CN114045326A - Diarrhea-type irritable bowel syndrome intestinal microbial marker and application thereof - Google Patents
Diarrhea-type irritable bowel syndrome intestinal microbial marker and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of microorganisms, and particularly relates to a diarrhea-predominant irritable bowel syndrome intestinal microbial marker and application thereof. The intestinal microbial markers include genus Staphylococcus, Akkermansia, Enterobacter, Dubosiella, and Lactobacillus. The invention discovers the level change of intestinal flora in mice with diarrhea-predominant irritable bowel syndrome for the first time, realizes the early diagnosis of diarrhea-predominant irritable bowel syndrome, and has the advantages of high specificity, strong sensitivity, simple and easy acquisition and processing of detection objects, no invasiveness, low cost and the like; the discovery of the marker provides reference for diagnosing the microbial diarrhea-predominant irritable bowel syndrome.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to a diarrhea-predominant irritable bowel syndrome intestinal microbial marker and application thereof.
Background
Diarrhea-predominant irritable bowel syndrome (IBS-D) is a gastrointestinal disorder with Diarrhea, abdominal distension and abdominal pain as the main symptoms, and recurrent attacks are often observed. The etiology and pathogenesis of IBS-D are not completely clear, and the clinical manifestations lack specificity, making diagnosis difficult. The identification of diagnostic biomarkers in irritable bowel syndrome has been a problem of interest and may lead to a rapid diagnosis of IBS-D in the future. At present, the diagnosis of IBS-D is mainly based on clinical symptoms, so the diagnosis is subjective and easy to miss diagnosis and misdiagnosis. With the progress of basic and clinical research, some potential IBS-related biomarkers are receiving extensive attention in order to find objective indicators to assist IBS diagnosis. Although gut kinetics, stool characteristics, autonomic reactivity, stool trypsin levels, etc. have been used as potential biological markers for IBS-D, such indicators have limited value for diagnosing IBS-D. Current research has found that the occurrence and development of IBS-D are closely related to the occurrence and development of intestinal microecological disorders. In recent years, intestinal biological disorders have been found to be associated with the pathogenesis of diarrhea-predominant irritable bowel syndrome, and a number of genera, some of which may have potential diagnostic value, are closely related to the pathogenesis of IBS-D.
Both animal and human derived studies have evidence to support the critical role of the gastrointestinal microbiota in the development and persistence of irritable bowel syndrome. First, the sterile mouse model provides direct evidence that intestinal microbiota can induce local intestinal dysfunction, leading to alterations in microbiota and characteristics of irritable bowel syndrome, such as visceral hypersensitivity in 4-week mice. Behavioral changes were also found in transplanted mice, suggesting that biological disorders may be responsible for the behavioral symptoms and colonic motor dysfunction of irritable bowel syndrome. Differences in the gut membrane and mucosal gastrointestinal microbiome of IBS-D from the control group were reported in all bacterial classifications using a series of qualitative and quantitative microbiological methods. The diversity, composition and function of intestinal microflora are strongly related to the symptoms of irritable bowel syndrome. According to these characteristics, the use of enterobacteria as food preparations has been studied for improving and promoting the healthy development of animal organisms. The symptoms of irritable bowel syndrome in animals are also strongly related to intestinal microorganisms, which cause anxiety and depression symptoms of irritable bowel syndrome in the host through the gut-brain axis. In addition, the biological species diversity of intestinal microorganisms of the diarrhea-predominant irritable bowel syndrome is obviously lower than that of healthy individuals, the diarrhea-predominant irritable bowel syndrome and the healthy individuals can be well identified according to the preference of intestinal bacteria, the accuracy can reach 90%, and the feasibility of finding the intestinal microorganisms which are obviously related to the diarrhea-predominant irritable bowel syndrome is further demonstrated.
In fact, the discovery and provision of microbial markers that are significantly associated with the symptoms of diarrhea-predominant irritable bowel syndrome are of great significance in both mouse model studies and in the treatment of IBS-D. In the future, the coliform bacteria can also provide reference materials for developing and improving IBS-D type microbial products, and can also be used for microecological preparations for treating IBS-D.
Disclosure of Invention
Aiming at the defects generally existing in the prior art, the invention provides a diarrhea-predominant irritable bowel syndrome intestinal microbial marker and application thereof. The microbial marker provided by the invention has the advantages of high specificity, strong sensitivity, simple and easy acquisition and processing of a detection object, no invasion, low cost and the like; the discovery of the marker provides reference for diagnosing the microbial diarrhea-predominant irritable bowel syndrome.
In order to achieve the purpose, the invention adopts the technical scheme that:
a diarrhea-predominant intestinal microbial marker for irritable bowel syndrome comprises a microorganism belonging to the genus Staphylococcus, Akkermansia, Enterobacter, Dubosiella, and Lactobacillus.
Preferably, the relative abundance of said Staphylococcus and Enterobacter flora shows an increase in the diarrheal irritable bowel syndrome animal model group, and the relative abundance of Akkermansia, Dubosiella and Lactobacillus flora decreases in the diarrheal irritable bowel syndrome animal model group.
Preferably, the relative abundance is calculated by analyzing species with obvious difference among groups, and further estimating the influence of the abundance of each component on the difference effect among intestinal flora groups by adopting linear discriminant analysis.
The invention also provides a method for screening the diarrhea-predominant irritable bowel syndrome intestinal microbial markers, which comprises the following steps:
s1, randomly dividing the C57 mice into 2 groups, namely a normal control group and a model group, and establishing an IBS-D mouse model by combining chronic constraint and lavage of senna leaves; after the IBS-D mouse model is successfully constructed (the stool dilution rate and the AWR score are used as indexes to indicate the successful establishment of the IBS-D mouse model), collecting the stools of all groups of mice in a sterile environment, extracting the total DNA of stool samples, detecting the stools of a diarrhea-predominant irritable bowel syndrome animal mouse model and a normal model mouse model by a 16S rRNA gene sequencing method, and comparing the difference of intestinal flora composition of the diarrhea-predominant irritable bowel syndrome animal model and the normal mouse model by data analysis;
s2, taxonomic analysis of species: comparing the representative sequence of the OTU with a microbial reference database to obtain species classification information corresponding to each OTU, further carrying out statistics on species composition of the intestinal flora of each pair of samples at each level, generating abundance tables of species in the intestinal flora at different classification levels by using QIIME software, and drawing a map of the species structure of the intestinal flora in the top ten of the microbial abundance of the samples at each taxonomy level by using an R language tool;
s3, screening intestinal microorganisms: LefSe analysis, namely analysis of species with obvious difference among groups, further estimating the influence of the abundance of each component on the difference effect among the intestinal flora groups by adopting linear discriminant analysis, and finding out the species with obvious difference in the abundance of the intestinal flora among the intestinal flora groups by adopting the analysis; the screening value of LDA Score is set to be 4.0 by default; screening five bacteria which are high in abundance and most obvious in difference in intestinal tracts of diarrhea-predominant irritable bowel syndrome C57 mice as markers to obtain the diarrhea-predominant irritable bowel syndrome C57 mice.
Preferably, the 16S rRNA gene sequencing method of step S1 is: collecting a microorganism sample of a healthy and diarrhea-predominant irritable bowel syndrome C57 mouse in a sterile environment, and then extracting microorganism genome DNA in a stool sample by using a PowerSoil DNA Isolation Kit, wherein the relevant operation is carried out according to the steps provided by the instruction of the PowerSoil DNA Isolation Kit; primers were designed for amplification based on the 16S rRNA V3-V4 region of bacteria followed by related high-throughput sequencing via the Illumina Miseq platform.
Preferably, the primer is V3-V4 (a specific primer with a sample identification marker (barcode) is synthesized according to a bacterial double hypervariable V3-V4 region), the sequence of an upstream primer is V3-V4-F, the sequence information is shown as SEQ ID NO.1, the sequence information of a downstream primer is V3-V4-R, and the sequence information is shown as SEQ ID NO. 2; the PCR amplification system for carrying out the amplification comprises: 40-60 ng of template DNA, 1.5 mu L of forward primer and reverse primer respectively, 0.2 mu L of Q5 High Fidelity DNA Polymerase, 10 mu L of High GC Enhancer, 10 mu L of Reaction Buffer and 1 mu L of dNTP Mix, and supplementing the total system to 50 mu L by using sterilized double distilled water; the reaction conditions are as follows: pre-denaturation at 95 ℃ for 5 min; 1min at 95 ℃, 1min at 50 ℃ and 1min at 72 ℃ for 15 cycles; finally, the extension is carried out at 72 ℃ for 7min, and the product is stored at 4 ℃.
5'-ACTCCTACGGGAGGCAGCA-3'(SEQ ID NO.1)；
5'-GGACTACHVGGGTWTCTAAT-3'(SEQ ID NO.2)；
Preferably, the data analysis process of step S1 includes data preprocessing, Alpha diversity analysis, Beta diversity analysis.
Preferably, the levels of step S2 include phylum, class, order, family, genus, species.
The invention also provides application of the diarrhea-predominant irritable bowel syndrome intestinal microbial marker in preparation or screening of diarrhea-predominant irritable bowel syndrome detection products.
In the present invention, the specific taxonomic features of the genus Staphylococcus are: (ii) Bacteria; firmicutes; bacillus; bacillales; staphylococcaceae; staphylococcus; s __ uncultred _ bacterium _ g _ Staphylococcus; the specific taxonomic features of the Akkermansia genus are: (ii) Bacteria; verrucomicrobia; verrucomicrobia; verrucomicrobiales; verrucomicrobiaceae; akkermansia; uncultured _ bacterium _ g _ Akkermansia; the specific taxonomic features of the genus Dubosiella are: (ii) Bacteria; firmicutes; erysipelotrichia; erysipelothecales erysipelothecaceae; dubosiella; s __ uncultured _ bacterium _ g _ Dubosiella; the specific taxonomic features of the genus Enterobacter are: (ii) Bacteria; firmicutes; proteobacteria; enterobacterials; enterobacteriaceae; enterobacter; the specific taxonomic features of the genus Lactobacillus are: (ii) Bacteria; firmicutes; bacillus; lactobacillus; lactobacillus acid; lactobacillus; s __ uncultured _ bacterium _ g _ Lactobacillus.
In the course of the test, the chronic restraint used In the invention is a world famous Irritable Bowel Syndrome model, Williams C L et al Stress-induced changes In the internal transit a model for the Irritable Bowel Syndrome [ J ]. Gastroenterology,1988,94(3):611-621. Chronic restraint is combined with the irrigation of the folium sennae to establish IBS-D as one of the world famous Diarrhea type Irritable Bowel Syndrome models, Li L et al Synergic Effect of Berberine-Based Medicine analyzed nanostructure on Diarrhenia-Predominant phase Irrit Bowenl Syndrome In Vivo [ J ]. Front. 2020,11:1210.
Compared with the prior art, the diarrhea-predominant irritable bowel syndrome intestinal microbial marker provided by the invention has the following advantages: by utilizing the characteristics that the intestinal microorganisms of diarrhea-predominant irritable bowel syndrome C57 mice are different from the intestinal microorganisms of healthy C57 mice, intestinal bacteria markers which can be used for diarrhea-predominant irritable bowel syndrome are screened. The marker is enriched in the intestinal tract of diarrhea-predominant irritable bowel syndrome C57 mice, and the mice show diarrhea, reduced dietary intake, mental fatigue, weight loss, withered fur, starch relaxation and dullness, and the discovery of the marker provides a reference for diagnosing the microbial diarrhea-predominant irritable bowel syndrome, and can be used for developing a microbial preparation for improving the diarrhea-predominant irritable bowel syndrome.
Description of the drawings:
FIG. 1 is a graph showing the results of an evaluation of a diarrhea-predominant irritable bowel syndrome model;
FIG. 2 shows the results of Alpha diversity analysis of diarrhea-predominant irritable bowel syndrome model and normal mice and healthy intestinal microorganisms;
FIG. 3 is a graph showing the results of analysis of the diversity of Beta in a diarrhea-predominant irritable bowel syndrome model and normal mouse intestinal microorganisms;
FIG. 4 is a graph showing the results of a taxonomic analysis of species of intestinal microorganisms in a diarrhea-predominant irritable bowel syndrome model and normal mice;
FIG. 5 is a graph showing the results of screening and analyzing intestinal microorganisms of a diarrhea-predominant irritable bowel syndrome model and normal mouse intestinal microorganisms;
FIG. 6 is a graph showing the results of the abundance of Staphylococcus, Akkermansia, Enterobacter, Dubosiella, and Lactobacillus in the intestinal tracts of the Con group and the Model group.
Detailed Description
The present invention is further explained with reference to the following specific examples, but it should be noted that the following examples are only illustrative of the present invention and should not be construed as limiting the present invention, and all technical solutions similar or equivalent to the present invention are within the scope of the present invention. The method and the device are operated according to the conventional technical method and the content of the instrument instruction, wherein the specific technology or condition is not indicated in the embodiment; the reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.
Example 1 Chronic restraint in combination with lavage of senna leaves
SPF grade C57BL/6J mice 16, body weights (20 ± 2) g, males, provided by the guangdong provincial medical animal center, animal certification numbers: SCXK (Yue) 2018-. Before experiment, the animals are placed in the laboratory adaptive environment and freely eat and drink water at room temperature (22 +/-2) DEG C and normal circadian rhythm. The raising management and the experimental operation process of the experimental animals in the experimental process both comply with the relevant regulations of the experimental animal center of Guangdong university of medicine.
60g of senna leaf is weighed and decocted in 300mL of distilled water. Filtering with double layer gauze after 10min to obtain supernatant. After decocting at high temperature, concentrating the mass concentration to 0.6g/mL, preserving in a refrigerator at-20 ℃, heating in water bath at 25 ℃ before administration every day, administering the drug twice in the stomach of a mouse with 10mL/kg every day, and receiving acute constraint stress for 28 days to establish an IBS-D model.
Example 2 evaluation of model
The total number of the particles discharged from the mice 6h after the model building and the number of the particles in the loose feces were calculated by a filter paper blotting method. The judgment of loose stool was made by the presence or absence of stains on the filter paper. The percentage (%) of loose stool is the number of loose stool particles/total number of stool particles × 100%. Classifying the thin excrement according to the diameter of the stain on filter paper of the thin excrement discharged by the mice after molding: grade 1, the diameter of the stain is less than 1 cm; 2, the diameter of the stain is 1-2 cm; grade 3, wherein the diameter of the stain is 2-3 cm; 4 grade, the diameter of the stain is 3-4 cm; grade 5, and the diameter of the stain is 4-5 cm. Diarrhea index is the rate of loose stool x the grade of loose stool. After fasting for 24h, the 6Fr catheter lubricated by paraffin oil is inserted through the anus, the tail end of the saccule is placed about 1.0cm away from the anus, and the catheter and the root of the tail of the mouse are fixed by a medical adhesive tape. The mouse is placed on a platform, water is gradually injected to expand the balloon after the mouse adapts to the environment, the expansion volume is respectively 0.25mL, 0.35mL and 0.50mL, rectal expansion lasts for 30s each time, and abdominal evacuation reflex (AWR) evaluation is carried out by an observer. This was repeated 3 times and the data averaged. AWR scoring criteria: 0 minute: no obvious behavioral response to the expansion; 1 minute: only a brief head movement at the beginning of stimulation followed by a cessation of movement; and 2, dividing: there is a slight contraction of the abdominal muscles, but the abdomen cannot be lifted off the platform; and 3, dividing: strong abdominal muscle contraction can lift the abdomen away from the platform; and 4, dividing: the abdominal muscles are kept rigid and contracted, so that the body becomes arched, the abdomen is raised, the pelvis structure is upright, and the clonorchis sinensis is reflected.
As shown in FIG. 1, the stool dilution rate and the AWR score were above grade 2 and above grade 5, respectively, indicating the successful establishment of IBS-D mouse model. Wherein, the Con group and the Model group refer to normal group mice and diarrhea-predominant irritable bowel syndrome Model group mice respectively. A) A condition of diarrhea; B) visceral sensitivity.
Example 3 fecal Collection and detection
Selecting and moldingFunctional IBS-D mice and healthy mice were 6 each, and stool samples were from an adult male C57 mouse model housed in an SPF animal house. Placing naturally discharged fresh mouse model feces into sterile dry collection tube with sterile forceps, placing in liquid nitrogen, returning to laboratory within 30min, and storing in-80 deg.C refrigerator. Extracting microbial genome DNA in each group of mouse excrement samples by using a PowerSoil DNA Isolation Kit, designing and obtaining a primer according to a conserved region, namely the primer shown in SEQ ID NO.1-2 of the application, adding a sequencing joint at the tail end of the primer, carrying out PCR amplification, purifying, quantifying, homogenizing, purifying, quantifying and homogenizing the product (the PCR product is subjected to column purification by using an OMEGA DNA purification column after being mixed according to the mass ratio of 1:1 according to the result of electrophoresis (ImageJ software), then using 1.8% agarose gel, cutting target fragments after 120V 40min electrophoresis, and recovering) to form a sequencing library, wherein the whole library construction process is as follows: target region PCR, SolexaPCR, Nanodrop quantification and polyculture, column purification, gel cutting recovery, library quality inspection of the built library, dry ice transportation of the qualified library to Baimaike sequencing company for sequencing with Illumina HiSeq 2500. The target region PCR system is prepared into 10 mu L: 50 ng. + -. 20% of genomic DNA, 0.3. mu.L of Vn F (10. mu.M), 0.3. mu.L of Vn R (10. mu.M), 2. mu.L of KOD FX Neo Buffer 5. mu. L, dNTP (2mM each), and 2. mu.L of KOD FX Neo 0.2. mu. L, ddH2O is supplemented to 10 mu L; the PCR reaction condition is 95 ℃ for 5 min; 95 ℃ for 30s, 50 ℃ for 30s, 72 ℃ for 40s, 25 cycles; 7min at 72 ℃; infinity at 4 ℃. The Solexa PCR system was formulated with 20. mu.L: target region PCR purified product 5. mu.L, 2.5. mu.L; the PCR reaction conditions are as follows: 30s at 98 ℃; 10s at 98 ℃, 30s at 65 ℃, 30s at 72 ℃ and 10 cycles; 5min at 72 ℃.
An original image data file obtained by high-throughput sequencing (such as a sequencing platform of Illumina HiSeq and the like) is analyzed and converted into an original sequencing sequence (Sequenced Reads) through Base recognition (Base Calling), and a result is stored in a FASTQ (fq for short) file format, wherein the sequence information of the sequencing sequence (Reads) and the corresponding sequencing quality information are contained.
Example 4 bacterial population analysis
The following analyses were performed on the sequencing results: (1) data preprocessing: according to the Overlap relation between PE Reads, splicing double-end sequence data obtained by Illumina Hiseq sequencing (Merge) to construct a sequence Tags, and simultaneously performing quality control filtration on the quality of Reads and the effect of Merge. Mainly comprises the following 3 steps: splicing the PE reads: performing overlap splicing on reads of each sample by using FLASH v1.2.7 software, wherein the finally obtained spliced sequence is original Tags data (Raw Tags); ② Tags filtration: filtering the spliced Raw Tags by using Trimmomatic v0.33 software to obtain high-quality tag data (Clean Tags); removing chimera: by using UCHIME v4.2 software, chimeric sequences were identified and removed, resulting in final valid data (Effective Tags). The information analysis content is as follows: OTU division, diversity and difference analysis;
(2) alpha diversity analysis: analyzing dilution curves of cecal contents of each group of mice by using an Alpha diversity analysis method (the number of sequencing strips is used as an abscissa, and the number of OTUs obtained after OTU clustering based on the number of sequencing strips is used as an ordinate), a Shannon index curve (the number of sequencing strips is used as an abscissa, and the Shannon index is used as an ordinate), a grade abundance curve (the number of OTUs is used as an abscissa, and the relative abundance is used as an ordinate; if the curve decreases smoothly, the sample diversity is high, and if the curve decreases steeply, the sample diversity is low)
(3) Beta diversity analysis: the Principal coordinate analysis (PCoA) is a reduced-dimension sorting method similar to PCA, and by having data for measuring the difference or distance between different samples, a rectangular coordinate system can be found by the method, the N samples are expressed into N points, the square of the Euclidean distance between the points is exactly equal to the original difference data, quantitative conversion of qualitative data is realized, and the most important elements and structures are extracted from multi-dimensional data. The classification of a plurality of samples can be realized through the principal coordinate analysis, and the species diversity difference among the samples is further displayed. The closer the distance between the two samples, the more similar the composition between the intestinal flora of the two samples. The PCoA analysis graph was drawn using the R language tool.
(4) Species taxonomic analysis: species classification information corresponding to each OTU can be obtained by comparing the representative sequence of the OTU with a microorganism reference database, further statistics is carried out on species composition of the intestinal flora of each pair of samples at each level (phylum, class, order, family, genus and species), a QIIME software is utilized to generate abundance tables of species in the intestinal flora at different classification levels, and then an R language tool is utilized to draw a map of the species structure of the intestinal flora of the samples at each taxonomy level.
(5) Screening intestinal microorganisms: LefSe analysis, a species analysis with significant differences between groups (which may be referred to as biomarker analysis), uses Linear Discriminant Analysis (LDA) to estimate how much the abundance of each component (species) has an effect on the difference in abundance between groups of intestinal flora, and uses this analysis to find species with significant differences in abundance between groups of intestinal flora.
The analysis and comparison aims to find out the difference of the microbial composition in the excrement of the IBS-D mouse model and the healthy mouse model and the dominant species in each group of samples, namely, the intestinal bacteria genus Staphylococcus, Akkermansia, Enterobacter, Dubosiella and Lactobacillus; the specific results are shown in FIGS. 2 to 6, wherein, in FIG. 5, A represents species difference analysis of fecal microorganisms of Con group and Model group at phylum, class, order, family, genus and species level (from inside to outside); b represents marker screening/LEfSe assay results: in the case of LDA >4.0, 2 genera in the Model panel were expressed more and 3 genera were expressed less at the genus level (P < 0.05).
It should be noted that although the above embodiments have been described, once the basic inventive concept is obtained, other variations and modifications can be made to these embodiments by those skilled in the art, so that the above embodiments are only examples of the present invention, and not to limit the scope of the present invention, and all the modifications made by the equivalent structures or equivalent processes in the present specification, or directly or indirectly applied to other related technical fields are included in the scope of the present invention.
SEQUENCE LISTING
<110> university of Guangdong department of pharmacy
<120> diarrhea-predominant irritable bowel syndrome intestinal microbial marker and application thereof
<130> 2021.11.25
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 19
<212> DNA
<213> V3-V4-F
<400> 1
actcctacgg gaggcagca 19
<210> 2
<211> 20
<212> DNA
<213> V3-V4-R
<400> 2
Claims (9)
1. A diarrhea predominant irritable bowel syndrome intestinal microbial marker, which comprises the genera Staphylococcus, Akkermansia, Enterobacter, Dubosiella and Lactobacillus.
2. The diarrhea predominant irritable bowel syndrome intestinal microbial marker according to claim 1, wherein the relative abundance of the genus Staphylococcus and Enterobacter shows an increase in the diarrhea predominant irritable bowel syndrome animal model group, and the relative abundance of the genus Akkermansia, Dubosiella, and Lactobacillus decreases in the diarrhea predominant irritable bowel syndrome animal model group.
3. The diarrhea-predominant irritable bowel syndrome intestinal microbial marker according to claim 2, wherein the relative abundance is calculated by inter-group differential significance species analysis, and further wherein the magnitude of the effect of the abundance of each component on the differential effect between the groups of intestinal flora is estimated by using linear discriminant analysis.
4. The method for screening intestinal microbial markers of irritable bowel syndrome with diarrhea according to claim 1, comprising the steps of:
s1, randomly dividing the C57 mice into 2 groups, namely a normal control group and a model group, and establishing an IBS-D mouse model by combining chronic constraint and lavage of senna leaves; after the IBS-D mouse model is successfully constructed, detecting the excrement of a diarrhea-predominant irritable bowel syndrome animal mouse model and a normal model mouse model by a 16S rRNA gene sequencing method, and comparing the difference of intestinal flora composition of the diarrhea-predominant irritable bowel syndrome animal model and the normal mouse model by analyzing data;
s2, taxonomic analysis of species: comparing the representative sequence of the OTU with a microbial reference database to obtain species classification information corresponding to each OTU, further carrying out statistics on species composition of the intestinal flora of each pair of samples at each level, generating abundance tables of species in the intestinal flora at different classification levels by using QIIME software, and drawing a map of the species structure of the intestinal flora in the top ten of the microbial abundance of the samples at each taxonomy level by using an R language tool;
s3, screening intestinal microorganisms: LefSe analysis, namely analysis of species with obvious difference among groups, further estimating the influence of the abundance of each component on the difference effect among the intestinal flora groups by adopting linear discriminant analysis, and finding out the species with obvious difference in the abundance of the intestinal flora among the intestinal flora groups by adopting the analysis; the screening value of LDA Score is set to be 4.0 by default; screening five bacteria which are high in abundance and most obvious in difference in intestinal tracts of diarrhea-predominant irritable bowel syndrome C57 mice as markers to obtain the diarrhea-predominant irritable bowel syndrome C57 mice.
5. The screening method of claim 4, wherein the 16S rRNA gene sequencing method of step S1 is: collecting the feces of each group of mice in a sterile environment, extracting the total DNA of a feces sample, designing primers according to the 16S rRNA V3-V4 region of bacteria for amplification, and then carrying out related high-throughput sequencing through an Illumina Miseq platform.
6. The screening method of claim 5, wherein the primer is V3-V4, the sequence of the upstream primer is V3-V4-F, the sequence information is shown as SEQ ID No.1, the sequence information is V3-V4-R, and the sequence information is shown as SEQ ID No. 2; the PCR amplification system for carrying out the amplification comprises: 40-60 ng of template DNA, 1.5 mu L of forward primer and reverse primer respectively, 0.2 mu L of Q5 High Fidelity DNA Polymerase, 10 mu L of High GC Enhancer, 10 mu L of Reaction Buffer and 1 mu L of dNTP Mix, and supplementing the total system to 50 mu L by using sterilized double distilled water; the reaction conditions are as follows: pre-denaturation at 95 ℃ for 5 min; 1min at 95 ℃, 1min at 50 ℃ and 1min at 72 ℃ for 15 cycles; finally, the extension is carried out at 72 ℃ for 7min, and the product is stored at 4 ℃.
7. The screening method of claim 4, wherein the data analysis process of step S1 includes data preprocessing, Alpha diversity analysis, Beta diversity analysis.
8. The screening method according to claim 4, wherein each level of step S2 includes phylum, class, order, family, genus, species.
9. Use of the diarrhea-predominant irritable bowel syndrome intestinal microbial marker of claim 1 in the preparation or screening of diarrhea-predominant irritable bowel syndrome assay products.
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