Microbial marker for evaluating visceral obesity and application thereof

Abstract

The invention belongs to the technical field of biological engineering, and discloses a microbial marker for evaluating visceral obesity and application of the microbial marker. The microbial marker is characterized by further comprising one or two or more of the following components: bacillus subtilis, bacillus subtilis, bacillus subtilis, bacillus subtilis, bacillus subtilis, bacillus subtilis, bacillus subtilis, bacillus subtilis, bacillus subtilis, bacillus subtilis, bacillus subtilis, bacillus subtilis, bacillus subtilis, bacillus subtilis and bacillus subtilis, and bacillus subtilis, bacillus subtilis, bacillus subtilis, bacillus subtilis, bacillus subtilis, bacillus subtilis, bacillus subtilis, bacillus subtilis, bacillus subtilis, bacillus subtilis, bacillus subtilis, bacillus subtilis, bacillus subtilis, bacillus subtilis, bacillus subtilis, bacillus subtilis, bacillus subtilis, bacillus subtilis, bacillus subtilis, bacillus subtilis, bacillus subtilis, bacillus subtilis, bacillus subtilis, bacillus subtilis, bacillus subtilis, bacillus subtilis, bacillus subtilis, bacillus subtilis, bacillus subtilis, bacillus subtilis and bacillus subtilis. The microbial marker can be used as a non-invasive biomarker, is used for rapidly detecting visceral obesity individuals, and is low in determination cost, simple and safe in operation method, sensitive and accurate.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering technology, and in particular to a microbial marker for evaluating visceral obesity and an application thereof. Background Art

[0002] Obesity is a global health crisis characterized by excessive accumulation of body fat and is associated with multiple metabolic disorders. A particularly harmful form of obesity involves the accumulation of visceral fat, which is stored around internal organs in the abdominal cavity. Visceral fat is more metabolically active than subcutaneous fat, and its excessive accumulation has been associated with an increased risk of cardiovascular disease, type 2 diabetes, and other obesity-related diseases.

[0003] Measurement of visceral fat is essential for assessing these health risks. Advanced imaging techniques, such as computed tomography (CT), can accurately quantify visceral fat by determining the area and volume of visceral fat. In addition, the ratio of visceral fat to subcutaneous fat obtained by CT scans is a valuable indicator for assessing the severity of visceral obesity and its potential health effects.

[0004] Recent studies have highlighted the important role of the gut microbiota in obesity and metabolic health. The gut microbiota consists of trillions of microorganisms that reside in the gastrointestinal tract and influence host metabolism, immune function, and energy homeostasis. Emerging evidence suggests that specific changes in the composition of the gut microbiota are associated with obesity and its complications. In particular, the relationship between visceral fat and the gut microbiota has received increasing attention, with certain microbial signatures associated with visceral fat accumulation and metabolic disorders.

[0005] Understanding the interaction between visceral fat and the gut microbiota could pave the way for new diagnostic and therapeutic approaches to obesity and its related diseases. Gut microbiota analysis of individuals with high visceral fat may reveal specific microbial signatures that contribute to or are caused by visceral fat accumulation. These microbial markers could serve as diagnostic tools to identify individuals at risk for visceral obesity and its associated complications, helping to improve metabolic health and mitigate the risks associated with visceral obesity.

[0006] Recent studies have begun to elucidate the complex relationship between visceral fat and the gut microbiota. It has been observed that individuals with higher visceral fat content exhibit different gut microbiota signatures compared to individuals with lower visceral fat content. These differences in microbial composition are thought to influence the accumulation of visceral fat and subsequent metabolic outcomes. For example, certain bacterial taxa, such as members of the phylum Firmicutes, were positively correlated with visceral fat accumulation, while Bacteroidetes were negatively correlated. The potential to use the gut microbiota as a diagnostic tool for visceral obesity is an exciting avenue of research. At the same time, understanding the relationship between visceral fat and the gut microbiota is critical for developing novel diagnostic and therapeutic strategies for obesity and its related diseases. Summary of the invention

[0007] The purpose of the present invention is to solve the problem that the current determination of visceral obesity still mainly relies on CT, which is expensive, takes a long time, has complicated procedures, and has radiation problems. Therefore, a microbial marker for evaluating visceral obesity and its application are provided. The microbial marker can be used as a non-invasive biomarker for rapid detection of individuals with visceral obesity. The determination cost is low, the operation method is simple and safe, and it is sensitive and accurate.

[0008] In order to achieve the above-mentioned object, in a first aspect, the present invention provides a microbial marker for evaluating visceral obesity, wherein the microbial marker contains a specific bacterial group Blautia; The microbial markers also contain one or two or more of Subdoligranulum, Bacteroides, Megasphaera, Ruminococcus, Roseburia and Ruminococcus_gauvreauii_groupp.

[0009] In a second aspect, the present invention provides a use of the microbial marker as described in the first aspect in the preparation of a product for assessing visceral obesity.

[0010] In a third aspect, the present invention provides a kit for assessing visceral obesity, the kit comprising one or two or more oligonucleotides that can specifically hybridize with a target nucleotide sequence from Blautia; The kit also includes one or two or more oligonucleotides that can specifically hybridize with one or two or more of the target nucleotide sequences from Subdoligranulum, the target nucleotide sequence from Bacteroides, the target nucleotide sequence from Megasphaera, the target nucleotide sequence from Ruminococcus, the target nucleotide sequence from Roseburia, and the target nucleotide sequence from Ruminococcus_gauvreauii_groupp.

[0011] Preferably, the kit further comprises reagents for isolating nucleic acid from the sample.

[0012] Preferably, the oligonucleotide is detectably labeled.

[0013] Preferably, the target nucleotide sequence of Blautia, the target nucleotide sequence of Subdoligranulum, the target nucleotide sequence of Bacteroides, the target nucleotide sequence of Megasphaera, the target nucleotide sequence of Ruminococcus, the target nucleotide sequence of Roseburia and the target nucleotide sequence of Ruminococcus_gauvreauii_groupp are fragments of the 16S ribosomal ribonucleic acid gene.

[0014] Preferably, the oligonucleotide is selected from one or two or more of a probe that specifically recognizes a target nucleotide sequence of Blautia, a probe that specifically recognizes a target nucleotide sequence of Subdoligranulum, a probe that specifically recognizes a target nucleotide sequence of Bacteroides, a probe that specifically recognizes a target nucleotide sequence of Megasphaera, a probe that specifically recognizes a target nucleotide sequence of Ruminococcus, a probe that specifically recognizes a target nucleotide sequence of Roseburia, and a probe that specifically recognizes a target nucleotide sequence of Ruminococcus_gauvreauii_groupp.

[0015] Preferably, the oligonucleotide is selected from one or two or more of a primer that specifically amplifies a target nucleotide sequence of Blautia, a primer that specifically amplifies a target nucleotide sequence of Subdoligranulum, a primer that specifically amplifies a target nucleotide sequence of Bacteroides, a primer that specifically amplifies a target nucleotide sequence of Megasphaera, a primer that specifically amplifies a target nucleotide sequence of Ruminococcus, a primer that specifically amplifies a target nucleotide sequence of Roseburia, and a primer that specifically amplifies a target nucleotide sequence of Ruminococcus_gauvreauii_groupp.

[0016] Preferably, the specific amplification method is PCR and / or RT-PCR.

[0017] Preferably, the primers for specific amplification are primers carrying a labeled sequence.

[0018] Preferably, the primers for specific amplification are: The nucleotide sequence of the upstream primer is shown in SEQ.ID.NO:1; The nucleotide sequence of the downstream primer is shown in SEQ.ID.NO:2.

[0019] In the above technical scheme, the present invention performs 16S sequencing on the feces of people with visceral obesity, and finds the differential flora of people with visceral obesity, such as Blautia, Faecalibacterium, Subdoligranulum, Bacteroides, Megasphaera, Ruminococcus, Roseburia and Ruminococcus_gauvreauii_groupp, and evaluates the correlation of these flora with visceral obesity. Through different ROC curve analyses, it is found that Blautia as a detection variable has higher sensitivity and accuracy for the diagnosis of visceral obesity than other flora, with an AUC value of 0.84, and can be used as a detection target for the diagnosis of visceral obesity.

[0020] At the same time, Blautia can also be combined with other bacterial flora or triglyceride values ​​as a detection target for the diagnosis of visceral obesity to make the results more accurate.

[0021] Other features and advantages of the present invention will be described in detail in the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the following specific embodiments, they are used to explain the present invention but do not constitute a limitation of the present invention. In the accompanying drawings: Figure 1 A Wilcoxon rank sum test result diagram of the ACE index of two obesity types involved in the present invention; Figure 2 It is a principal coordinate analysis diagram of species level for two obesity types involved in the present invention; Figure 3 A Venn diagram of two types of obesity involved in the present invention; Figure 4 This is a diagram showing the results of the Wilcoxon rank sum test of two obesity types involved in the present invention at the genus level; Figure 5 The relative abundance distribution of the microbial communities of the two obesity types involved in the present invention at the genus level; Figure 6 Heatmap of Spearman correlations between different genera and various metabolic and clinical indicators; Figure 7 Figure 2 ROC curves of the performance of Blautia abundance and triglyceride (TG) level alone and in combination for predicting individuals with visceral obesity at the genus level. DETAILED DESCRIPTION

[0023] The specific embodiments of the present invention are described in detail below. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention.

[0024] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0025] In order to evaluate whether the composition of intestinal flora can be used as a predictor of visceral obesity, the present invention collects fecal samples from people with visceral obesity and people with subcutaneous fat obesity, performs 16S rRNA sequencing, and uses bioinformatics to perform statistics on sequencing data, thereby screening out intestinal flora related to visceral obesity, integrating intestinal flora with disease information, and predicting visceral obesity patients to the greatest extent. The present invention, through 16S rRNA sequencing, discovered for the first time that Blautia showed significant differences between people with visceral obesity and people with subcutaneous fat obesity, indicating that Blautia can be used as a predictor of visceral obesity.

[0026] In the embodiments of the present invention, many conventional techniques in molecular biology, protein biochemistry, cell biology, immunology, microbiology and recombinant DNA are used, and these techniques are well known.

[0027] As used herein, the term "fragment" refers to a polynucleotide that is at least about 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 200, 300, 400, 500, 1000 nucleotides in length or more.

[0028] The term "nucleic acid" as used herein refers broadly to: a segment of a chromosome; a segment or portion of DNA, cDNA and / or RNA. Nucleic acids can be obtained or acquired from a nucleic acid sample initially isolated from any source (e.g., isolated from, purified from, amplified, cloned or reverse transcribed from sample DNA or RNA).

[0029] As used herein, the term "oligonucleotide" refers to a short polymer consisting of deoxyribonucleotides, ribonucleotides or any combination thereof. The length of an oligonucleotide is generally between 10 nucleotides and about 100 nucleotides. An oligonucleotide preferably has a length of 15 nucleotides to 70 nucleotides, and most commonly 20 nucleotides to 26 nucleotides. An oligonucleotide can be used as a primer or a probe.

[0030] An oligonucleotide is "specific" for a nucleic acid if, when the oligonucleotide and nucleic acid are aligned, the oligonucleotide has at least 50% sequence homology with a portion of the nucleic acid. An oligonucleotide specific for a nucleic acid is one that hybridizes to the target of interest and does not substantially hybridize to nucleic acids not of interest under appropriate hybridization or washing conditions. Higher degrees of sequence homology are preferred and include sequence homology of at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%.

[0031] As used herein, the term "hybridization" or "specific hybridization" refers to the annealing of two complementary nucleic acid strands to each other under appropriately stringent conditions. Hybridization is usually performed using nucleic acid molecules of the length of a probe. Nucleic acid hybridization techniques are well known in the art. Those skilled in the art understand how to estimate and adjust the stringency of hybridization conditions so that sequences with at least a desired degree of complementarity will stably hybridize, while sequences with less complementarity will not stably hybridize.

[0032] The term "amplification" used herein means one or more methods known in the prior art for replicating a target nucleic acid and thus increasing the number of copies of a selected nucleic acid sequence. Amplification can be exponential or linear. The target nucleic acid can be a DNA or RNA. The sequence amplified in this way forms an "amplicon". Although the exemplary method described below involves amplification using polymerase chain reaction ("PCR"), many other methods (e.g., isothermal method, rolling circle method, etc.) for amplifying nucleic acids are known in the prior art. It will be appreciated by those skilled in the art that these other methods can be used in place of the PCR method or can be used together with the PCR method.

[0033] The term "target nucleic acid" or "target nucleotide" as used herein refers to a fragment of a chromosome for which a probe or primer is designed, a complete gene with or without an intergenic sequence, a fragment or part of a gene with or without an intergenic sequence, or a nucleic acid sequence. The target nucleic acid may include: a wild-type sequence; a nucleic acid sequence containing a mutation, deletion, or duplication; a tandem repeat; a gene of interest; a region of a gene of interest or any upstream or downstream region thereof. The target nucleic acid may represent an alternative sequence or allele of a specific gene. The target nucleic acid may be obtained from genomic DNA, cDNA, or RNA. The target nucleic acid used herein may be natural DNA or a product amplified by PCR. In one embodiment, the target nucleic acid is a fragment of a 16S ribosomal RNA gene from a bacterial population.

[0034] In a first aspect, the present invention provides a microbial marker for assessing visceral obesity, wherein the microbial marker is characterized by the abundance of a specific bacterial community, Blautia; The microbial marker also contains one or two or more of Subdoligranulum, Bacteroides, Megasphaera, Ruminococcus, Roseburia and Ruminococcus_gauvreauii_groupp. For example, the microbial marker contains Blautia and Subdoligranulum; the microbial marker contains Blautia and Bacteroides; the microbial marker contains Blautia and Megasphaera; the microbial marker contains Blautia and Ruminococcus; the microbial marker contains Blautia, Subdoligranulum and Bacteroides; the microbial marker contains Blautia, Subdoligranulum, Bacteroides, Megasphaera and Ruminococcus; the microbial marker contains combinations of Blautia, Subdoligranulum, Bacteroides, MegasphaeraRuminococcus, Roseburia and Ruminococcus_gauvreauii_groupp.

[0035] In a second aspect, the present invention provides a use of the microbial marker as described in the first aspect in the preparation of a product for assessing visceral obesity.

[0036] In a third aspect, the present invention provides a kit for assessing visceral obesity, the kit comprising one or two or more oligonucleotides that can specifically hybridize with a target nucleotide sequence from Blautia; The kit also includes one or two or more oligonucleotides that can specifically hybridize with one or two or more of the target nucleotide sequences from Subdoligranulum, the target nucleotide sequence from Bacteroides, the target nucleotide sequence from Megasphaera, the target nucleotide sequence from Ruminococcus, the target nucleotide sequence from Roseburia, and the target nucleotide sequence from Ruminococcus_gauvreauii_groupp.

[0037] In a preferred embodiment of the present invention, the kit further comprises a reagent for isolating nucleic acid from a sample.

[0038] In a preferred embodiment of the invention, the oligonucleotide is detectably labeled.

[0039] In a preferred embodiment of the present invention, the target nucleotide sequence of Blautia, the target nucleotide sequence of Subdoligranulum, the target nucleotide sequence of Bacteroides, the target nucleotide sequence of Megasphaera, the target nucleotide sequence of Ruminococcus, the target nucleotide sequence of Roseburia and the target nucleotide sequence of Ruminococcus_gauvreauii_groupp are fragments of the 16S ribosomal ribonucleic acid gene.

[0040] In a preferred embodiment of the present invention, the oligonucleotide is selected from one or two or more of a probe that specifically recognizes a target nucleotide sequence of Blautia, a probe that specifically recognizes a target nucleotide sequence of Subdoligranulum, a probe that specifically recognizes a target nucleotide sequence of Bacteroides, a probe that specifically recognizes a target nucleotide sequence of Megasphaera, a probe that specifically recognizes a target nucleotide sequence of Ruminococcus, a probe that specifically recognizes a target nucleotide sequence of Roseburia, and a probe that specifically recognizes a target nucleotide sequence of Ruminococcus_gauvreauii_groupp.

[0041] In a preferred embodiment of the present invention, the oligonucleotide is selected from one or two or more of primers for specifically amplifying a target nucleotide sequence of Blautia, primers for specifically amplifying a target nucleotide sequence of Subdoligranulum, primers for specifically amplifying a target nucleotide sequence of Bacteroides, primers for specifically amplifying a target nucleotide sequence of Megasphaera, primers for specifically amplifying a target nucleotide sequence of Ruminococcus, primers for specifically amplifying a target nucleotide sequence of Roseburia, and primers for specifically amplifying a target nucleotide sequence of Ruminococcus_gauvreauii_groupp.

[0042] In a preferred embodiment of the present invention, the specific amplification method is PCR and / or RT-PCR.

[0043] In a preferred embodiment of the present invention, the primers for specific amplification are primers carrying a labeled sequence.

[0044] In a preferred embodiment of the present invention, the primers for specific amplification are: The nucleotide sequence of the upstream primer is shown in SEQ.ID.NO:1, SEQ.ID.NO:1 5′-ACTCCTACGGGAGGCAGCAG-3′; The nucleotide sequence of the downstream primer is shown in SEQ.ID.NO:2. SEQ.ID.NO:2 5′-GGACTACHVGGGTWTCTAAT-3′.

[0045] The present invention will be described in detail below by way of examples. The experimental methods and equipment described in the following examples are conventional methods and equipment unless otherwise specified.

[0046] Example 1

[0047] This example is used to screen for intestinal flora associated with visceral obesity (1) Research subjects and sample collection: 64-slice spiral CT (Somatom definition AS, German) was used to diagnose people with visceral obesity and subcutaneous fat obesity, and their stool samples were collected; (2) 16S rRNA sequencing: a. DNA extraction: The total genomic DNA of the microbial community was extracted according to the instructions of the genomic DNA extraction kit (PF Mag-Bind Stool DNA Kit (OmegaBio-tek, Georgia, US)). The integrity of the extracted genomic DNA was detected by 1% agarose gel electrophoresis, and the DNA concentration and purity were determined using NanoDrop2000 (Thermo Scientific, USA); b. PCR amplification: Using the DNA extracted in step a as a template, PCR amplification of the V3-V4 variable region of the 16S rRNA gene was performed using the upstream primer 338F (5'-ACTCCTACGGGAGGCAGCAG-3') and the downstream primer 806R (5'-GGACTACHVGGGTWTCTAAT-3') carrying the Barcode sequence, wherein: The amplification program was as follows: 95°C pre-denaturation for 3 min, 27 cycles (95°C denaturation for 30 s, 55°C annealing for 30 s, 72°C extension for 30 s), then 72°C stable extension for 10 min, and finally storage at 4°C (PCR instrument: ABI GeneAmp® 9700); PCR reaction system: 5× TransStart FastPfu Buffer 4 μL, 2.5mM dNTPs 2 μL, Upstream Primer (5uM) 0.8 μL, Downstream Primer (5uM) 0.8 μL, TransStart FastPfu 0.4 μL DNA polymerase, 10 ng template DNA, make up to 20 μL; Each sample was replicated three times. The PCR products of the same sample were mixed and recovered using 2% agarose gel. The PCR products were purified and the size of the band fragments was detected by 2% agarose gel electrophoresis. The recovered products were detected and quantified using Quantus™ Fluorometer (Promega, USA).

[0048] (3) Sequencing library construction: The purified PCR products were used to construct libraries using the NEXTFLEX Rapid DNA-Seq Kit (Bioo Scientific, Austin, Texas, USA): a. Connector link; b. Use magnetic beads to screen and remove the self-ligated fragments; c. Enrichment of library templates using PCR amplification; d. Recover the PCR product with magnetic beads to obtain the final library.

[0049] Sequencing was performed using the Illumina PE300 / PE250 platform (Shanghai Meiji Biopharmaceutical Technology Co., Ltd.).

[0050] (4) High-throughput sequencing data analysis Use fastp (https: / / github.com / OpenGene / fastp, version 0.19.6) software to perform quality control on the double-end raw sequencing sequences, and use FLASH (http: / / www.cbcb.umd.edu / software / flash, version 1.2.11) software for splicing: a. Filter the bases with a quality value of less than 20 at the tail of the reads, set a 50bp window, and if the average quality value in the window is less than 20, cut off the back end bases from the window, filter the reads below 50bp after quality control, and remove the reads containing N bases; b. According to the overlap relationship between PE reads, the paired reads are spliced ​​(merged) into a sequence, with a minimum overlap length of 10bp; c. The maximum mismatch ratio allowed in the overlap region of the spliced ​​sequence is 0.2, and non-compliant sequences are screened; d. Differentiate samples based on the barcodes and primers at both ends of the sequence and adjust the sequence direction. The allowed number of mismatches for the barcode is 0, and the maximum number of mismatches for the primers is 2.

[0051] Based on the default parameters, the optimized sequences after quality control splicing were denoised using the DADA2 plug-in in the Qiime2 process. The sequences after DADA2 denoising are usually called ASVs (i.e., amplicon sequence variants). The chloroplast and mitochondrial sequences annotated to all samples were removed (if the samples were contaminated with chloroplasts and mitochondria, it is recommended to remove them). In order to minimize the impact of sequencing depth on subsequent Alpha diversity and Beta diversity data analysis, the number of sequences of all samples was flattened to 20,000. After flattening, the average sequence coverage (Good's coverage) of each sample could still reach 99.09%. Based on the Sliva 16S rRNA gene database (v 138), the Naive bayes classifier in Qiime2 was used to perform species taxonomy analysis on ASVs. PICRUSt2 (version 2.2.0) software was used for 16S function prediction analysis.

[0052] Example 2

[0053] Verify the correlation between the flora involved in the present invention and visceral obesity The data obtained in Example 1 were analyzed. All data analyses were performed on the MajorBio cloud platform (https: / / cloud.majorbio.com), as follows: The mothur software (http: / / www.mothur.org / wiki / Calculators, version 1.30.2) was used to calculate the alpha diversity index Chao1, Shannon index, etc., and the Wilxocon rank sum test was used to analyze the inter-group differences in alpha diversity; PCoA analysis (principal coordinate analysis) based on the Bray-Curtis distance algorithm was used to test the similarity of the microbial community structure between samples, and the PERMANOVA non-parametric test was combined to analyze whether the differences in microbial community structure between sample groups were significant; Welch's confidence inverted method was used to calculate the effect size of different species, and the confidence interval was calculated using the Welch T test method.

[0054] LEfSe analysis (Linear discriminant analysis Effect Size) (http: / / huttenhower.sph.harvard.edu / LEfSe) (LDA>2, P<0.05) was used to determine the bacterial groups with significant differences in abundance from phylum to genus levels among different groups; Distance-based redundancy analysis (db-RDA) was used to investigate the impact of clinical indicators on the structure of intestinal bacterial communities; The correlation between clinical parameters and Blautia was analyzed using the MaAsLin2 package; The random forest algorithm was combined to screen out genera with high diagnostic value, and its diagnostic efficacy was verified by ROC curve.

[0055] The main results are as follows Figure 1-7 shown.

[0056] Depend on Figure 1 The data showed that there was a difference in the Ace index between the visceral obesity group and the subcutaneous obesity group at the ASV (Amplicon Sequence Variant) level. The Ace index of the subcutaneous obesity group was significantly higher than that of the visceral obesity group (Wilcoxon rank sum test, ** indicates P value < 0.01), indicating that subcutaneous obesity is associated with higher microbial diversity.

[0057] Depend on Figure 2 The PCoA (principal coordinate analysis) diagram shows that at the genus level, the clustering of the microbial communities between the visceral obesity group and the subcutaneous obesity group, the two groups are separated along the first two principal components PC1 (22.77%) and PC2 (14.12%). The analysis showed that there were significant differences in the microbial composition of the two obesity types (R = 0.2608, P = 0.001), and each group showed a different clustering pattern.

[0058] Figure 3 The Venn diagram shows the number of common and unique genera in the microbial composition of visceral obesity and subcutaneous obesity groups. There are 38 unique genera in the visceral obesity group, 61 unique genera in the subcutaneous obesity group, and 148 genera in common between the two groups, indicating that there are differences in microbial diversity between the two obesity types, and there are also significant common microbial communities.

[0059] Depend on Figure 4 It can be seen that at the genus level, the Wilcoxon rank sum test results between the visceral obesity and subcutaneous obesity groups, the bar graph on the left shows the relative abundance ratio of each genus in the two groups, and the forest plot on the right shows the difference in relative abundance between the two groups and the 95% confidence interval; the significance of the P value is marked as * (P < 0.05) and ** (P < 0.01), indicating that there are significant differences in the abundance of these genera between different groups.

[0060] Depend on Figure 5 The bar chart shows the relative abundance distribution of microbial communities at the genus level in the visceral obesity and subcutaneous obesity groups. Each color represents a specific genus, and the height of different colors indicates the relative abundance ratio of the genus in the group. Figure 5 The figure shows the difference in microbial community composition between the two types of obesity. The abundance distribution of various genera varies between the two groups. This comparison helps to understand the diversity of intestinal microorganisms and potential specific genera under different obesity types. Among them, the Blautia genus (red) has the largest difference in proportion, indicating that the Blautia genus can be used as a microbial marker to distinguish the two types of obesity.

[0061] Figure 6 The Spearman correlation heatmap shows the correlations between different genera and various metabolic and clinical indicators.

[0062] The colors indicate the strength and direction of the correlation, red represents positive correlation, blue represents negative correlation, and the * symbol indicates the significance level of the correlation. It can be clearly seen that the Blautia genus is significantly positively correlated with VFA (visceral fat area) and triglyceride (TG) levels, which indicates that Blautia is associated with these metabolic or clinical indicators, plays an important role in certain metabolic or health states, and has the potential to be used as a marker to detect these metabolic or clinical indicators.

[0063] Figure 7 The ROC curves show the efficacy of Blautia abundance and triglyceride (TG) level alone and in combination in predicting visceral obesity at the genus level.

[0064] Among them, the blue curve indicates that the AUC value of Blautia is 0.84 (95% CI: 0.68-0.99), the orange curve indicates that the AUC value of TG is 0.54 (95% CI: 0.29-0.79), and the red curve indicates that the AUC value of Blautia combined with TG is 0.85 (95% CI: 0.7-1). It can be seen that when Blautia is used alone as an indicator to distinguish the two types of obesity, it has a higher sensitivity and specificity, and when combined with TG as a common indicator to distinguish the two types of obesity, it has a higher sensitivity and specificity.

[0065] In summary, the microbial markers for assessing visceral obesity of the present invention can be used as non-invasive biomarkers for rapid detection of individuals with visceral obesity, with low measurement cost, simple and safe operation method, and high sensitivity and accuracy.

[0066] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0067] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

[0068] In addition, various embodiments of the present invention may be arbitrarily combined, and as long as they do not violate the concept of the present invention, they should also be regarded as the contents disclosed by the present invention.

Claims

1. A microbial marker for evaluating visceral obesity, characterized in that: The microbial marker contains a specific bacterial group, Blautia; The microbial markers also contain one or two or more of Subdoligranulum, Bacteroides, Megasphaera, Ruminococcus, Roseburia and Ruminococcus_gauvreauii_groupp.

2. Use of the microbial marker according to claim 1 in the preparation of a product for assessing visceral obesity.

3. A kit for evaluating visceral obesity, characterized in that: The kit includes one or two or more oligonucleotides that can specifically hybridize with a target nucleotide sequence from Blautia; The kit also includes one or two or more oligonucleotides that can specifically hybridize with one or two or more of a target nucleotide sequence from Subdoligranulum, a target nucleotide sequence from Bacteroides, a target nucleotide sequence from Megasphaera, a target nucleotide sequence from Ruminococcus, a target nucleotide sequence from Roseburia, and a target nucleotide sequence from Ruminococcus_gauvreauii_groupp; And / or, the kit further comprises a reagent for isolating nucleic acid from a sample.

4. The kit according to claim 3, characterized in that The oligonucleotide is detectably labeled.

5. The kit according to claim 3, characterized in that The target nucleotide sequence of Blautia, the target nucleotide sequence of Subdoligranulum, the target nucleotide sequence of Bacteroides, the target nucleotide sequence of Megasphaera, the target nucleotide sequence of Ruminococcus, the target nucleotide sequence of Roseburia and the target nucleotide sequence of Ruminococcus_gauvreauii_groupp are fragments of the 16S ribosomal ribonucleic acid gene.

6. The kit according to claim 3, characterized in that The oligonucleotide is selected from one or two or more of a probe that specifically recognizes a target nucleotide sequence of Blautia, a probe that specifically recognizes a target nucleotide sequence of Subdoligranulum, a probe that specifically recognizes a target nucleotide sequence of Bacteroides, a probe that specifically recognizes a target nucleotide sequence of Megasphaera, a probe that specifically recognizes a target nucleotide sequence of Ruminococcus, a probe that specifically recognizes a target nucleotide sequence of Roseburia, and a probe that specifically recognizes a target nucleotide sequence of Ruminococcus_gauvreauii_groupp; and / or, The oligonucleotide is selected from one or two or more of a primer that specifically amplifies a target nucleotide sequence of Blautia, a primer that specifically amplifies a target nucleotide sequence of Subdoligranulum, a primer that specifically amplifies a target nucleotide sequence of Bacteroides, a primer that specifically amplifies a target nucleotide sequence of Megasphaera, a primer that specifically amplifies a target nucleotide sequence of Ruminococcus, a primer that specifically amplifies a target nucleotide sequence of Roseburia, and a primer that specifically amplifies a target nucleotide sequence of Ruminococcus_gauvreauii_groupp.

7. The kit according to claim 6, characterized in that The specific amplification method is PCR and / or RT-PCR.

8. The kit according to claim 7, characterized in that The primers for specific amplification are primers carrying a labeled sequence; and / or, The primers for the specific amplification are: The nucleotide sequence of the upstream primer is shown in SEQ.ID.NO:1; The nucleotide sequence of the downstream primer is shown in SEQ.ID.NO:2.

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