CN113832079A

CN113832079A - Intestinal microorganism combination and application thereof as systemic lupus erythematosus marker

Info

Publication number: CN113832079A
Application number: CN202111303496.6A
Authority: CN
Inventors: 谢宝钊; 黄菁梅; 李水贤; 赖春桃; 林智明; 杨柳; 陆雪; 钟金河; 卢品伶
Original assignee: Wuzhou Gongren Hospital
Current assignee: Wuzhou Gongren Hospital
Priority date: 2021-11-05
Filing date: 2021-11-05
Publication date: 2021-12-24
Anticipated expiration: 2041-11-05
Also published as: CN113832079B

Abstract

The invention discloses an intestinal microorganism combination and application thereof as a systemic lupus erythematosus marker, and particularly relates to a method for determining sponibacillus (Sporobacter), vibrio butyricum (Butyricimonas) and bacillus coralis (Phascolecatobacter) with different levels in SLE patients by respectively carrying out 16s rRNA sequencing on intestinal microorganism samples from an individual with systemic lupus erythematosus and a healthy individual, and defining the relationship between the level difference and the systemic lupus erythematosus. These intestinal microorganisms with level differences can serve as markers of systemic lupus erythematosus.

Description

Intestinal microorganism combination and application thereof as systemic lupus erythematosus marker

Technical Field

The invention relates to microbiology, in particular to an intestinal microorganism combination and application thereof as a systemic lupus erythematosus marker.

Background

Systemic Lupus Erythematosus (SLE) is a common chronic autoimmune connective tissue disease with multiple system involvement. SLE patients are mainly young women, the ratio of women to men is 9:1, and the peak onset age is 20-40 years. It is now generally accepted that the causes of this disease are associated with T lymphocyte depletion, reduced T-reg function, over-activation of B cells and dysfunction, and that large amounts of autoantibodies are produced leading to multiple organ and system damage. At present, the clinical diagnosis of the systemic lupus erythematosus mainly adopts the examination means of clinical symptoms, serological laboratory autoantibodies and the like, and lacks of a specific auxiliary disease judgment method and a related detection kit.

The intestinal micro-ecosystem is the largest ecosystem of the human body, and more than 10 species are planted in the intestinal tract¹⁴Orders of magnitude bacteria, the number of genes in the gut flora genome is about 150 times the total number of genes in the human genome. The intestinal flora is interdependent with human body, affects functions including intestinal nervous system, intestinal endocrine system, immune system, intestinal permeability, etc., and plays an important role in food digestion, vitamin nutrition, resistance to invasion of foreign pathogenic microorganisms, stimulation of immunity, etc. The balance of the intestinal micro-ecosystem is subject to factors including food,Under the influence of a series of external factors such as medicines, antibiotics, pathogens and the like can break the balance of intestinal flora, so as to cause a series of related diseases, including various gastrointestinal diseases, hepatic diseases, inflammatory diseases, rheumatoid arthritis, multiple sclerosis and other immune system diseases, diabetes, metabolic syndrome, obesity and other metabolic system diseases and the like. Researches in recent years show that specific intestinal flora can influence the health state of a host through an immune system, and has the effects of regulating immunity, improving defense function, inhibiting tumor formation, promoting normal cell metabolism, promoting bone health, inhibiting bone calcium loss, delaying aging and the like. With the introduction of the concept of "gut-liver axis" and "brain-gut axis", more and more research has been focused on the species and function of the intestinal flora. Manipulation of intestinal bacterial composition and local metabolites through the use of probiotics has been explored as a promising approach for therapeutic intervention in disease.

Aiming at the systemic lupus erythematosus, at present, no very effective early detection method exists in clinic, so that a method with high sensitivity and economy needs to be established to meet the requirements of clinical diagnosis, prevention or treatment of systemic lupus erythematosus diseases.

Disclosure of Invention

The invention aims to provide an intestinal microorganism combination which can be used as a marker of systemic lupus erythematosus disease.

In a first aspect of the present invention there is provided an enteral microbial combination comprising one or more microorganisms selected from the group consisting of: bacillus sporogenes (Sporobacter), Vibrio butyricum (Butyricimans), and Bacillus coralis (Phascolatobacter).

In another preferred embodiment, the intestinal microbial combination comprises at least: bacillus sporogenes (Sporobacter), Vibrio butyricum (Butyricimans), and Bacillus coralis (Phascolatobacter).

In another preferred embodiment, the intestinal microbial combination further comprises one or more microbes selected from the group consisting of: rational bacteria (Alisipes), Prevotella (Alloprovella), Deuterobacter (Odoribacter), and Prevotella (Prevotella).

In another preferred embodiment, the gut microorganism is used in combination for diagnosis (including early diagnosis and/or adjuvant diagnosis) and treatment (including adjuvant treatment) of systemic lupus erythematosus; or preparing a kit or reagent for diagnosing (including early diagnosis and/or auxiliary diagnosis) and treating (including auxiliary treatment) the systemic lupus erythematosus.

In another preferred embodiment, the detection of the respective microbial biomarkers is performed by PCR; preferably, the PCR comprises QPCR, or RT-QPCR.

In another preferred embodiment, the gut microbial composition is a gene combination comprising gene nucleic acid fragments specific for each microorganism.

In a second aspect of the present invention, there is provided a reagent set for diagnosing (including early diagnosis and/or assisted diagnosis) systemic lupus erythematosus, the reagent set comprising reagents for detecting each microorganism in the combination of intestinal microorganisms according to the first aspect of the present invention.

In another preferred embodiment, each of said agents is a primer, probe, antisense oligonucleotide, aptamer or antibody specific for each microorganism.

In another preferred embodiment, the reagent is a PCR detection reagent; preferably including primers that specifically amplify genes of each microorganism; more preferably, a probe that specifically binds to each microorganism gene is also included.

In another preferred embodiment, the PCR detection reagent comprises a multiplex PCR detection system, the multiplex PCR detection system comprises a primer pair group, and the primer pair group comprises:

a first primer pair comprising a forward primer set forth as SEQ ID No. 4; and, a reverse primer as set forth in SEQ ID No. 5;

a second primer pair comprising a forward primer set forth in SEQ ID No. 8; and, a reverse primer as set forth in SEQ ID No. 9; and

a third primer pair comprising a forward primer set forth in SEQ ID No. 13; and, a reverse primer as set forth in SEQ ID No. 14.

In another preferred embodiment, the primer set group comprises:

a fourth primer pair comprising a forward primer set forth in SEQ ID No. 10; and, a reverse primer as set forth in SEQ ID No. 11.

In another preferred embodiment, the multiplex PCR detection system further comprises a probe set, wherein the probe set comprises:

a first probe, wherein the sequence of the first probe is shown as SEQ ID No. 12;

a second probe, wherein the sequence of the second probe is shown as SEQ ID No. 15;

a third probe, wherein the sequence of the third probe is shown as SEQ ID No. 16; and/or

And a fourth probe, wherein the sequence of the fourth probe is shown as SEQ ID No. 17.

In a third aspect of the invention, there is provided a kit comprising a combination of gut microorganisms according to the first aspect of the invention, and/or a set of reagents according to the second aspect of the invention.

In another preferred embodiment, each microorganism of the combination of gut microorganisms according to the first aspect of the present invention is used as a standard.

In another preferred embodiment, the kit further comprises a PCR buffer, and/or a PCR reaction enzyme system.

In a fourth aspect of the present invention, there is provided a use of a combination of intestinal microorganisms or a detection reagent thereof for preparing a kit for diagnosing (including early diagnosis and/or assisted diagnosis) systemic lupus erythematosus, wherein the combination of intestinal microorganisms comprises one or more microorganisms selected from the group consisting of: bacillus sporogenes (Sporobacter), Vibrio butyricum (Butyricimans), and Bacillus coralis (Phascolatobacter).

In another preferred embodiment, the diagnosis comprises the steps of:

(1) providing a sample from a subject to be tested, and detecting the level of each microorganism in the intestinal microorganism combination in the sample by using a PCR method;

(2) comparing the level measured in step (1) with the corresponding level of microorganisms in a normal population.

In another preferred example, in step (1), 16S rDNA of each microorganism is detected by using a PCR method.

In another preferred embodiment, the sample is selected from feces.

In a fifth aspect of the invention, there is provided a medicament for use in the treatment (including co-treatment) of systemic lupus erythematosus, said medicament having the ability to modulate a combination of gut microbes according to the first aspect of the invention in the gut of a patient.

In a sixth aspect of the invention, there is provided a method of screening for a drug for use in the treatment (including co-treatment) of systemic lupus erythematosus, the method comprising the steps of:

(1) in the test group, administering a test compound to a subject to be tested, and detecting the level L1 of each microorganism in said combination of gut microorganisms in a sample derived from said subject in the test group; in a control group, administering a blank control (including vehicle) to the subject to be tested, and detecting the level L2 of each microorganism in the combination of gut microorganisms in a sample derived from the subject in the control group;

(2) comparing the level L1 and the level L2 detected in the previous step to determine whether the test compound is a candidate compound for treating systemic lupus erythematosus;

wherein the gut microbiome combination comprises one or more selected from the group consisting of: bacillus sporogenes (Sporobacter), Vibrio butyricum (Butyricimans), and Bacillus coralis (Phascolatobacter).

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Drawings

Figure 1 shows the results of comparing the abundance and diversity of intestinal flora in SLE patients and healthy people:

(a) the alpha diversity was assessed using the Chao1 index; (b) evaluating alpha diversity using Shannon index; (P <0.001, Kruskal-Wallis test).

Figure 2 shows the results of assessing the structural diversity of the intestinal flora in SLE patients and healthy people:

(a) PCOA visualization based on Bray-Curtis distance; (b) graph of PCA analysis.

FIG. 3 shows the ROC curve of Sporobacter bacteria.

FIG. 4 shows the ROC curve of the bacterium Butyricimonas.

FIG. 5 shows a ROC curve of Phascolarcotobacterium bacteria.

Detailed Description

The present inventors have conducted intensive studies to determine the level differences of various intestinal microorganisms present in SLE patients by 16s rRNA sequencing of intestinal microorganism samples from individual systemic lupus erythematosus and healthy individuals, respectively, and to determine the relationship between these level differences and systemic lupus erythematosus. These intestinal microorganisms having level difference can be used for a composition or a kit for preventing, diagnosing/treating systemic lupus erythematosus.

The invention confirms that SLE patients are obviously different from healthy people in the classification of intestinal bacteria species, the difference (P <0.05) exists in the levels of various microorganisms, the expression levels of sporobacillus (Sporobacter), vibrio butyricum (Butyricimonas) and bacillus colatobacter (Phascolecobacterium) of the patients are obviously lower than those of the healthy people, the difference has obvious statistical significance, and the expression levels of the indexes are closely related to the incidence of systemic lupus erythematosus.

"16S rRNA" refers to rRNA constituting a 30S small subunit of a prokaryotic ribosome, which has a base sequence of which most part is highly conserved on the one hand, and a part of the region showing high base sequence diversity on the other hand. In particular, since there is little diversity between species and there is diversity between species, it is possible to efficiently identify prokaryotes by comparing the sequences of 16S rRNA.

In a preferred embodiment of the present invention, the sequence of the target gene of each strain 16s rrna is as follows:

representative sequence of Sporobacter (SEQ ID No.: 1):

GACGAACGCTGGCGGCGTGCCTAACACATGCAAGTCGAACGGAGACAATTGGTTCGCTGATTGTCTTAGTGGCGGACGGGTGAGTAACGCGTGAGCAATCTGCCCTTCGGAGGGGGACAACAGCTGGAAACGGCTGCTAATACCGCATAATGTATATTCAAGGCATCTTGGATATACCAAAGATTTATCGCCGAAGGATGAGCTCGCGTCTGATTAGCTAGTTGGTGAGGTAAAGGCTCACCAAGGCTGCGATCAGTAGCCGGACTGAGAGGTTGAACGGCCACATTGGAACTGAGATACGGGCCAGACTCCTACGGGAGGGAGCAGTGGGGAATTTTGGNCAATGGGGGAAAGCCNTACCCAGCAACGCCGCGTGAAGGAAGAAGGCCTTCGGGTTGTAAACTTCTTTGACCAGGGACGAAACAAATGACGGTACCTGGAAAACAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTATTACGTAGGTGGCAAGCGTTGTCCGGATTTACTGGGTGTAAAGGGCGCGTAGGCGGGAGTACAAGTCAGATGTGAAATCTGGGGGCTTAACCCTCAAACTGCATTTGAAACTGTATTTCTTGAGTATCGGAGAGGCAGGCGGAATTCCTAGTGTAGCGGTGAAATGCGTTGATATTAGGAGGAACACCAGTGGCGAAGGCGGCCTGCTGGACGACAACTGACTCTGAGGCGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCTGTAAACGATGAATACTAGGTGTGGGGGGACTGACCCCCTCCGTGCCGGAGTTAACACAATAAGTATTCCACCTGGGGAGTACGNCCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCAGTGGATTATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGGCTTGACATCGTACTAACGAAGCAGAGATGCATTAGGTGCCCTTCCGGGGAAAGTATAGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCCTATTGTNATTTGCTACNCGAGANCACTCTAGCGAGGCTGCCGATGACAAACCGGAGGAAGGTGGGGACGACGTCAAATCATCATGCCCCTTATGTCCTGGGCTACACACGTAATACAATGTCTCTCACAGAGGGAAGCAAGACCGCGAGGTGGAGCAAATCCCTAAAATGCGTCTCAGTTCAGATTGCAGGCTGCAACTCGCCTGCATGAAGTCGGAATTGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGAGAGCCGGGAACACCCGAAGTCCGTAGTCTAACCGCAAGGGGGACGCGGCCGAAGGTGGGTTTGGTAATTGGGGTG

representative sequences of the bacterium Butyricimonas are as follows (SEQ ID No.: 2):

AGAGTTTGATCCTGGCTCAGGATGAACGCTAGCGACAGGCTTAACACATGCAAGTCGAGGGGCAGCACGGTGTAGCAATACACTGGTGGCGACCGGCGCACGGGTGAGTAACACGTGTGCAACCAACCCCGTACCGGGAGATAACCCGCGGAAACGTGGACTAACATCCCATGATACTCGAGAGCCGCATGGCTCTCGATTTAAAATTCCGGTGGTACGGGACGGGCACGCGCGACATTAGGTAGTTGGCGGGGTAACGGCCCACCAAGCCGACGATGTCTAGGGGTTCTGAGAGGAAGGTCCCCCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGAGGAATATTGGTCAATGGGCGAGAGCCTGAACCAGCCAAGTCGCGTGAGGGAAGAATGGTCTATGGCCTGTAAACCTCTTTTGTCAGGGAAGAATAAGGATGACGAGTCATTCGATGCCAGTACTTGACGAATAAGCATCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGGGGATGCGAGCGTTATCCGGATTTATTGGGTTTAAAGGGCGCGTAGGCGGGACGTCAAGTCAGCGGTAAAAGACTGCAGCTAAACTGTAGCACGCCGTTGAAACTGGCGCCCTGGAGACGAGACGAGGGAGGCGGAACAAGTGAAGTAGCGGTGAAATGCATAGATATCACTTGGAACCCCGATAGCGAAGGCAGCTTCCCAGGCTCGTTCTGACGCTGATGCGCGAGAGCGTGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGCTCACTGGATCTTGGCGATACACTGCCAGGGTTCAAGCGAAAGTATTAAGTGAGCCACCTGGGGAGTACGTCGGCAACGATGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGAGGAACATGTGGTTTAATTCGATGATACGCGAGGAACCTTACCCGGGTTTAAATGTAGATTGCATGAGGTGGAAACGCTTCTTCCCTTCGGGGCTATTTACAAGGTGCTGCATGGTTGTCGTCAGCTCGTGCCGTGAGGTGTCGGGTTAAGTCCCATAACGAGCGCAACCCCTATCGCCAGTTGCCATCGGTTGAAGCCGGGCACTCTGTCGAGACTGCCACCGTAAGGTGCGAGGAAGGCGGGGATGACGTCAAATCAGCACGGCCCTTACACCCGGGGCGACACACGTGTTACAATGGCCGGTACAGAGGGCAGCCACGGGGTGACCCGGAGCGAATCTCTAAAGCCGGTCGTAGTTCGGACTGGAGTCTGCAACCCGACTCCACGAAGTTGGATTCGCTAGTAATCGCGCATCAGCCATGGCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCAAGCCATGGAAGCCGGGAGTACCTGAAGATCGTGACCGCGAGGAACGGGCTAGGGTAATACCGGTAACTGGGGCTAAGTCGTAACAAGGTAACC

representative sequences of Phascolarcotobacterium are as follows (SEQ ID NO: 3):

GACGAACGCTGGCGGCGTGCCTAACACATGCAAGTCGNACGGAGAATTTTATTTCGGTAGAATTCTTAGTGGCGAACGGGTGAGTAACGCGTAGGCAACCTACCCTTTAGACGGGGACAACATTCCGAAAGGAGTGCTAATACCGGATGTGATCATCGTGCCGCATGGCAGGATGAAGAAAGATGGCCTCTACAAGTAAGCTATCGCTAAAGGATGGGCCTGCGTCTGATTAGCTAGTTGGTAGTGTAACGGACTACCAAGGCGATGATCAGTAGCCGGTCTGAGAGGATGAACGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTGGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGATTTCGGTCTGTAAAGCTCTGTTGTTTATGACGAACGTGCAGTGTGTGAACAATGCATTGCAATGACGGTAGTAAACGAGGAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCGAGCGTTGTCCGGAATTATTGGGCGTAAAGAGCATGTAGGCGGCTTAATAAGTCGAGCGTGAAAATGCGGGGCTCAACCCCGTATGGCGCTGGAAACTGTTAGGCTTGAGTGCAGGAGAGGAAAGGGGAATTCCCAGTGTAGCGGTGAAATGCGTAGATATTGGGAGGAACACCAGTGGCGAAGGCGCCTTTCTGGACTGTGTCTGACGCTGAGATGCGAAAGCCAGGGTAGCGAACGGGATTAGATACCCCGGTAGTCCTGGCCGTAAACGATGGGTACTAGGTGTAGGAGGTATCGACCCCTTCTGTGCCGGAGTTAACGCAATAAGTACCCCGCCTGGGGAGTACGGCCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGTATGTGGTTTAATTCGACGCAACGCGAAGAACCTTACCAAGGCTTGACATTGATTGAACGCTCTAGAGATAGAGATTTCCCTTCGGGGACAAGAAAACAGGTGGTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCCTATCCTATGTTACCAGCAAGTAAAGTTGGGGACTCATGGGAGACTGCCAGGGACAACCTGGAGGAAGGCGGGGATGACGTCAAGTCATCATGCCCCTTATGTCTTGGGCTACACACGTACTACAATGGTCGGAAACAGAGGGAAGCGAAGCCGCGAGGCAGAGCAAACCCCAGAAACCCGATCTCAGTTCGGATCGCAGGCTGCAACCCGCCTGCGTGAAGTCGGAATCGCTAGTAATCGCAGGTCAGCATACTGCGGTGAATACGTNCCCGGGCCTTGTACACACCGCCCGTCACACCACGAAAGTTGGTAACACCCGAAGCCGGTGAGGTAACCTATTAGGAGCCAGCCGTCTAAGGTGGNGCCGATGATTGGGGTG

the invention provides application of an intestinal microorganism combination to preparing or screening a systemic lupus erythematosus detection product, wherein the intestinal microorganism combination comprises one or more microorganisms selected from the following groups: bacillus sporogenes (Sporobacter), Vibrio butyricum (Butyricimans), and Bacillus coralis (Phascolatobacter).

The inventor researches and discovers that in systemic lupus erythematosus patients, the level of the intestinal microorganisms in the excrement of the patients is remarkably reduced. Wherein the level of Sporobacter (Sporobacter) is reduced by about 55%, the level of Vibrio butyricum (Butyricimonas) is reduced by about 50%, and the level of Lactobacillus (Phascolatobacterium) is reduced by about 10% compared to healthy population.

Therefore, the intestinal microorganisms can be used as biomarkers for diagnosing and detecting the systemic lupus erythematosus.

In one embodiment, the systemic lupus erythematosus test product comprises a microbial abundance test reagent for the enteric microbes; preferably as described for each microorganism.

In one embodiment, the specific recognition reagent is selected from a primer, probe, antisense oligonucleotide, aptamer or antibody specific for each of said microorganisms.

In one embodiment, the specific primer is capable of detecting a primer for each of the microorganisms that includes 16s rrna and other gene sequences that recognize the particular microorganism.

In the present invention, the term "primer" is a 7-50 bp sequence that is capable of forming a base pair complementary to a template strand and functions as an initiation point for replication of the template strand. The primers are generally synthesized, but naturally occurring nucleic acids may also be used. The sequence of the primer does not necessarily need to be completely identical to the sequence of the template, and may be sufficiently complementary to hybridize with the template. Additional features that do not alter the basic properties of the primer may be incorporated. Examples of additional features that may be incorporated include, but are not limited to, methylation, capping, substitution of more than one nucleic acid with a homolog, and modification between nucleic acids.

In one embodiment, the primer can be used to amplify 16S rRNA of the corresponding microorganism and other gene sequences that can recognize the microorganism, and after the sequences are amplified, the presence of the microorganism can be detected or the level of the microorganism can be measured by the presence or absence of the production of a desired product.

The sequence amplification method using the primer may use various methods known in the art. For example, Polymerase Chain Reaction (PCR), reverse transcription-polymerase chain reaction (RT-PCR), multiplex PCR, nested (nested) PCR, real-time fluorescent quantitative PCR, and the like can be used.

The specific recognition reagent combination of the intestinal microorganisms provided by the invention can be used for preparing a systemic lupus erythematosus early-stage detection kit. Preferably, the kit is a PCR detection kit; the kit may comprise a test tube or other suitable container, reaction buffer, base-triphosphate deoxynucleotides (dNTPs), enzymes such as Taq-polymerase reverse transcriptase, SYBR Green fluorescent dye, DEPC-water, and the like.

The invention also provides a gene chip for detecting the systemic lupus erythematosus, which comprises a solid phase carrier and oligonucleotide probes fixed on the solid phase carrier, wherein the oligonucleotide probes specifically identify gene sequences of all microorganisms in the intestinal microorganism combination.

In the present invention, the term "probe" refers to a molecule that is capable of binding to a specific sequence or subsequence or other portion of another molecule. Unless otherwise indicated, the term "probe" generally refers to a polynucleotide probe that is capable of binding to another polynucleotide (often referred to as a "target polynucleotide") by complementary base pairing. Depending on the stringency of the hybridization conditions, a probe can bind to a target polynucleotide that lacks complete sequence complementarity to the probe. The probe may be directly or indirectly labeled, and includes within its scope a primer. Hybridization modalities, including, but not limited to: solution phase, solid phase, mixed phase or in situ hybridization assays.

The probe has a base sequence complementary to a specific base sequence of the target gene. Here, the term "complementary" may or may not be completely complementary as long as it is a hybrid. These polynucleotides usually have a homology of 90% or more, preferably 95% or more, more preferably 100% with respect to the specific nucleotide sequence. These probes may be DNA, RNA, or a polynucleotide obtained by replacing a nucleotide in a part or all of them with an artificial Nucleic Acid such as PNA (peptide Nucleic Acid), LNA (locked Nucleic Acid, bridge Nucleic Acid, crosslinked Nucleic Acid), ENA (2 '-O, 4' -C-Ethylene-Bridged Nucleic acids), GNA (glycerol Nucleic Acid), TNA (Threose Nucleic Acid ), or the like.

The present invention will be described in further detail with reference to the following examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures for conditions not specified in detail in the following examples are generally carried out under conventional conditions such as those described in molecular cloning, A laboratory Manual (Huang Petang et al, Beijing: scientific Press, 2002) by Sambrook. J, USA, or under conditions recommended by the manufacturer. Unless otherwise indicated, percentages and parts are by weight. The test materials and reagents used in the following examples are commercially available without specific reference.

Example 1 study Subjects and sample Collection

With informed consent, fecal samples from 300 subjects, including 120 SLE patients and 180 healthy controls, were collected at southern hospital, southern medical university.

To be eligible for inclusion in the study, the individual must meet the following stool sample collection criteria:

1) no antibiotic or immunomodulator treatment, no special diet (diabetic, vegetarian, etc.) and normal lifestyle (no additional stress) for at least 3 months;

2) at least 3 months after any medical intervention;

3) non-pregnancy, tumour, infection or autoimmune disease or rheumatic disease

In order to distribute a fecal sample collection tube for a research subject and inform a specific collection method, the subject takes 10-15g of fresh feces and puts the fresh feces into the collection tube with a fecal genome protective solution, and the feces are quickly delivered to a hospital laboratory at normal temperature and stored in a refrigerator at 4 ℃ until the feces and the feces are used.

Example 216S rRNA analysis of intestinal microflora

2.1DNA extraction

DNA extraction was performed using a fecal microbial genome extraction kit (Guansoutheast core medical science and technology Co., Ltd.) according to the manufacturer's instructions.

The extract is treated with DNase-free RNase to eliminate RNA contamination. The DNA content was determined using a NanoDrop spectrophotometer, a Qubit fluorometer.

2.2DNA library construction and sequencing

Performing two-round 16S rRNA gene PCR amplification on the purified genome DNA by using the bacterial universal primer to complete the construction of a sequencing sample library, and using

2.0Fluorometer (Invitrogen, USA) assay concentration, Agilent2100(Agilent, USA) assay library size; measurement Using Illumina MiSeq (Illumina, San Diego, USA)The sequencer is used for sequencing the sequence of the amplified 16S rRNA hypervariable region, and the sequencing region is V3-V4 region.

2.3 data analysis

Using Trimmomatic v.0.32 software to trim and filter original sequencing data (FASTQ), and clustering high-quality sequence files by 97% similarity standard to obtain operation classification units (OTU). Alpha and beta diversity analysis was then performed using Qiime2 v.2017.12 software and R software.

The intestinal flora alpha diversity was assessed using Chao1, Shannon diversity index, and the intestinal flora beta diversity was assessed using principal coordinate analysis (PCoA) and PCA analyses. Groups of samples were analyzed for the class of significant differences using an analytical method based on linear discriminant analysis effect measures (LEfSe).

2.4 results

Intestinal microflora information of healthy population and SLE patients was obtained by 16S rRNA-based analysis, confirming that intestinal microflora diversity and abundance of SLE patients are significantly reduced compared to healthy population (fig. 1).

The diversity of intestinal flora beta is evaluated by PCoA analysis and PCA analysis, and as a result, SLE patients and healthy people have obvious separation tendency, which indicates that the intestinal flora structure of SLE patients is different from that of HC group (figure 2).

In addition, it was confirmed that SLE patients were significantly different from healthy people in the classification of intestinal bacteria species, and had differences in the levels of various intestinal microorganisms (P <0.05), and that the patients had significantly lower expression levels of Sporobacter bacteria, Butyricimonas bacteria, and phascolarcotobacterium bacteria than healthy people, and the differences had significant statistical significance, suggesting that the expression levels of these indices were closely related to the onset of systemic lupus erythematosus (table 1).

TABLE 1 different species

Example 3 validation of clinical assays for intestinal flora in SLE patients

The above studies indicate that SLE patients are significantly different from healthy people in the classification of intestinal bacteria species, and have differences in the level of multiple intestinal microorganisms, and that the expression levels of Sporobacter, Butyricimonas and Phascolarcotobacterium of the patients are significantly lower than those of the healthy people (P < 0.05).

Based on the correlation of the relevant flora with SLE, SLE can be diagnosed by detecting the abundance of the relevant flora in a patient sample.

Thus, 100 stool samples from healthy and SLE patients were collected and numbered for testing in this example. Specific fluorescent quantitative PCR primer probes are designed aiming at the 16S rRNA gene sequence of the target microorganism, and an internal standard is set.

The sequences of primers specific to Real-time fluorescent Quantitative PCR (Quantitative Real-time PCR) for detecting the abundance of Sporobacter, Butyrimonas, and Phascolarcotacterium in a sample are shown in Table 2.

TABLE 2

A fluorescent quantitative PCR amplification detection system: mu.l of each strain upstream and downstream primer, 5. mu.l of template DNA, and 6. mu.l of SYBR Green. The fluorescent quantitative PCR reaction conditions are as follows: 10min at 95 ℃, 15s at 95 ℃ and 2min at 60 ℃ for 40 cycles.

Each sample is used for detecting 9 bacteria, and an internal standard gene and a pure water control sample are arranged.

PCR reaction is carried out on an ABI7500 fluorescence real-time quantitative PCR instrument, and the expression abundance of each target gene in the sample is calculated by adopting a delta CT method (CT value comparison method).

Analyses were performed using a logistic regression model and ROC curves were plotted to take into account the diagnostic performance of the species. The ROC curve is a curve drawn based on a series of different two classification methods (cut-off values or decision thresholds) with true positive rate (sensitivity) as ordinate and false positive rate (1-specificity) as abscissa. The closer the ROC curve is to the upper left corner, the higher the diagnostic accuracy of the marker, the point of the ROC curve closest to the upper left corner being the best threshold with the least error and the least total number of false positives and false negatives. Calculating the area under the ROC curve (AUC) of each potential marker can judge the diagnostic value of the potential marker, and the diagnostic value is higher when the AUC is higher.

TABLE 3 diagnostic potential analysis

The test results showed that the intestinal flora of SLE patients showed significant differences (P <0.05) among Sporobacter, butyricmonas and phascolarcotobacterium bacteria compared to healthy individuals. The results are consistent with the results of sequencing the 16s rrna library.

The ROC curves of the respective flora are shown in fig. 3 to fig. 5, and the results show that the detected flora has higher sensitivity and specificity, so that the ROC curves can be used for the auxiliary diagnosis of SLE patients.

Therefore, when one or more indexes of Sporobacter, Butyricimonas and Phascolarcotobacterium in the intestinal flora of the tested people are lower than those of normal people, the probability of suffering from the systemic lupus erythematosus is higher, thereby being helpful for judging the disease condition and being a potential target point for treating the systemic lupus erythematosus.

EXAMPLE 4 preparation of assay kit

This example provides a Real-time fluorescent Quantitative PCR (Quantitative Real-time PCR) assay kit based on the detection of abundance of Sporobacter, Butyricimonas, and Phascolatobacter bacteria in a sample.

In order to improve the efficiency of sample detection, the present inventors performed multiple rounds of design and experimental verification on the PCR amplification primers of the above strains during the research process to obtain a multiplex detection system capable of performing multiplex detection on the above strains. A plurality of pairs of primers and probes are artificially designed, optimized selection and verification are carried out on the primers and the probes, and finally different screened primers and probes aiming at various floras are subjected to combined test, so that the problem of mutual interference inhibition among the primers of a multiple fluorescence PCR system is solved well, and a multiple detection system with better specificity and sensitivity is obtained, wherein the sequence information of the primers and the probes in the system is as follows, wherein F is a forward primer, R is a reverse primer, and P is a probe.

TABLE 4 multiple detection lines

The contents of the components in the PCR reaction system in the using process are as follows:

5×PCR buffer	5μL
		each primer-F (50. mu. mol/L)	0.3μL
Each primer-R (50. mu. mol/L)	0.3μL
		Each probe-P (50. mu. mol/L)	0.1μL
20mmol/L dNTPs (containing dUTP)	0.3μL
		PCR reaction enzyme system	3μL
Template nucleic acid	5μL
		DEPC water	Make up to 25. mu.L

Performing amplification reaction in a real-time fluorescent PCR instrument, wherein the fluorescent channel is selected from FAM, VIC, Texas red and Cy5, and the PCR amplification procedure is as follows;

50 ℃, 2min, 95 ℃, 15 min; 1 cycle

94 ℃, 15sec, 55 ℃, 45sec (fluorescence collected); 40 cycles.

And calculating the expression abundance of each target gene in the sample by adopting a delta CT method (CT value comparison method) through different fluorescence channel curves and Ct values after the PCR is finished.

Each of the 100 healthy and SLE patient samples from example 3 were tested using the multiplex assay system described above. The test results were compared with the single test results in example 3 to determine the clinical compliance rate. The results show that the detection results of the three multi-detection systems can completely correspond to the single detection result of the embodiment 3, and the clinical compliance rate reaches 100%.

Comparative example 1

The inventors designed pairs of primers and probes for each target sequence after deep comparison and analysis of the 16sRNA gene sequence of the target bacteria, and it was difficult to obtain effective multiplex PCR amplification primers and probe sequences due to competitive inhibition between primers in a multiplex reaction system, primer specificity differences, inconsistent annealing temperatures, primer dimers, and the like. Through a large number of experiments, designed primers and probes are optimally selected and verified, and the sequences and the combination of the primers and the probes which can be used for multiplex PCR amplification are finally determined.

In experiments, it was found that even in the case where primer pairs and probe sequences for respective target nucleic acids have been substantially determined, there is a significant difference in the effect of multiplex amplification with different primer pair combinations.

For example, in the multiplex PCR step, the primer sequences of Butyricimonas in the above multiplex reaction system were replaced with the control primer pair 1:

F1：GGTCCAGACTCCTACGGGAG(SEQ ID NO.:6)

R1：GCAGCACCTTGTAAATAGCC(SEQ ID NO.:7)

the other components were unchanged.

The control primer pair 1 can normally detect the target nucleic acid sequence of the Butyricimonas in the single PCR detection.

The detection results of 20 samples in the multiplex detection system show that the multiplex detection system containing the control primer pair 1 detects the target butyricolomonas bacteria only in 13 samples, has poor accuracy and cannot meet the clinical detection requirements.

Replacing the primer sequence of the Sporobacter in the multiple reaction system with a control primer pair 2:

F2：GCGGCGTGCCTAACACATG(SEQ ID NO.:18)

R2：CATAATCCACTGCTTGTGCG(SEQ ID NO.:19)

the other components were unchanged.

The control primer pair 2 can normally detect the target nucleic acid sequence of the Sporobacter in the single PCR detection.

The results of detection of 20 samples among the multiple detection systems showed that the detection of the target Sporobacter bacteria among 16 samples alone in the multiple detection system including the control primer set 2 did not satisfy the clinical detection requirements with accuracy.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Sequence listing

<110> Sterculia city worker hospital

<120> intestinal microorganism combination and application thereof as systemic lupus erythematosus marker

<130> P210347

<160> 19

<170> PatentIn version 3.5

<210> 1

<211> 1436

<212> DNA

<213> Sporobacter (Sporobacter)

<220>

<221> misc_feature

<222> (341)..(341)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (357)..(357)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (843)..(843)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (1074)..(1074)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (1084)..(1084)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (1090)..(1090)

<223> n is a, c, g, or t

<400> 1

gacgaacgct ggcggcgtgc ctaacacatg caagtcgaac ggagacaatt ggttcgctga 60

ttgtcttagt ggcggacggg tgagtaacgc gtgagcaatc tgcccttcgg agggggacaa 120

cagctggaaa cggctgctaa taccgcataa tgtatattca aggcatcttg gatataccaa 180

agatttatcg ccgaaggatg agctcgcgtc tgattagcta gttggtgagg taaaggctca 240

ccaaggctgc gatcagtagc cggactgaga ggttgaacgg ccacattgga actgagatac 300

gggccagact cctacgggag ggagcagtgg ggaattttgg ncaatggggg aaagccntac 360

ccagcaacgc cgcgtgaagg aagaaggcct tcgggttgta aacttctttg accagggacg 420

aaacaaatga cggtacctgg aaaacaagcc acggctaact acgtgccagc agccgcggta 480

ttacgtaggt ggcaagcgtt gtccggattt actgggtgta aagggcgcgt aggcgggagt 540

acaagtcaga tgtgaaatct gggggcttaa ccctcaaact gcatttgaaa ctgtatttct 600

tgagtatcgg agaggcaggc ggaattccta gtgtagcggt gaaatgcgtt gatattagga 660

ggaacaccag tggcgaaggc ggcctgctgg acgacaactg actctgaggc gcgaaagcgt 720

ggggagcaaa caggattaga taccctggta gtccacgctg taaacgatga atactaggtg 780

tggggggact gaccccctcc gtgccggagt taacacaata agtattccac ctggggagta 840

cgnccgcaag gttgaaactc aaaggaattg acgggggccc gcacaagcag tggattatgt 900

ggtttaattc gaagcaacgc gaagaacctt accagggctt gacatcgtac taacgaagca 960

gagatgcatt aggtgccctt ccggggaaag tatagacagg tggtgcatgg ttgtcgtcag 1020

ctcgtgtcgt gagatgttgg gttaagtccc gcaacgagcg caacccctat tgtnatttgc 1080

tacncgagan cactctagcg aggctgccga tgacaaaccg gaggaaggtg gggacgacgt 1140

caaatcatca tgccccttat gtcctgggct acacacgtaa tacaatgtct ctcacagagg 1200

gaagcaagac cgcgaggtgg agcaaatccc taaaatgcgt ctcagttcag attgcaggct 1260

gcaactcgcc tgcatgaagt cggaattgct agtaatcgcg gatcagcatg ccgcggtgaa 1320

tacgttcccg ggccttgtac acaccgcccg tcacaccatg agagccggga acacccgaag 1380

tccgtagtct aaccgcaagg gggacgcggc cgaaggtggg tttggtaatt ggggtg 1436

<210> 2

<211> 1486

<212> DNA

<213> Vibrio butyricum (Butyricimonas)

<400> 2

agagtttgat cctggctcag gatgaacgct agcgacaggc ttaacacatg caagtcgagg 60

ggcagcacgg tgtagcaata cactggtggc gaccggcgca cgggtgagta acacgtgtgc 120

aaccaacccc gtaccgggag ataacccgcg gaaacgtgga ctaacatccc atgatactcg 180

agagccgcat ggctctcgat ttaaaattcc ggtggtacgg gacgggcacg cgcgacatta 240

ggtagttggc ggggtaacgg cccaccaagc cgacgatgtc taggggttct gagaggaagg 300

tcccccacac tggaactgag acacggtcca gactcctacg ggaggcagca gtgaggaata 360

ttggtcaatg ggcgagagcc tgaaccagcc aagtcgcgtg agggaagaat ggtctatggc 420

ctgtaaacct cttttgtcag ggaagaataa ggatgacgag tcattcgatg ccagtacttg 480

acgaataagc atcggctaac tccgtgccag cagccgcggt aatacggggg atgcgagcgt 540

tatccggatt tattgggttt aaagggcgcg taggcgggac gtcaagtcag cggtaaaaga 600

ctgcagctaa actgtagcac gccgttgaaa ctggcgccct ggagacgaga cgagggaggc 660

ggaacaagtg aagtagcggt gaaatgcata gatatcactt ggaaccccga tagcgaaggc 720

agcttcccag gctcgttctg acgctgatgc gcgagagcgt gggtagcgaa caggattaga 780

taccctggta gtccacgccg taaacgatgc tcactggatc ttggcgatac actgccaggg 840

ttcaagcgaa agtattaagt gagccacctg gggagtacgt cggcaacgat gaaactcaaa 900

ggaattgacg ggggcccgca caagcggagg aacatgtggt ttaattcgat gatacgcgag 960

gaaccttacc cgggtttaaa tgtagattgc atgaggtgga aacgcttctt cccttcgggg 1020

ctatttacaa ggtgctgcat ggttgtcgtc agctcgtgcc gtgaggtgtc gggttaagtc 1080

ccataacgag cgcaacccct atcgccagtt gccatcggtt gaagccgggc actctgtcga 1140

gactgccacc gtaaggtgcg aggaaggcgg ggatgacgtc aaatcagcac ggcccttaca 1200

cccggggcga cacacgtgtt acaatggccg gtacagaggg cagccacggg gtgacccgga 1260

gcgaatctct aaagccggtc gtagttcgga ctggagtctg caacccgact ccacgaagtt 1320

ggattcgcta gtaatcgcgc atcagccatg gcgcggtgaa tacgttcccg ggccttgtac 1380

acaccgcccg tcaagccatg gaagccggga gtacctgaag atcgtgaccg cgaggaacgg 1440

gctagggtaa taccggtaac tggggctaag tcgtaacaag gtaacc 1486

<210> 3

<211> 1481

<212> DNA

<213> Bacillus calcoaceticus (Phascolarcotobacterium)

<220>

<221> misc_feature

<222> (38)..(38)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (1370)..(1370)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (1465)..(1465)

<223> n is a, c, g, or t

<400> 3

gacgaacgct ggcggcgtgc ctaacacatg caagtcgnac ggagaatttt atttcggtag 60

aattcttagt ggcgaacggg tgagtaacgc gtaggcaacc taccctttag acggggacaa 120

cattccgaaa ggagtgctaa taccggatgt gatcatcgtg ccgcatggca ggatgaagaa 180

agatggcctc tacaagtaag ctatcgctaa aggatgggcc tgcgtctgat tagctagttg 240

gtagtgtaac ggactaccaa ggcgatgatc agtagccggt ctgagaggat gaacggccac 300

attgggactg agacacggcc caaactccta cgggaggcag cagtggggaa tcttccgcaa 360

tggacgaaag tctgacggag caacgccgcg tgagtgatga aggatttcgg tctgtaaagc 420

tctgttgttt atgacgaacg tgcagtgtgt gaacaatgca ttgcaatgac ggtagtaaac 480

gaggaagcca cggctaacta cgtgccagca gccgcggtaa tacgtaggtg gcgagcgttg 540

tccggaatta ttgggcgtaa agagcatgta ggcggcttaa taagtcgagc gtgaaaatgc 600

ggggctcaac cccgtatggc gctggaaact gttaggcttg agtgcaggag aggaaagggg 660

aattcccagt gtagcggtga aatgcgtaga tattgggagg aacaccagtg gcgaaggcgc 720

ctttctggac tgtgtctgac gctgagatgc gaaagccagg gtagcgaacg ggattagata 780

ccccggtagt cctggccgta aacgatgggt actaggtgta ggaggtatcg accccttctg 840

tgccggagtt aacgcaataa gtaccccgcc tggggagtac ggccgcaagg ttgaaactca 900

aaggaattga cgggggcccg cacaagcggt ggagtatgtg gtttaattcg acgcaacgcg 960

aagaacctta ccaaggcttg acattgattg aacgctctag agatagagat ttcccttcgg 1020

ggacaagaaa acaggtggtg catggctgtc gtcagctcgt gtcgtgagat gttgggttaa 1080

gtcccgcaac gagcgcaacc cctatcctat gttaccagca agtaaagttg gggactcatg 1140

ggagactgcc agggacaacc tggaggaagg cggggatgac gtcaagtcat catgcccctt 1200

atgtcttggg ctacacacgt actacaatgg tcggaaacag agggaagcga agccgcgagg 1260

cagagcaaac cccagaaacc cgatctcagt tcggatcgca ggctgcaacc cgcctgcgtg 1320

aagtcggaat cgctagtaat cgcaggtcag catactgcgg tgaatacgtn cccgggcctt 1380

gtacacaccg cccgtcacac cacgaaagtt ggtaacaccc gaagccggtg aggtaaccta 1440

ttaggagcca gccgtctaag gtggngccga tgattggggt g 1481

<210> 4

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 4

ggttcgctga ttgtcttagt 20

<210> 5

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 5

accacataat ccactgcttg 20

<210> 6

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 6

ggtccagact cctacgggag 20

<210> 7

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 7

gcagcacctt gtaaatagcc 20

<210> 8

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 8

ctttagacgg ggacaacatt 20

<210> 9

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 9

cgaagggaaa tctctatctc 20

<210> 10

<211> 19

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 10

cccttcattg agaccaagt 19

<210> 11

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 11

gtacttgagc atgtaggcct 20

<210> 12

<211> 18

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 12

tgaaggaaga aggccttc 18

<210> 13

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 13

aactgagaca cggtccagac 20

<210> 14

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 14

cttcctccgt tttgtcaacg 20

<210> 15

<211> 19

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 15

acgtgccagc agccgcggt 19

<210> 16

<211> 19

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 16

aaagtctgac ggagcaacg 19

<210> 17

<211> 18

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 17

ccgggatctg gcattacg 18

<210> 18

<211> 19

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 18

gcggcgtgcc taacacatg 19

<210> 19

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 19

cataatccac tgcttgtgcg 20

Claims

1. An enteral microbial composition comprising one or more microbes selected from the group consisting of:

bacillus sporogenes (Sporobacter), Vibrio butyricum (Butyricimans), and Bacillus coralis (Phascolatobacter).

2. The intestinal microbial combination of claim 1, wherein said intestinal microbial combination comprises: bacillus sporogenes (Sporobacter), Vibrio butyricum (Butyricimans), and Bacillus coralis (Phascolatobacter).

3. The gut microorganism combination according to claim 1, wherein the gut microorganism combination is used for the diagnosis (including early diagnosis and/or assisted diagnosis) and treatment (including assisted treatment) of systemic lupus erythematosus; or preparing a kit or reagent for diagnosing (including early diagnosis and/or auxiliary diagnosis) and treating (including auxiliary treatment) the systemic lupus erythematosus.

4. A reagent set for diagnosing systemic lupus erythematosus comprising reagents for detecting each microorganism in the intestinal microorganism combination of claim 1.

5. The reagent set of claim 4, wherein each of the reagents is a primer, probe, antisense oligonucleotide, aptamer, or antibody specific for each microorganism;

preferably, the reagent is a PCR detection reagent; more preferably, the PCR detection reagent comprises a multiplex PCR detection system comprising:

6. A kit comprising the gut microorganism combination of claim 1 and/or the set of reagents of claim 4.

7. Use of a combination of gut microorganisms or a detection reagent thereof for the preparation of a kit for the diagnosis of systemic lupus erythematosus, wherein said combination of gut microorganisms comprises at least: bacillus sporogenes (Sporobacter), Vibrio butyricum (Butyricimans), and Bacillus coralis (Phascolatobacter).

8. The use according to claim 7, wherein said diagnosis comprises the steps of:

9. A medicament for treating systemic lupus erythematosus which has the ability to modulate the intestinal microbial combination of claim 1 in the intestines of a patient.

10. A method of screening for a drug for treatment of systemic lupus erythematosus, the method comprising the steps of:

(1) in the test group, administering a test compound to a subject to be tested, and detecting the level L1 of each microorganism in said combination of gut microorganisms in a sample derived from said subject in the test group; in a control group, administering a blank control to the subject to be tested, and detecting the level L2 of each microorganism in the combination of gut microorganisms in a sample derived from the subject in the control group;

wherein the intestinal microbial combination at least comprises one or more microbes selected from the group consisting of: bacillus sporogenes (Sporobacter), Vibrio butyricum (Butyricimans), and Bacillus coralis (Phascolatobacter).