysis based on 16S rRNA gene sequencing can quickly identify strains of the genus Fusicatenibacter in the gut microbiota and evaluate their abundance and diversity. In addition, single-cell sorting techniques based on fluorescence in situ hybridization (FISH) and flow cytometry can isolate target strains from complex microbial communities. The application of these techniques provides new ideas for the enrichment and screening of Fusicatenibacter strains. However, existing methods still have certain limitations. For example, although metagenomic analysis can identify the presence of strains, it cannot directly obtain live strains for functional studies. Traditional culturing methods can obtain live strains, but their efficiency is low and it is difficult to meet the requirements of high-throughput screening. Therefore, developing a new enrichment and screening method that combines molecular biology and traditional culturing techniques is of great significance for improving the isolation efficiency and functional research of Fusicatenibacter strains. Summary of the Invention

[0003] Aiming at the above technical problems, the object of the present invention is to enrich and isolate gut microorganisms of the genus Fusicatenibacter by using unique devices, procedures and culture medium formulations, combining molecular biology and traditional culturing techniques, in the case of the lack of professional instrument equipment such as anaerobic workstations in ordinary laboratories. The specific steps are as follows: (1) Preparation of basic anaerobic culture medium; (2) Preparation of enrichment culture; (3) Enrichment culture; (4) Extraction of nucleic acids from gut bacteria; (5) High-throughput sequencing and analysis; (6) Isolation and culture of strains; (7) Molecular identification of gut bacteria; Preferably, the preparation of the basic anaerobic culture medium in step (1) includes the following steps: ① Prepare the basic culture medium components according to the growth rules of most gut bacteria and adjust the pH value; these culture media generally contain necessary sugars, proteins, vitamins, and some short-chain fatty acids; ② Add resazurin at a concentration of one-thousandth as an oxygen indicator, fill with nitrogen, and sterilize by autoclaving or filter sterilization. Although the oxygen indicator is not necessary for the culturing of all gut bacteria, it provides an important basis for the operation of culturing various gut bacteria. When the oxygen indicator shows red, the culture medium conditions cannot meet the growth of strict anaerobes, but can still meet the growth of facultative anaerobes and microaerophiles; ③ Prepare a cysteine solution with a concentration of more than 1% separately, fill it with nitrogen, sterilize it by high pressure or filtration, and use it before inoculation. Cysteine is a commonly used water-soluble deoxidizer. Cysteine can be directly added, or cysteine hydrochloride can be added, and their effects are equivalent; Preferably, the preparation of the special enrichment culture in step (2) is carried out according to the following steps: ① Mix 1-5 g of fresh walnut-like dried fruits with 100 ml of sterile distilled water, and use a mortar to grind at a normal speed for 5 min; dissolve about 50 mg of α-amylase (activity 380 U / mg) in 20 ml of filtered and sterilized CaCl2 solution (1 m mol / L, pH = 7.0); place 5 ml of the amylase-CaCl2 solution and the ground mixture into a 500-ml beaker, and incubate the mixture without stirring in a 37°C water bath for 30 min; ② Acidify the mixture in ① above to pH = 2.8 (±0.1) with 6 mol / L HCL solution, add about 80 mg of pepsin (activity > 3000 U / mg) to the mixture, and further incubate with shaking at 110 rpm at 37°C for 2-4 h; ③ Add 6 mol / L NaOH solution to the above solution to adjust the mixture in ② above to pH = 5.5. Dissolve 0.1 g of pancreatin (mainly trypsin and pancreatic lipase with an activity > 227 U / mg) and 0.25 g of bile extract in 10 mL of 0.5 mol / L NaHCO3 solution, inject it into the above 100-ml mixture, adjust the mixture to pH = 6.5 (±0.1), and shake and incubate at 110 rpm at 37°C for 3-5 h; Preferably, the enrichment culture in step (3) includes the following steps: ① Add 1 / 2 pore volume of the basic liquid medium in step 1) to a 5-ml vial in sequence, then add 1 / 20 pore volume of the mixture in step 2) above and 5 μl of polymyxin B solution (0.5 mg / ml); ② Add 1 / 10 volume of silicone oil, oil-soluble antioxidant, and liquid paraffin respectively, and let them naturally stratify to block oxygen. The oil-soluble antioxidant can be vitamin E and unsaturated fatty acids; ③ Add 1 / 10 volume of 1% cysteine solution, seal the cap, and place it in the refrigerator or at room temperature for more than 12 hours, waiting for the red color of the oxygen indicator to fade; ④ Take a total of 1 g of fresh feces from multiple people, add it to 10 ml of the basic medium solution containing 1% cysteine deoxidizer, shake and disperse and mix well, and let it stand for 3-5 min; ⑤ Add 1 / 20 volume of the supernatant of the mixed bacterial solution to the above vial, culture it in a 37°C incubator, and after the bacteria grow, directly dispense it into 1-ml tubes, add glycerol with a final concentration of 15% and freeze and store; Preferably, the intestinal bacteria nucleic acid extraction in step (4) includes the following steps: ① Take 1 ml of the above intestinal bacteria liquid sample, centrifuge at 4000 g for 5 min and retain the precipitate. Add 95% zirconia grinding beads. Add 500 μl of CTAB DNA extraction solution to the centrifuge tube with grinding beads and grind on a grinder for 5 min; ② Place it in a 65 °C water bath for incubation for 15 min, centrifuge at 12000 g for 1 min; Take 400 μl of the supernatant into the adsorption column, and add 200 μl of absolute ethanol to the adsorption column and mix well; After centrifuging at 12000 g for 1 min, replace the collection tube; ③ Add 500 μl of 70% ethanol, centrifuge at 12000 g for 1 min, replace the collection tube, and repeat this step once; Replace the collection tube again, place the washed adsorption column AC, drop 50 μl of elution buffer EB, centrifuge at 12000 g for 1 min in a centrifuge, collect the DNA after elution, and perform electrophoresis and imaging scanning on the extracted DNA sample; Preferably, the high-throughput sequencing and analysis in step (5) includes the following steps: ① Design for the 16s rRNA v3-v4 region, perform high-throughput sequencing, and estimate the proportion of each genus of bacteria by sequence alignment; ② Analyze the proportion of the sample containing Fusicatenibacter bacteria. If it is less than 5%, take out the cryotube and repeat steps (3) enrichment culture and (4) intestinal bacteria nucleic acid extraction process; ③ If the proportion of Fusicatenibacter bacteria is greater than 5%, continue to step (6) strain isolation; Preferably, the strain isolation and culture in step (6) includes the following steps: ① Prepare in advance a solid medium plate containing the components of the above steps (1) and (2), and a ready-to-use anaerobic bag for coating the bacterial liquid ("A ready-to-use anaerobic bag for anaerobic plate separation and culture", patent number: ZL201920214535.7) Prepare a ready-to-use anaerobic bag for coating the bacterial liquid; ② Take 50 μl of the frozen intestinal bacteria sample, dilute it 10 times and coat it on the solid medium plate, and then culture it at 37 °C for 1-2 days; ③ Pick colonies and perform anaerobic culture as described in step (3) above. After the bacteria grow, use them for subsequent nucleic acid extraction; Preferably, the intestinal bacteria molecular identification in step (7) includes the following steps: ① Extract the nucleic acid of the single colony culture one by one according to the nucleic acid extraction method in step (4) above; ② Perform PCR experiments according to the following formula: The reaction volume is 20 μl, 2 μl of 10X Taq Buffer, 1.6 μl of 2.5 mM dNTP mixture, 1 U of Taq, 0.8 μl of each 10 μM primer. The upstream primer is FusLP: TCAACAGCGCATCCTGCA, and the downstream primer FusRP is: TGTTGTCGAGGTTCGGCTG. The template volume is 1 μl, and the rest is made up with pure water; The PCR reaction program is: 95 °C for 300 s; 94 °C for 15 s, 56 °C for 45 s, 72 °C for 45 s, for a total of 35 - 40 cycles; 72 °C for 300 s; ③ If a characteristic band of 250 bp is detected by electrophoresis of the product, it is a positive colony. The bacterium to which this positive band belongs is the Fusicatenibacter bacterium. Compared with the prior art, the present invention has the following beneficial effects: 1) It can cultivate Fusicatenibacter bacteria under ordinary aerobic experimental conditions, breaking through the harsh growth conditions at low cost; 2) It can find samples suitable for screening Fusicatenibacter bacteria through enrichment and sequencing, overcoming the sample complexity; 3) It has found a pharmaceutical formulation that can induce the large-scale growth of Fusicatenibacter bacteria, reducing the screening workload. Description of the Drawings

[0004] Figure 1 . Positive band for screening Fusicatenibacter bacteria. M is the marker of DL2000. Detailed Embodiments

[0005] Example 1. Preparation of the Component Ratio of YCFA Anaerobic Medium Preparation steps: (1) Weigh and add reagents according to the formula of YCFA liquid medium (1 L), and at the same time prepare a stock solution of resazurin with a concentration of one-thousandth; (2) Add 1 ml of the resazurin stock solution (1000 times) to 1 L of the liquid medium, so that the final concentration of resazurin in the medium is 1 ppm; (3) Pour the liquid medium into an anaerobic bottle, pass high-purity nitrogen for 1 min, immediately cover the cap, and reserve it after autoclaving; (4) Prepare a stock solution of 1% cysteine as an oxygen scavenger, pass high-purity nitrogen for 1 min, immediately cover the cap, and reserve it after autoclaving; Example 2. Preparation of the Enriched Culture of Fusicatenibacter bacteria Using raw pecan kernels as materials, configure a culture plate suitable for culturing and transferring intestinal bacteria according to the following steps: (1) Mix 5 g of preliminarily chopped raw pecan kernels with 100 ml of sterile distilled water, and grind them in a mortar at a normal speed for 5 min until the diameter of the kernel particles is <1 mm; dissolve approximately 50 mg of α-amylase (activity 380 U / mg) in 20 ml of filtered and sterilized CaCl2 solution (1 mmol / L, pH = 7.0); place 5 ml of the amylase-CaCl2 solution and the kernel mixture into a 500-ml beaker, and incubate the mixture without stirring in a 37°C water bath for 30 min; (2) Acidify the kernel mixture with 6 mol / L HCl solution to pH = 2.8 (±0.1), add approximately 80 mg of pepsin to the kernel mixture, and further incubate it with shaking at 110 rpm at 37°C for 2 h; (3) Add 6 mol / L NaOH solution to adjust the kernel mixture to pH = 5.5. Dissolve 0.1 g of pancreatin (mainly trypsin and pancreatic lipase with an activity >227 U / mg) and 0.25 g of bile extract in 10 mL of 0.5 mol / L NaHCO3 solution, inject it into 100 ml of the kernel mixture, adjust the kernel mixture to pH = 6.5 (±0.1), and incubate it with shaking at 110 rpm at 37°C for 3 h; Example 3. Enrichment culture of Fusicatenibacter ① In a 5-ml vial, sequentially add 1 / 2 of the pore volume of the basic liquid medium in step 1), then add 1 / 20 of the pore volume of the enrichment culture mixture in Example 2 above and 5 μl of polymyxin B (0.5 mg / ml); ② Add 1 / 20 - 1 / 10 of the volume of silicone oil, oil-soluble antioxidant, and liquid paraffin respectively, and let them naturally stratify to block oxygen. The oil-soluble antioxidant can be vitamin E and unsaturated fatty acids; ③ Add 1 / 20 - 1 / 10 of the volume of 1% cysteine solution, seal the cap, and place it in the refrigerator or at room temperature for more than 12 h until the red color of the oxygen indicator fades; ④ Take a total of 1 g of fresh feces from multiple people, add it to 10 ml of the basic medium solution containing 1% cysteine deoxidizer, shake and disperse it evenly, and let it stand for 3 - 5 min; ⑤ Add 1 / 20 of the volume of the supernatant of the mixed fecal bacteria solution to the above vial, culture it in a 37°C incubator, and after the bacteria grow, directly aliquot it into 1-ml tubes and add glycerol with a final concentration of 15% and store it at -80°C; Example 4. Extraction of intestinal bacteria nucleic acid ① Take 1 ml of the above intestinal bacterial liquid sample, centrifuge at 4000 g for 5 min and retain the precipitate. Add 95% zirconia grinding beads. Add 500 μl of CTAB DNA extraction solution to the centrifuge tube with grinding beads, and grind on a grinder for 5 min;; ② Place it in a 65°C water bath for incubation for 15 min, centrifuge at 12000 g for 1 min; Take 400 μl of the supernatant into the adsorption column, and add 200 μl of absolute ethanol to the adsorption column, and mix well; After centrifuging at 12000 g for 1 min, replace the collection tube; ③ Add 500 μl of 70% ethanol, centrifuge at 12000 g for 1 min, replace the collection tube, and repeat this step once; Replace the collection tube again, place the washed adsorption column AC, drop 50 μl of elution buffer EB, centrifuge at 12000 g for 1 min in a centrifuge, collect the DNA after elution, and perform electrophoresis and imaging scanning on the extracted DNA sample; Example 5. High-throughput sequencing and analysis of microbial samples ① For the V4-V5 region of 16S rRNA, synthesize specific primers with barcodes for high-throughput sequencing. PCR uses TransGen AP221-02: TransStart Fastpfu DNA Polymerase; PCR instrument: ABI Model 9700. After mixing the PCR products, detect them by 2% agarose gel electrophoresis, and use the AxyPrep DNA Gel Extraction Kit (AXYGEN) to cut the gel and recover the PCR products; ② After removing the primer adapter sequences and low-quality bases (Phred Quality Score = 20) from the raw sequencing data, splice them. After removing non-specific amplification sequences and chimeras, obtain the effective sequence data of each sample. Use the Mothur software package to divide the 16S sequences into operational taxonomic units (OTUs) with a threshold of 97%. Use RDP classifier 2.2 to perform taxonomic analysis on the OTU representative sequences at a similarity level of 97%, obtain the species classification information corresponding to each OTU, and statistically analyze the bacterial community composition of each sample at the levels of kingdom, phylum, class, order, family, and genus. The results after experimental testing are as follows: Only the basic medium (CK) and the microbial flora of the basic medium containing the Fusicatenibacter enrichment (RA) had the proportions shown in Table 1. With a 5% bacterial content as the dominant bacterium, the dominant bacterium Fusicatenibacter did not appear in the CK group, and the content in the RA group reached the dominant bacterium level, indicating that the RA medium had successfully induced the genus Fusicatenibacter. Actually, through further analysis, it was found that the abundance level of the genus Fusicatenibacter in the RA group increased by more than a thousand times compared with the control; Table 1 The composition of the dominant bacterial genera in the intestinal flora affected by the RA medium Example 6. Isolation and culture of Fusicatenibacter strains Preparation of solid plates and screening of bacteria: ① Autoclave 15% agar alone. When it cools down to a temperature that is not too hot to touch, add the preheated liquid media of Example 1 and Example 2 above, mix well, pour the plates, air-dry in the laminar flow hood for 10 min, and place them in a ready-to-use anaerobic bag prepared in advance for coating with bacterial liquid (utility model patent product "A ready-to-use anaerobic bag for anaerobic plate separation and culture", patent number: ZL201920214535.7); ② After the color of the plates fades, perform operations and coating according to the requirements of the ready-to-use anaerobic bag, and culture overnight at 37 °C; ③ Pick colonies and culture them according to the liquid medium in 1) above. Finally, add the culture to a final concentration of 15% glycerol for cryopreservation for future testing; Example 7. Molecular identification of intestinal bacteria ① Take 1 ml of the preserved intestinal bacterial liquid sample, centrifuge at 4000 g for 5 min and retain the precipitate. Add 95% zirconia grinding beads. Add 500 μl of CTAB DNA extraction solution to the centrifuge tube with grinding beads, and place it on a MM400 grinder to grind at 25 HZ for 5 min; ② Place it in a 65 °C water bath for incubation for 15 min, and centrifuge at 12000 g for 1 min; Take 400 μl of the supernatant and transfer it to an adsorption column, and add 200 μl of absolute ethanol to the adsorption column, and mix well; After centrifuging at 12000 g for 1 min, replace the collection tube; ③ Add 500 μl of 70% ethanol, centrifuge at 12000 g for 1 min, replace the collection tube, and repeat this step once; Replace the collection tube again, place the washed adsorption column AC, drop 50 μl of elution buffer EB, centrifuge at 12000 g for 1 min in a centrifuge, collect the DNA after elution, and perform electrophoresis and imaging scanning on the extracted DNA sample; ④Perform PCR experiments according to the following formula: The reaction volume is 20 μl, 2 μl of 10X Taq Buffer, 1.6 μl of 2.5 mM dNTP mixture, 1 u of Taq, 0.8 μl of each 10 μM primer. The upstream primer is FusLP: TCAACAGCGCATCCTGCA, and the downstream primer FusRP is: TGTTGTCGAGGTTCGGCTG. The template volume is 1 μl, and the rest is made up with pure water; The PCR reaction program is: 95 °C for 300 s; 94 °C for 15 s, 56 °C for 45 s, 72 °C for 45 s, for a total of 35 - 40 cycles; 72 °C for 300 s. The PCR instrument is genepro Thermal Cycler (Bioer), the electrophoresis device is the Beijing Liuyi horizontal electrophoresis device, the electrophoresis marker M is DL2000 (Takara), and the screenshot is as shown in the figure. Figure 1 The bacterial sample with the 250 bp band appearing is the Fusicatenibacter bacterium.

Claims

1. Enrichment and screening Fusicatenibacter The method of claim 1, wherein: The method comprises the following steps: (1) Preparation of basic anaerobic culture medium: Prepare the basic culture medium components according to the growth rules of most intestinal bacteria and adjust the pH value. These culture media generally contain necessary sugars, proteins, vitamins, and some short-chain fatty acids. Add 1 / 1000 concentration of resazurin as an oxygen indicator, fill with nitrogen, autoclave or filter sterilize; prepare a cysteine ​​solution with a concentration of more than 1%, fill with nitrogen, autoclave or filter sterilize, and use it before inoculation; (2) Preparation of enrichment culture: ① Mix 1-5g of fresh walnuts with 100ml of sterile distilled water and grind them at normal speed for 5min in a grinding mortar; dissolve about 50mg of amylase (activity 380 U / mg) in 20ml of filtered and sterilized CaCl2 solution (1m mol / L, pH=7.0); place 5ml of amylase-CaCl2 solution and the ground mixture in a 500ml beaker and incubate the mixture without stirring for 30min in a 37℃ water bath; ② Acidify the mixture in ① above to pH = 2.8 (± 0.1) with 6 mol / L HCL solution, add about 80 mg of pepsin (activity > 3000 U / mg) to the mixture, and further incubate at 37°C with shaking at 110 rpm for 2-4 h; ③ Add 6 mol / L NaOH solution to the above solution to adjust the pH of the above ② mixture to 5.5, dissolve 0.1g pancreatic enzyme (mainly trypsin and pancreatic lipase with activity >227U / mg) and 0.25g bile extract in 10mL 0.5 mol / L NaHCO3 solution, inject into the above 100ml mixture, adjust the mixture to pH = 6.5 (±0.1), and incubate at 37℃ with shaking at 110rpm for 3-5h; (3) Enrichment culture: ① In a 5 ml vial, add 1 / 2 pore volume of the basic liquid culture medium in step 1), then add 1 / 20 pore volume of the mixed solution in step (2) above and 5 ul of polymyxin B (0.5 mg / ml); ②Add 1 / 10 volume of silicone oil, oily antioxidant and liquid paraffin respectively, and let them naturally separate and block oxygen. Oily antioxidants can be vitamin E and unsaturated fatty acids; ③ Add 1 / 10 volume of 1% cysteine ​​solution, seal and cover, place in the refrigerator or at room temperature for more than 12 hours, and wait for the red color of the oxygen indicator to fade; ④ Take 1g of fresh feces from multiple people, add it to 10ml of basic culture medium solution containing 1% cysteine ​​deoxidizer, shake and mix, and let it stand for 3-5min; ⑤ Add 1 / 20 volume of the mixed bacterial solution supernatant to the above-mentioned vial, culture in a 37°C incubator, and after the bacteria grow, aliquot into 1 ml tubes, add glycerol at a final concentration of 15%, and freeze; (4) Extraction of intestinal bacterial nucleic acid ① Take 1 ml of the above intestinal bacterial liquid sample, centrifuge at 4000g for 5 minutes and retain the precipitate, add 95% zirconium oxide grinding beads, add 500ul CTAB DNA extraction solution to the centrifuge tube with grinding beads, and grind it in a grinder for 5 minutes; ② Incubate in a 65℃ water bath for 15min, centrifuge at 12000g for 1min; take 400ul of the supernatant into the adsorption column, add 200ul of anhydrous ethanol to the adsorption column, mix thoroughly; centrifuge at 12000g for 1min, and replace the collection tube; ③ Add 500ul of 70% ethanol, centrifuge at 12000g for 1min, replace the collection tube, and repeat this step once more; replace the collection tube again, put in the cleaned adsorption column AC, drop 50μl of elution buffer EB, centrifuge at 12000g for 1min, collect DNA after elution, and perform electrophoresis and imaging scanning on the extracted DNA sample; (5) High-throughput sequencing and analysis: ① Design high-throughput sequencing for the 16s rRNA v3-v4 region, and estimate the proportion of each bacterial genus through sequence alignment; ②Analysis samples include Fusicatenibacter If the proportion of bacteria is less than 5%, take out the cryopreserved tube and repeat the culture and nucleic acid extraction process in step (3); ③If Fusicatenibacter If the bacterial ratio is greater than 5%, continue with the strain separation in step (5); (6) Strain isolation and cultivation: ① Prepare in advance the solid culture medium plates containing the ingredients of step (1) and step (2) above, and the ready-to-use anaerobic bags for bacterial liquid coating; ② Take 50ul of the frozen intestinal bacteria sample, dilute it 10 times, and then spread it on the solid culture medium in the above step (2), and then culture it at 37°C for 1-2 days; ③ Pick the clones and carry out anaerobic culture as described in step (3) above. After the bacteria grow out, they are used for subsequent nucleic acid extraction; (7) Molecular identification of intestinal bacteria: ① Extract nucleic acid from single colony cultures one by one according to the nucleic acid extraction method in step 3) above; ②Perform PCR experiment according to the following formula: reaction volume 20ul, 10X Taq Buffer 2ul, 2.5 mM dNTPmixture 1.6ul, Taq 1u, 10uM primer 0.8ul each, upstream primer is FusLP: TCAACAGCGCATCCTGCA, downstream primer FusRP is: TGTTGTCGAGGTTCGGCTG, template volume is 1ul, and the rest is made up with pure water; PCR reaction program: 95°C 300s; 94°C 15s, 56°C 45s, 72°C 45s, a total of 35-40 cycles; 72°C 300s; ③ The product is detected by electrophoresis. If there is a 200bp characteristic band, it is a positive colony. The bacteria to which the positive band belongs are Fusicatenibacter bacteria.

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