CN113832079B - Intestinal microorganism combination and application thereof as systemic lupus erythematosus marker - Google Patents

Abstract

The invention discloses an intestinal microorganism combination and application thereof as a systemic lupus erythematosus marker, and in particular relates to a method for determining spore bacillus (Sporobacter), vibrio butyricum (Butyricimonas) and kola bacillus (Phascolobacter) with level differences in SLE patients by respectively carrying out 16s rRNA sequencing on intestinal microorganism samples from systemic lupus erythematosus individuals and healthy individuals, and determining the relationship between the level differences and systemic lupus erythematosus. These gut microorganisms with level differences can be used as markers for 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 (systemic lupus erythematosus, SLE) is a common chronic autoimmune connective tissue disease with multiple system involvement. SLE patients mainly comprise young females, the ratio of females to males is 9:1, and the peak onset age is 20-40 years. It is currently widely believed that the pathogenesis is related to T lymphocyte depletion, reduced T-reg function, B cell overactivation and dysfunction, with the production of large amounts of autoantibodies resulting in multiple organ and multiple system damage. At present, clinical diagnosis of systemic lupus erythematosus mainly adopts clinical symptoms, laboratory autoantibodies of serology and other detection means, and lacks a specific auxiliary disease judgment method and a related detection kit.

The intestinal microecological system is the largest ecological system of human body, and more than 10 intestinal tract implants ¹⁴ The number of genes in the genome of the intestinal flora of bacteria of the order of magnitude is about 150 times the total number of genes in the human genome. The intestinal flora and human body interdependence affect functions including intestinal nervous system, intestinal endocrine system, immune system, intestinal permeability, etc., and plays an important role in food digestion, vitamin nutrition, resisting invasion of exogenous pathogenic microorganisms, stimulating immunity, etc. The balance of the intestinal micro-ecological system is affected by a series of external factors including foods, medicines and the like, and antibiotics, pathogens and the like can break the balance of intestinal flora, so that a series of related diseases are caused, including various gastrointestinal diseases, liver diseases, inflammatory diseases, rheumatic arthritis, immune system diseases such as multiple sclerosis and the like, diabetes, metabolic syndrome, obesity and the like. In recent years, researches show that the 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 proposition of the concepts of "intestinal-hepatic axis" and "brain-intestinal axis", more and more research is beginning to focus on the variety and function of intestinal flora. Manipulation of enterobacterial composition and local metabolites through the use of probiotics has been explored as a promising approach for disease therapeutic intervention.

For systemic lupus erythematosus, no effective early detection method exists clinically at present, so that a method with high sensitivity and economy is needed to be established so as 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 systemic lupus erythematosus disease marker.

In a first aspect of the present invention there is provided an intestinal microbial combination comprising one or more microorganisms selected from the group consisting of: spore bacillus (Sporobacter), butyric acid vibrio (butyl bacteria), kola bacillus (Phascoloarcobacterium).

In another preferred embodiment, the intestinal microorganism combination comprises at least: spore bacillus (Sporobacter), vibrio butyrate (butyl bacteria), and kola bacillus (Phascoloarcobacterium).

In another preferred embodiment, the intestinal microbial combination further comprises one or more microorganisms selected from the group consisting of: examples of bacteria include, but are not limited to, bacillus species (Alistipes), prevotella species (Alloprvotella), enterobacter species (Odoribacter), and Prevotella species (Prevotella).

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

In another preferred embodiment, each microbial biomarker is detected by PCR; preferably, the PCR comprises QPCR, or RT-QPCR.

In another preferred embodiment, the intestinal microorganism combination is a gene combination comprising specific gene nucleic acid fragments of the respective microorganisms.

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

In another preferred embodiment, each of the reagents 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 for specifically amplifying the genes of each microorganism; more preferably, probes that specifically bind to genes of each microorganism are also included.

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

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

a second primer pair comprising a forward primer as 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 as 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 pair group includes:

a fourth primer pair comprising a forward primer as 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 comprising:

a first probe having a sequence as set forth in SEQ ID NO. 12;

a second probe having a sequence as set forth in SEQ ID NO. 15;

a third probe having a sequence set forth in SEQ ID NO. 16; and/or

And a fourth probe, the fourth probe sequence is shown as SEQ ID NO. 17.

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

In another preferred embodiment, each microorganism in the intestinal microorganism combination according to the first aspect of the 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, the invention provides the use of an intestinal microbiota or detection reagent thereof for the manufacture of a kit for diagnosing (including early diagnosis and/or co-diagnosis) systemic lupus erythematosus, wherein the intestinal microbiota comprises one or more microorganisms selected from the group consisting of: spore bacillus (Sporobacter), butyric acid vibrio (butyl bacteria), kola bacillus (Phascoloarcobacterium).

In another preferred embodiment, the intestinal microbial combination further comprises one or more microorganisms selected from the group consisting of: examples of bacteria include, but are not limited to, bacillus species (Alistipes), prevotella species (Alloprvotella), enterobacter species (Odoribacter), and Prevotella species (Prevotella).

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 microorganism level in a normal population.

In another preferred embodiment, the 16SrDNA of each microorganism is detected in step (1) using a PCR method.

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

In a fifth aspect of the invention, there is provided a medicament for the treatment (including adjuvant treatment) of systemic lupus erythematosus, said medicament having the ability to modulate the combination of intestinal microorganisms according to the first aspect of the invention in the intestinal tract of a patient.

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

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

(2) Comparing the level L1 detected in the previous step with the level L2, thereby determining whether the test compound is a candidate compound for treating systemic lupus erythematosus;

wherein the intestinal microbial combination comprises one or more selected from the group consisting of: spore bacillus (Sporobacter), butyric acid vibrio (butyl bacteria), kola bacillus (Phascoloarcobacterium).

In another preferred embodiment, the intestinal microbial combination further comprises one or more microorganisms selected from the group consisting of: examples of bacteria include, but are not limited to, bacillus species (Alistipes), prevotella species (Alloprvotella), enterobacter species (Odoribacter), and Prevotella species (Prevotella).

It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.

Drawings

Figure 1 shows comparison of intestinal flora richness and diversity in SLE patients and healthy people:

(a) Evaluating alpha diversity by using a Chao1 index; (b) evaluating alpha diversity using Shannon index; p <0.001, kruskal-Wallis test).

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

(a) PCOA visualizations based on Bray-Curtis distances; (b) PCA analysis chart.

FIG. 3 shows the ROC curve of Sporobacter bacteria.

FIG. 4 shows the ROC curve of Butyricimonas bacteria.

FIG. 5 shows the ROC curve of Phascoloarcobacterium.

Detailed Description

The inventors of the present invention conducted intensive studies to determine the differences in levels of various intestinal microorganisms present in SLE patients by sequencing the intestinal microorganism samples from individuals with systemic lupus erythematosus and healthy individuals, respectively, and determined the relationship between these differences in levels and systemic lupus erythematosus. These gut microorganisms with level differences can be used in compositions or kits for preventing, diagnosing/treating systemic lupus erythematosus diseases.

The invention confirms that SLE patients are obviously different from healthy people in intestinal bacteria species classification, and have differences (P < 0.05) in various microorganism levels, and the expression level of spore bacillus (Sporobacter), vibrio (butyl bacteria) and kola bacillus (Phascolobacter) of the patients is obviously lower than that of the healthy people, and the differences have obvious statistical significance, so that the expression level of the indexes is closely related to the incidence of systemic lupus erythematosus.

"16S rRNA" refers to rRNA constituting a 30S small subunit of a prokaryotic ribosome, which on the one hand is highly preserved in a large part of the base sequence and on the other hand shows a high base sequence diversity in a partial region. Particularly, there is little diversity among the isoforms and diversity among the isoforms is exhibited, so that by comparing the sequences of 16SrRNA, prokaryotes can be efficiently identified.

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

sporobacter representative sequence (SEQ ID NO.: 1):

GACGAACGCTGGCGGCGTGCCTAACACATGCAAGTCGAACGGAGACAATTGGTTCGCTGATTGTCTTAGTGGCGGACGGGTGAGTAACGCGTGAGCAATCTGCCCTTCGGAGGGGGACAACAGCTGGAAACGGCTGCTAATACCGCATAATGTATATTCAAGGCATCTTGGATATACCAAAGATTTATCGCCGAAGGATGAGCTCGCGTCTGATTAGCTAGTTGGTGAGGTAAAGGCTCACCAAGGCTGCGATCAGTAGCCGGACTGAGAGGTTGAACGGCCACATTGGAACTGAGATACGGGCCAGACTCCTACGGGAGGGAGCAGTGGGGAATTTTGGNCAATGGGGGAAAGCCNTACCCAGCAACGCCGCGTGAAGGAAGAAGGCCTTCGGGTTGTAAACTTCTTTGACCAGGGACGAAACAAATGACGGTACCTGGAAAACAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTATTACGTAGGTGGCAAGCGTTGTCCGGATTTACTGGGTGTAAAGGGCGCGTAGGCGGGAGTACAAGTCAGATGTGAAATCTGGGGGCTTAACCCTCAAACTGCATTTGAAACTGTATTTCTTGAGTATCGGAGAGGCAGGCGGAATTCCTAGTGTAGCGGTGAAATGCGTTGATATTAGGAGGAACACCAGTGGCGAAGGCGGCCTGCTGGACGACAACTGACTCTGAGGCGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCTGTAAACGATGAATACTAGGTGTGGGGGGACTGACCCCCTCCGTGCCGGAGTTAACACAATAAGTATTCCACCTGGGGAGTACGNCCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCAGTGGATTATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGGCTTGACATCGTACTAACGAAGCAGAGATGCATTAGGTGCCCTTCCGGGGAAAGTATAGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCCTATTGTNATTTGCTACNCGAGANCACTCTAGCGAGGCTGCCGATGACAAACCGGAGGAAGGTGGGGACGACGTCAAATCATCATGCCCCTTATGTCCTGGGCTACACACGTAATACAATGTCTCTCACAGAGGGAAGCAAGACCGCGAGGTGGAGCAAATCCCTAAAATGCGTCTCAGTTCAGATTGCAGGCTGCAACTCGCCTGCATGAAGTCGGAATTGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGAGAGCCGGGAACACCCGAAGTCCGTAGTCTAACCGCAAGGGGGACGCGGCCGAAGGTGGGTTTGGTAATTGGGGTG

representative sequences of the bacteria Butyricimonas are as follows (SEQ ID NO.: 2):

AGAGTTTGATCCTGGCTCAGGATGAACGCTAGCGACAGGCTTAACACATGCAAGTCGAGGGGCAGCACGGTGTAGCAATACACTGGTGGCGACCGGCGCACGGGTGAGTAACACGTGTGCAACCAACCCCGTACCGGGAGATAACCCGCGGAAACGTGGACTAACATCCCATGATACTCGAGAGCCGCATGGCTCTCGATTTAAAATTCCGGTGGTACGGGACGGGCACGCGCGACATTAGGTAGTTGGCGGGGTAACGGCCCACCAAGCCGACGATGTCTAGGGGTTCTGAGAGGAAGGTCCCCCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGAGGAATATTGGTCAATGGGCGAGAGCCTGAACCAGCCAAGTCGCGTGAGGGAAGAATGGTCTATGGCCTGTAAACCTCTTTTGTCAGGGAAGAATAAGGATGACGAGTCATTCGATGCCAGTACTTGACGAATAAGCATCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGGGGATGCGAGCGTTATCCGGATTTATTGGGTTTAAAGGGCGCGTAGGCGGGACGTCAAGTCAGCGGTAAAAGACTGCAGCTAAACTGTAGCACGCCGTTGAAACTGGCGCCCTGGAGACGAGACGAGGGAGGCGGAACAAGTGAAGTAGCGGTGAAATGCATAGATATCACTTGGAACCCCGATAGCGAAGGCAGCTTCCCAGGCTCGTTCTGACGCTGATGCGCGAGAGCGTGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGCTCACTGGATCTTGGCGATACACTGCCAGGGTTCAAGCGAAAGTATTAAGTGAGCCACCTGGGGAGTACGTCGGCAACGATGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGAGGAACATGTGGTTTAATTCGATGATACGCGAGGAACCTTACCCGGGTTTAAATGTAGATTGCATGAGGTGGAAACGCTTCTTCCCTTCGGGGCTATTTACAAGGTGCTGCATGGTTGTCGTCAGCTCGTGCCGTGAGGTGTCGGGTTAAGTCCCATAACGAGCGCAACCCCTATCGCCAGTTGCCATCGGTTGAAGCCGGGCACTCTGTCGAGACTGCCACCGTAAGGTGCGAGGAAGGCGGGGATGACGTCAAATCAGCACGGCCCTTACACCCGGGGCGACACACGTGTTACAATGGCCGGTACAGAGGGCAGCCACGGGGTGACCCGGAGCGAATCTCTAAAGCCGGTCGTAGTTCGGACTGGAGTCTGCAACCCGACTCCACGAAGTTGGATTCGCTAGTAATCGCGCATCAGCCATGGCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCAAGCCATGGAAGCCGGGAGTACCTGAAGATCGTGACCGCGAGGAACGGGCTAGGGTAATACCGGTAACTGGGGCTAAGTCGTAACAAGGTAACC

a representative sequence of Phascoloarcobacterium is as follows (SEQ ID NO.: 3):

GACGAACGCTGGCGGCGTGCCTAACACATGCAAGTCGNACGGAGAATTTTATTTCGGTAGAATTCTTAGTGGCGAACGGGTGAGTAACGCGTAGGCAACCTACCCTTTAGACGGGGACAACATTCCGAAAGGAGTGCTAATACCGGATGTGATCATCGTGCCGCATGGCAGGATGAAGAAAGATGGCCTCTACAAGTAAGCTATCGCTAAAGGATGGGCCTGCGTCTGATTAGCTAGTTGGTAGTGTAACGGACTACCAAGGCGATGATCAGTAGCCGGTCTGAGAGGATGAACGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTGGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGATTTCGGTCTGTAAAGCTCTGTTGTTTATGACGAACGTGCAGTGTGTGAACAATGCATTGCAATGACGGTAGTAAACGAGGAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCGAGCGTTGTCCGGAATTATTGGGCGTAAAGAGCATGTAGGCGGCTTAATAAGTCGAGCGTGAAAATGCGGGGCTCAACCCCGTATGGCGCTGGAAACTGTTAGGCTTGAGTGCAGGAGAGGAAAGGGGAATTCCCAGTGTAGCGGTGAAATGCGTAGATATTGGGAGGAACACCAGTGGCGAAGGCGCCTTTCTGGACTGTGTCTGACGCTGAGATGCGAAAGCCAGGGTAGCGAACGGGATTAGATACCCCGGTAGTCCTGGCCGTAAACGATGGGTACTAGGTGTAGGAGGTATCGACCCCTTCTGTGCCGGAGTTAACGCAATAAGTACCCCGCCTGGGGAGTACGGCCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGTATGTGGTTTAATTCGACGCAACGCGAAGAACCTTACCAAGGCTTGACATTGATTGAACGCTCTAGAGATAGAGATTTCCCTTCGGGGACAAGAAAACAGGTGGTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCCTATCCTATGTTACCAGCAAGTAAAGTTGGGGACTCATGGGAGACTGCCAGGGACAACCTGGAGGAAGGCGGGGATGACGTCAAGTCATCATGCCCCTTATGTCTTGGGCTACACACGTACTACAATGGTCGGAAACAGAGGGAAGCGAAGCCGCGAGGCAGAGCAAACCCCAGAAACCCGATCTCAGTTCGGATCGCAGGCTGCAACCCGCCTGCGTGAAGTCGGAATCGCTAGTAATCGCAGGTCAGCATACTGCGGTGAATACGTNCCCGGGCCTTGTACACACCGCCCGTCACACCACGAAAGTTGGTAACACCCGAAGCCGGTGAGGTAACCTATTAGGAGCCAGCCGTCTAAGGTGGNGCCGATGATTGGGGTG

the present invention provides the use of an intestinal microbial combination comprising one or more microorganisms selected from the group consisting of: spore bacillus (Sporobacter), butyric acid vibrio (butyl bacteria), kola bacillus (Phascoloarcobacterium).

The inventors have found that in patients with systemic lupus erythematosus, the level of intestinal microorganisms in the patient's feces is significantly reduced. Wherein the sporobacillus (Sporobacter) level is reduced by about 55%, the vibrio butyricum (butyl bacteria) level is reduced by about 50%, and the kola bacillus (Phascolobacter) level is reduced by about 10% compared to healthy people.

Thus, the intestinal microorganisms can be used as biomarkers for diagnosis and detection of systemic lupus erythematosus.

In one embodiment, the systemic lupus erythematosus detection product comprises a microbial abundance detection reagent for the intestinal microorganism; preferably a specific recognition reagent for each microorganism as described.

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

In one embodiment, the specific primer is capable of detecting primers of each microorganism that include 16SrRNA and other gene sequences that recognize the particular microorganism.

In the present invention, the term "primer" is a sequence of 7 to 50bp which is capable of forming a base pair complementary to a template strand and functions as a starting point for copying the template strand. Primers are usually synthesized, but naturally occurring nucleic acids may also be used. The sequence of the primer need not be exactly the same as the sequence of the template, but may be sufficiently complementary to hybridize with the template. Additional features may be incorporated that do not alter the basic properties of the primer. Examples of additional features that can be incorporated include methylation, capping, substitution of one or more nucleic acids with homologs, and modification between nucleic acids, but are not limited thereto.

In one embodiment, the primer may be used to amplify 16SrRNA of a corresponding microorganism and other gene sequences that recognize the microorganism, and the presence of the microorganism or the level of the microorganism can be detected by the presence or absence of a desired product after the amplification of the sequences.

A variety of methods known in the art can be used for sequence amplification methods using primers. For example, polymerase Chain Reaction (PCR), reverse transcription-polymerase chain reaction (RT-PCR), multiplex PCR, nested (nested) PCR, real-time fluorescent quantitative PCR, etc. can be used.

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

The invention also provides a gene chip for detecting systemic lupus erythematosus, which comprises a solid phase carrier and an oligonucleotide probe fixed on the solid phase carrier, wherein the oligonucleotide probe specifically recognizes the gene sequence of each microorganism 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, the probe is able to bind to a target polynucleotide that lacks complete sequence complementarity with the probe. Probes may be labeled directly or indirectly, and include primers. Hybridization means, 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. The term "complementary" as used herein is not limited to being completely complementary as long as it is hybridized. These polynucleotides generally have homology of 90% or more, preferably 95% or more, more preferably 100% or more with respect to the specific base sequence. These probes may be DNA or RNA, or may be polynucleotides obtained by replacing part or all of the nucleotides with artificial nucleic acids such as PNA (Polyamide nucleicacid, peptide nucleic acid), LNA (locked nucleic acid, bridged Nucleic Acid, crosslinked nucleic acid), ENA (2 '-O,4' -C-ethyl-bridged nucleic acids), GNA (Glycerolnucleic acid ), TNA (Threose nucleic acid, threose nucleic acid), or the like.

The present invention will be described in further detail with reference to the following examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The following examples are not to be construed as limiting the details of the experimental procedure, and are generally carried out under conventional conditions such as those described in the guidelines for molecular cloning laboratory, sambrook.J.et al, (Huang Peitang et al, beijing: scientific Press, 2002), or as recommended by the manufacturer. Percentages and parts are by weight unless otherwise indicated. The experimental materials and reagents used in the following examples were obtained from commercial sources unless otherwise specified.

EXAMPLE 1 study subject and sample collection

Stool samples from 300 subjects, including 120 SLE patients and 180 healthy controls, were collected at southern hospitals at southern medical universities with informed consent.

To qualify for inclusion in the study, individuals must meet the following fecal sample collection criteria:

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

2) At least 3 months after any medical intervention;

3) No pregnancy, tumor, infection or autoimmune or rheumatic diseases

The fecal sample collection tube is dispatched to the study subjects and the specific collection method is informed, the subjects take 10-15g of fresh feces and put into the collection tube with fecal genome protection liquid, and the feces are delivered to a hospital laboratory at normal temperature and stored in a refrigerator at 4 ℃ until use.

EXAMPLE 2 16S rRNA analysis of intestinal microflora

2.1DNA extraction

DNA extraction was performed using fecal microorganism genome extraction kit (cantonese medical science co.) according to the manufacturer's instructions.

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

2.2 construction of DNA library and sequencing

Performing two rounds of 16S rRNA gene PCR amplification on the purified genome DNA by using bacterial universal primers to complete the construction of a sequencing sample library, and using2.0Fluorometer (Invitrogen, USA) to detect concentration, agilent2100 (Agilent, USA) to detect library size; the amplified 16S rRNA hypervariable region sequence was sequenced using an Illumina Miseq (Illumina, san Diego, USA) sequencer, with the sequencing region being the V3-V4 region.

2.3 data analysis

Raw sequencing data (FASTQ) were trimmed and filtered using trimmatic v.0.32 software, and the high quality sequence files were clustered by a 97% similarity criterion to give an Operational Taxon (OTU). Alpha and beta diversity analysis was then performed using Qiime2 v.2017.12 software and R software.

Intestinal flora alpha diversity was assessed using the Chao1, shannon diversity index, and intestinal flora beta diversity was assessed using principal coordinate analysis (PCoA) and PCA analysis. Groups of samples were analyzed for differences in significance using a linear discriminant analysis effect amount based analysis method (LEfSe).

2.4 results

Intestinal microbiota information of healthy people and SLE patients was obtained by 16S rRNA-based analysis, which confirmed that intestinal flora diversity and richness of SLE patients were significantly reduced compared to healthy people (fig. 1).

PCoA analysis and PCA analysis assessed intestinal flora beta diversity, and as a result, SLE patients and healthy people had a clear tendency to segregate, suggesting that SLE patients had different intestinal flora structures than HC groups (FIG. 2).

In addition, it was confirmed that SLE patients were significantly different from healthy individuals in the classification of intestinal bacteria species, and that there was a difference in the levels of various intestinal microorganisms (P < 0.05), and that the amounts of expression of Sporobacter, butyricimonas, phascolobacter bacteria were significantly lower than those of healthy individuals, and that the differences had significant statistical significance, suggesting that the amounts of expression of these indicators were closely related to the onset of systemic lupus erythematosus (Table 1).

TABLE 1 differential species

Example 3 verification of clinical detection of intestinal flora in SLE patients

The above study shows that SLE patients are significantly different from healthy people in the species classification of intestinal bacteria, and have differences in the levels of multiple intestinal microorganisms, and the expression level of Sporobacter, butyricimonas and Phascolobacter bacteria of the patients is significantly lower than that of 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 were collected for healthy and SLE patients in this example, and the samples were numbered and tested. Specific fluorescent quantitative PCR primer probes are designed aiming at the 16S rRNA gene sequence of target microorganisms, and internal standards are set.

The real-time fluorescent quantitative PCR (Quantitative Real-time PCR) specific primer sequences for detecting the abundance of Sporobacter, butyricimonas, phascolobacter bacteria in the samples are shown in Table 2.

TABLE 2

Fluorescent quantitative PCR amplification detection system: each strain was primed at 0.5. Mu.l upstream and downstream, template DNA was 5. Mu.l, SYBR Green 6. Mu.l. The fluorescent quantitative PCR reaction conditions are as follows: 95℃for 10min,95℃for 15s and 60℃for 2min, 40 cycles in total.

Each sample was tested for 9 bacteria, respectively, and an internal standard gene and a pure water control sample were set.

PCR reaction is carried out on an ABI7500 fluorescent 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).

Analysis was performed using logistic regression models and ROC curves were drawn to take into account the diagnostic properties of the species. ROC curves are curves plotted on the ordinate with true positive rate (sensitivity) and false positive rate (1-specificity) on the abscissa, according to a series of different classification schemes (demarcation values or decision thresholds). 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, with the least total number of false positives and false negatives. Calculating the area under the ROC curve (AUC) for each potential marker can determine the diagnostic value of the potential marker, with greater AUC being greater.

TABLE 3 diagnostic potential analysis

The results showed that the intestinal flora of SLE patients showed significant differences (P < 0.05) in Sporobacter, butyricimonas, phascolobacter, etc., relative to healthy individuals. This result is consistent with the 16SrRNA library sequencing result.

Figures 3 to 5 show ROC curves for each flora, which show that the detected flora has high sensitivity and specificity and can therefore be used for the assisted diagnosis of SLE patients.

Therefore, when one or more indexes of Sporobacter bacteria, butyricimonas bacteria and Phascolobacter bacteria in the intestinal flora of the tested population are lower than those of normal people, the probability of suffering from systemic lupus erythematosus is high, so that the method is favorable for assisting in judging the disease condition, and is also a potential target for treating the systemic lupus erythematosus.

Example 4 preparation of detection kit

The embodiment provides a real-time fluorescence quantitative PCR (Quantitative Real-time PCR) detection kit based on the abundance of Sporobacter, butyricimonas and Phascoloarcobacterium in a detection sample.

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

TABLE 4 multiplex assay systems

The content of each component in the PCR reaction system in the using process is 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 enzyme system	3μL
Template nucleic acid	5μL
		DEPC water	Make up to 25. Mu.L

Performing amplification reaction in a real-time fluorescence PCR instrument, wherein FAM, VIC, texas red and Cy5 are selected as fluorescence channels, and the PCR amplification procedure is as follows;

50 ℃,2min,95 ℃ for 15min;1 cycle

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

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

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

Comparative example 1

After deep comparison and analysis of 16sRNA gene sequences of target bacteria, the inventors designed a plurality of pairs of primers and probes for each target sequence, and it was difficult to obtain effective multiplex PCR amplification primers and probe sequences due to competition inhibition, primer specificity difference, inconsistent annealing temperature, primer dimer and the like among primers of a multiplex reaction system. Through a large number of experiments, the designed primers and probes are optimally selected and verified, and finally, the sequences of the primers and the probes and the combination thereof which can be used for multiplex PCR amplification are determined.

It was found in experiments that even in the case where the primer pairs and probe sequences for each target nucleic acid have been basically determined, there is a significant difference in the effect of multiplex amplification by different primer pair combinations.

For example, in the multiplex PCR step, the Butyricimonas primer sequence in the multiplex reaction system described above is replaced with the control primer pair 1:

F1：GGTCCAGACTCCTACGGGAG(SEQ ID NO.:6)

R1：GCAGCACCTTGTAAATAGCC(SEQ ID NO.:7)

the other components are unchanged.

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

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

The primer sequence of the Sporobacter bacteria in the multiple reaction system is replaced by a control primer pair 2:

F2：GCGGCGTGCCTAACACATG(SEQ ID NO.:18)

R2：CATAATCCACTGCTTGTGCG(SEQ ID NO.:19)

the other components are unchanged.

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

In the multiplex detection system, the detection results of 20 samples show that the accuracy of the multiplex detection system containing the control primer pair 2 can not meet the clinical detection requirement only by detecting target Sporobacter bacteria in 16 samples.

All documents mentioned in this application are incorporated by reference as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the claims appended hereto.

Sequence listing

<110> Chinese market workers Hospital

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

<130> P210347

<160> 19

<170> PatentIn version 3.5

<210> 1

<211> 1436

<212> DNA

<213> spore bacillus (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> Karaoke bacillus (Phascoloarcobacterium)

<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 (6)

1. Use of an intestinal microbiota or a detection reagent thereof for the preparation of a kit for the diagnosis of systemic lupus erythematosus, wherein the intestinal microbiota consists of sporobacilli (Sporobacter), vibrio butyricum (Butyricimonas) and kola bacteria (Phascolobacter).

2. The use according to claim 1, wherein the reagent is a PCR detection reagent.

3. The use of claim 2, wherein the PCR detection reagent comprises a multiplex PCR detection system comprising a primer pair set comprising:

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

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

4. The use of claim 3, wherein the primer pair set further comprises:

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

5. The use of claim 4, wherein the multiplex PCR detection system further comprises a probe set comprising:

a first probe having a sequence as set forth in SEQ ID NO. 12;

a second probe having a sequence as set forth in SEQ ID NO. 15;

a third probe having a sequence set forth in SEQ ID NO. 16; and

and a fourth probe, the fourth probe sequence is shown as SEQ ID NO. 17.

6. The use according to claim 1, wherein 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 microorganism level in a normal population.