JP2023020631A - Method for testing systemic lupus erythematosus - Google Patents

Abstract

To provide a novel biomarker for systemic lupus erythematosus and a method for use thereof.SOLUTION: A method for testing systemic lupus erythematosus includes the step (1) for detecting at least one biomarker selected from the group consisting of Streptococcus anginosus and Streptococcus intermedius in an enteric bacteria-containing sample taken from a subject.SELECTED DRAWING: None

Description

TECHNICAL FIELD The present invention relates to a method for testing systemic lupus erythematosus, and the like.

Many autoimmune diseases, including systemic lupus erythematosus, are difficult to diagnose with a single biomarker, and can only be diagnosed by combining multiple hematological and histological findings and clinical symptoms. However, there are many problems to be solved in diagnosis, such as the high degree of invasiveness of histological examination and the high degree of diagnostic expertise, and the discovery of new biomarkers is desired.

The intestinal microbiota has a significant impact on human immunity and metabolism, and it has been reported that it may be the etiology of various diseases such as inflammatory bowel disease, colon cancer, diabetes, and autism. is viewed as promising. In recent years, inventions relating to intestinal flora that contribute to diagnosis of autoimmune diseases have been reported (Patent Document 1).

Until now, screening using intestinal microbiota has been measured by bacterial species at the genus level and OTU units, which can be estimated from 16S rRNA, etc., and has been a method lacking precision. In recent years, a technique using next-generation sequencing called shotgun sequencing, which enables comprehensive evaluation and analysis of the entire DNA of microorganisms in feces, has been implemented. However, this method requires genome data obtained after performing complex analysis on a vast amount of genome data of bacterial flora, making it difficult to apply to general screening. Against this background, there has been a need for the development and adaptation of techniques for simpler and more accurate screening of bacterial flora.

International Publication No. 2016/050111

An object of the present invention is to provide a novel biomarker for systemic lupus erythematosus and a method for using the same. Preferably, the object is to provide a simple test technique for systemic lupus erythematosus.

As a result of intensive research, the present inventors have found a method for testing systemic lupus erythematosus, comprising (1) selecting from the group consisting of Streptococcus anginosus and Streptococcus intermedius in a sample containing intestinal bacteria collected from a subject; The present inventors have found that the above-described problems can be solved by a testing method comprising the step of detecting at least one biomarker obtained by the method. As a result of further research based on this knowledge, the present invention was completed. That is, the present invention includes the following aspects.

Section 1. A method of testing for systemic lupus erythematosus, comprising:
(1) detecting at least one biomarker selected from the group consisting of Streptococcus anginosus and Streptococcus intermedius in an intestinal bacterium-containing sample collected from a subject;
inspection methods, including;

Section 2. Furthermore, (2) determining that the subject is suffering from systemic lupus erythematosus when the amount or concentration of the biomarker detected in step (1) is equal to or greater than the cutoff value;
The inspection method according to Item 1, comprising:

Item 3. Item 3. The test method according to Item 1 or 2, wherein the biomarkers are Streptococcus anginosus and Streptococcus intermedius.

Section 4. Item 4. The inspection method according to any one of Items 1 to 3, wherein the intestinal bacteria-containing sample is feces.

Item 5. Item 5. The test method according to any one of Items 1 to 4, wherein the subject is a human.

Item 6. A diagnostic agent for systemic lupus erythematosus, comprising at least one biomarker-detecting agent selected from the group consisting of Streptococcus anginosus and Streptococcus intermedius.

Item 7. A test kit for systemic lupus erythematosus, comprising at least one biomarker-detecting agent selected from the group consisting of Streptococcus anginosus and Streptococcus intermedius.

Item 8. The amount or concentration of at least one biomarker selected from the group consisting of Streptococcus anginosus and Streptococcus intermedius in a sample containing intestinal bacteria collected from an animal treated with the test substance as an indicator A method for screening an active ingredient of a prophylactic or therapeutic agent for systemic lupus erythematosus.

Item 9. The amount or concentration of at least one biomarker selected from the group consisting of Streptococcus anginosus and Streptococcus intermedius in a sample containing intestinal bacteria collected from an animal treated with the test substance as an indicator A method for evaluating induction or exacerbation of systemic lupus erythematosus.

According to the present invention, biomarkers for systemic lupus erythematosus can be provided. By using the biomarker, systemic lupus erythematosus examination, screening of active ingredients of prophylactic or therapeutic agents for systemic lupus erythematosus, evaluation of producibility or exacerbation of systemic lupus erythematosus, etc. may become possible. Since the examination technique of the present invention can utilize a sample that can be easily collected, it can be easily implemented.

1 is a diagram showing screening results of Test Example 1. FIG. Plots represent each bacterium. The horizontal axis shows the logarithmic value obtained by dividing the expression level in the systemic lupus erythematosus patient group by the expression level in the control group, and the vertical axis shows the false discovery rate when denying the null hypothesis that there is no difference in the expression level. is logarithmically transformed to base 10 and given a negative sign.

As used herein, the expressions "contain" and "include" include the concepts "contain", "include", "consist essentially of" and "consist only of".

1. Method for testing systemic lupus erythematosus In one aspect of the present invention, there is provided a method for testing for systemic lupus erythematosus, comprising: (1) a sample containing intestinal bacteria collected from a subject, comprising Streptococcus anginosus and Streptococcus intermedius The present invention relates to a testing method (also referred to herein as the “systemic lupus erythematosus testing method of the present invention”), comprising the step of detecting at least one biomarker selected from the group. This will be explained below.

1-1. Process (1)
The type of systemic lupus erythematosus to be tested is not particularly limited. All classes, grades, and stages of systemic lupus erythematosus in various classification criteria for systemic lupus erythematosus are subject to testing. Also, the lesion site is not particularly limited.

A subject is a target organism of the test method of the present invention, and its species is not particularly limited. Species of the subject include, for example, various mammals such as humans, monkeys, mice, rats, dogs, cats, and rabbits, preferably humans.

The subject's condition is not particularly limited. Subjects include, for example, a subject whose presence or absence of systemic lupus erythematosus is unknown, a subject that has already been determined to have systemic lupus erythematosus by another method, and a subject that has already been determined not to have systemic lupus erythematosus. Examples include specimens that have been determined by the method of , and specimens that are undergoing treatment for systemic lupus erythematosus.

The enteric bacteria-containing sample is not particularly limited as long as it contains enteric bacteria. Samples containing intestinal bacteria include, for example, gastrointestinal contents such as intestinal juice and feces. Among these, stool is preferable from the viewpoint of the burden on the subject. The intestinal bacteria-containing sample may be used singly or in combination of two or more. A sample containing intestinal bacteria can be collected from a subject by methods known to those skilled in the art.

The detection target in step (1) is at least one biomarker selected from the group consisting of Streptococcus anginosus and Streptococcus intermedius (in the present specification, these may be collectively referred to as "target biomarkers"). is.

The target biomarker is a biomarker whose amount is changing in systemic lupus erythematosus, and systemic lupus erythematosus can be distinguished by using this as an indicator.

Streptococcus anginosus and Streptococcus intermedius are biomarkers of interest with higher abundance in systemic lupus erythematosus specimens than in healthy specimens.

The number of target biomarkers in step (1) may be only one, but by combining Streptococcus anginosus and Streptococcus intermedius, systemic lupus erythematosus can be tested more accurately.

Detection is typically performed by measuring the amount or concentration of the biomarker of interest. "Concentration" is not limited to absolute concentration, but also relative concentration, weight per unit volume, amount per total bacterial cell amount or total nucleic acid amount in sample, raw data measured to know absolute concentration, etc. It's okay.

A method for detecting a target biomarker is not particularly limited as long as it is a method capable of specifically detecting part or all of the target biomarker. Specific examples of detection methods include, for example, culturing intestinal bacteria in a pre-predicted selective medium and confirming the presence or absence of target intestinal bacterium colonies, a certain intestinal bacterium-specific gene or gene Southern hybridization method, Northern hybridization method, DNA microarray method and the like for detecting derived mRNA can be mentioned. A method using shotgun sequencing of DNA in a sample (for example, the method of Test Example 1 described later) can also be used. In addition, metabolic detection of bacterial metabolites, antibody-based methods of proteomic detection of bacterial proteins, and the like are also available.

In addition, as a means to measure bacteria in a sample, for example, a method of culturing intestinal bacteria in a selective medium predicted in advance and counting the number of bacteria, or a method of culturing intestinal bacteria in a selective liquid medium and measuring turbidity and absorbance. method, FISH method, real-time PCR method, RT-PCR method, and the like.

Here, the RT-PCR method will be explained. The analysis method using the RT-PCR method includes, for example, (1) a step of extracting the RNA of the target intestinal bacteria in the sample, (2) an RT using a nucleic acid fragment (primer) that hybridizes to the extracted RNA. - PCR can be performed, and (3) the step of detecting the DNA fragment amplified in step (2). A DNA fragment (PCR product) specific to the target intestinal bacterium can be obtained by combining the nucleic acid fragment with the template cDNA derived from the specimen and performing an amplification reaction. By observing the PCR product over time and specifying the number of PCR cycles when a certain amount of DNA is reached, it becomes possible to quantify the number of target intestinal bacteria in the sample.

Observation of the amplified PCR product over time can be performed by labeling the PCR product with an intercalating fluorescent dye such as SYBR (R) GreenI and measuring the fluorescence intensity at each PCR step. Since intercalating dyes have the property of increasing fluorescence intensity by intercalating into double-stranded nucleic acids, it is possible to accurately measure PCR products generated by PCR reaction from cDNA of target bacteria. (R)GreenI is preferably used.

By specifying the PCR cycle number (hereinafter referred to as the CT value) when an arbitrarily set fluorescence intensity (DNA amount) is reached, it is possible to quantify the target intestinal bacteria in the sample. TaqMan probes, MoleculerBeacons, etc. labeled with fluorescent dyes can also be used. TaqMan probes and Moleculer Beacons are probes in which a fluorescent dye and a quencher are bound to an oligonucleotide having homology to the internal sequence of the region amplified by PCR, and used together in the PCR reaction. Since the interaction between the fluorescent dye bound to the probe and the quencher emits fluorescence corresponding to the PCR amplification reaction, it is possible to observe the amplified PCR product over time by measuring the fluorescence intensity at each PCR step. can.

Quantification of target intestinal bacteria in a sample can be obtained from a calibration curve of the logarithmic value of the number of bacteria measured by the DAPI counting method, the culture method, or the like, and the CT value. That is, a standard curve is prepared in advance by plotting the logarithmic value of the number of target bacteria on the horizontal axis and the CT value on the vertical axis, and the CT value obtained as a result of the PCR reaction is applied to the standard curve, Quantify the target intestinal bacteria.

According to the testing method of the present invention including step (1), the amount and/or concentration of the target biomarker, which is a detection indicator for systemic lupus erythematosus, can be provided, thereby assisting the detection of systemic lupus erythematosus. be able to.

The test results of the test method of the present invention including step (1) are used to determine therapeutic effect, clarify the pathology of systemic lupus erythematosus, predict prognosis of systemic lupus erythematosus, stratify patients, select treatment methods (personalized medicine, treatment response It can be used for sex), etc.

1-2. Process (2)
As an aspect, the testing method of the present invention further includes (2) when the amount or concentration of the biomarker detected in the step (1) is a cutoff value or more, the subject has systemic lupus erythematosus determining to be affected;
is preferably included. According to the testing method of the present invention including step 2, systemic lupus erythematosus can be determined.

The cut-off value can be appropriately set by those skilled in the art from the viewpoint of sensitivity, specificity, positive predictive value, negative predictive value, etc. Based on the amount and/or concentration of the biomarker of interest in the endobacterium-containing sample, it can be a determined value on a case-by-case basis or a predetermined value. The cut-off value is, for example, the amount and/or concentration of the target biomarker in an intestinal bacterium-containing sample collected from a subject not suffering from systemic lupus erythematosus (when there are multiple subjects, the mean value, median value etc.), for example, 0.5 to 1.4 times, 0.6 to 1.3 times, 0.7 to 1.2 times, 0.8 to 1.1 times, 0.9 to 1 times.

In a preferred embodiment of step (2), if the subject is a subject under treatment for systemic lupus erythematosus, the cutoff value is, for example, the amount and / or concentration of the biomarker of interest in a past sample for the same subject. The therapeutic effect can be determined by using the value based on the above.

2. When the test method of the present invention including the step (2) of diagnosing systemic lupus erythematosus with higher accuracy determines that the subject has systemic lupus erythematosus, the test method of the present invention further includes systemic Systemic lupus erythematosus can be diagnosed with greater accuracy by combining the steps of applying a physician's diagnosis of lupus erythematosus. In addition, since the testing method of the present invention can more accurately detect systemic lupus erythematosus, by combining the above-described steps with the testing method of the present invention, it is possible to more efficiently and accurately detect "suffering from systemic lupus erythematosus." Diagnose.

3. If the test method of the present invention including the treatment step (2) for systemic lupus erythematosus determines that the subject is suffering from systemic lupus erythematosus, the test method of the present invention may additionally be treated with the above "2. Systemic lupus erythematosus. Diagnosis of Lupus Erythematosus with Higher Accuracy", when diagnosed as suffering from systemic lupus erythematosus, further for the combination of the test method of the present invention and the step of applying a diagnosis by a doctor, (3) By performing the step of treating a subject determined or diagnosed as having systemic lupus erythematosus, the disease of the subject can be treated. In addition, since the testing method of the present invention can more accurately detect systemic lupus erythematosus, the steps ( By combining 3), a subject suffering from systemic lupus erythematosus can be treated more efficiently and more reliably.

Methods for treating systemic lupus erythematosus are not particularly limited, but typically include drug therapy. Pharmaceuticals used for drug therapy are not particularly limited, but include, for example, non-steroidal anti-inflammatory analgesics, steroids, and the like. One, two, or a combination of three or more medicaments can be used.

Four. Test agent for systemic lupus erythematosus In one aspect of the present invention, an agent for detecting at least one biomarker selected from the group consisting of Streptococcus anginosus and Streptococcus intermedius (herein, "test agent of the present invention") The present invention relates to a test agent for systemic lupus erythematosus (also referred to herein as the “test agent of the present invention”). This will be explained below.

The detection agent of the present invention is not particularly limited as long as it can specifically detect the target biomarker. The detection agent includes, for example, primers, probes, antibodies, etc. directed to bacterial genes, which are target biomarkers, or their expression products.

The detection agent of the present invention may be modified as long as its function is not significantly impaired. Modifications include, for example, addition of labels such as fluorescent dyes, enzymes, proteins, radioisotopes, chemiluminescent substances, biotin, and the like.

As the fluorescent dye used in the present invention, those generally used for the detection and quantification of nucleic acids by labeling nucleotides can be suitably used. '-hexachloro-6-carboxylfluorescein, green fluorescent dye), fluorescein, NED (trade name, manufactured by Applied Biosystems, yellow fluorescent dye), or 6-FAM (trade name, manufactured by Applied Biosystems, yellow green fluorescent dye), rhodamin or its derivatives (eg, tetramethylrhodamine (TMR)). As a method for labeling nucleotides with a fluorescent dye, an appropriate known labeling method can be used [see Nature Biotechnology, 14, 303-308 (1996)]. A commercially available fluorescent labeling kit can also be used (eg, Oligonucleotide ECL 3'-oligo labeling system manufactured by Amersham Pharmacia).

The detection agent of the present invention can also be used by immobilizing it on any solid phase. Therefore, the test agent of the present invention can be provided in the form of a substrate on which a detecting agent is immobilized (for example, a microarray chip on which probes are immobilized, etc.).

The solid phase used for immobilization is not particularly limited as long as it can immobilize polynucleotides, etc. Examples include glass plates, nylon membranes, microbeads, silicon chips, capillaries, and other substrates. can be done. Immobilization of the detection agent to the solid phase is not particularly limited. Immobilization methods are well known in the art according to the type of immobilized probe, such as using a commercially available spotter (manufactured by Amersham, etc.) for microarrays [e.g., photolithographic technology (Affymetrix), in situ synthesis of oligonucleotides by inkjet technology (Rosetta Inpharmatics), etc.].

Primers, probes and the like are not particularly limited as long as they selectively (specifically) recognize target biomarkers and nucleic acids derived therefrom. Here, "selectively (specifically) recognize" means, for example, in the Northern blot method, the target biomarker can be specifically detected, and in the RT-PCR method, the target biomarker or a nucleic acid derived from it (cDNA, etc.) is specifically amplified, but is not limited to it, as long as a person skilled in the art can determine that the detected product or amplified product is derived from the target biomarker good.

Specific examples of primers and probes include the polynucleotides described in (a) below and the polynucleotides described in (b) below:
(a) a polynucleotide having at least 15 consecutive bases in the base sequence of the target biomarker and/or a polynucleotide complementary to the polynucleotide, and (b) the base sequence of the target biomarker or a base complementary thereto At least one selected from the group consisting of polynucleotides having at least 15 bases that hybridize to the sequence under stringent conditions.

Complementary polynucleotide or complementary base sequence (complementary strand, reverse strand) refers to the full-length sequence of the polynucleotide consisting of the base sequence of the target biomarker, or the base sequence of at least 15 consecutive bases in the base sequence Polynucleotides or bases that are in a base-complementary relationship based on base pair relationships such as A:T and G:C with respect to the partial sequences (herein, these are also referred to as "positive strands" for convenience) It means an array. However, such a complementary strand is not limited to the case where it forms a completely complementary sequence with the base sequence of the target positive strand, but also has a complementary relationship to the extent that it can hybridize with the target positive strand under stringent conditions. may have. Here, stringent conditions are conditions that bind complexes or probes as taught by Berger and Kimmel (1987, Guide to Molecular Cloning Techniques Methods in Enzymology, Vol. 152, Academic Press, San Diego Calif.). It can be determined based on the melting temperature (Tm) of the nucleic acid. For example, the conditions for washing after hybridization are usually about "1×SSC, 0.1% SDS, 37° C.". The complementary strand is preferably one that maintains a hybridized state with the target positive strand even after washing under such conditions. Although it is not particularly limited, a more stringent hybridization condition is about "0.5 x SSC, 0.1% SDS, 42°C", and an even more stringent hybridization condition is about "0.1 x SSC, 0.1% SDS, 65°C" for washing. can be done. Specifically, such complementary strands include a strand consisting of a base sequence that is completely complementary to the base sequence of the target positive strand, and at least 90%, preferably 95%, more preferably A strand consisting of a nucleotide sequence having 98% or more, more preferably 99% or more identity can be exemplified.

Primers, probes, and the like can be designed using various design programs, for example, based on the nucleotide sequence of the target biomarker. Specifically, candidate sequences for primers or probes obtained by subjecting the base sequences of the target biomarkers to a design program, or sequences containing at least the sequences as a part thereof, can be used as primers or probes.

The base length of primers, probes, etc. is not particularly limited as long as it has a length of at least 15 consecutive bases as described above, and can be appropriately set according to the application. Examples of base lengths include 15 to 35 bases when used as primers, and 15 to 35 bases when used as probes, for example.

The test agent of the present invention may contain other detecting agents (for example, probes for detecting other nucleic acids, antibodies, etc.) other than the detecting agent of the present invention. In this case, the test agent of the present invention may be a test agent capable of testing other diseases and conditions in addition to systemic lupus erythematosus. In this case, the detecting agent of the present invention is included as a detecting agent for testing for systemic lupus erythematosus. From this point of view, the test agent of the present invention, in one aspect thereof, is a test agent for systemic lupus erythematosus containing a detecting agent for systemic lupus erythematosus test consisting of the detecting agent of the present invention.

The test agent of the present invention may be in the form of a composition. The composition may contain other ingredients as needed. Other components include bases, carriers, solvents, dispersants, emulsifiers, buffers, stabilizers, excipients, binders, disintegrants, lubricants, thickeners, humectants, colorants, and perfumes. , chelating agents and the like.

The test agent of the present invention may be in the form of a kit. The kit may contain, in addition to the detection agent or the composition containing the same, those that can be used to detect the biomarker of interest in a sample containing intestinal bacteria of a subject. Specific examples of such materials include various reagents (eg, nucleic acid extraction reagents, buffer solutions, etc.), instruments (eg, instruments for purification and separation of intestinal bacteria-containing samples), and the like.

Five. Screening Method for Active Ingredients of Preventive or Curative Agents for Systemic Lupus Erythematosus In one aspect of the present invention, a sample containing intestinal bacteria collected from an animal treated with a test substance comprises Streptococcus anginosus and Streptococcus intermedius. A screening method for an active ingredient of a prophylactic or therapeutic agent for systemic lupus erythematosus, wherein the amount or concentration of at least one biomarker selected from the group is used as an index (herein referred to as the "active ingredient screening method of the present invention" ). This will be explained below.

The animal species is not particularly limited. Examples of animal species include various mammals such as humans, monkeys, mice, rats, dogs, cats, and rabbits.

A wide range of test substances can be used regardless of whether they are naturally occurring compounds or artificially produced compounds. In addition, not only purified compounds, but also compositions in which various compounds are mixed, and extracts of animals and plants can be used. Compounds include not only low-molecular-weight compounds, but also high-molecular-weight compounds such as proteins, nucleic acids, and polysaccharides.

More specifically, the active ingredient screening method of the present invention is characterized in that the value of the indicator is the amount or concentration of the corresponding biomarker in a sample containing intestinal bacteria collected from animals not treated with the test substance (control ), the test substance is selected as an active ingredient of a prophylactic or therapeutic agent for systemic lupus erythematosus (or a candidate substance for an active ingredient of a prophylactic or therapeutic agent for systemic lupus erythematosus).

A corresponding biomarker means the same bacterium as the target biomarker used as an indicator.

"Low" means, for example, that the index value is 1/2, 1/5, 1/10, 1/20, 1/50, 1/100 of the control value.

6. In one aspect of the present invention, a method for evaluating provocation or exacerbation of systemic lupus erythematosus from the group consisting of Streptococcus anginosus and Streptococcus intermedius in a sample containing intestinal bacteria collected from an animal treated with a test substance A method for evaluating the induction or exacerbation of systemic lupus erythematosus, which uses the amount or concentration of at least one selected biomarker as an index (herein, may be referred to as the "toxicity evaluation method of the present invention". ). This will be explained below.

More specifically, in the toxicity evaluation method of the present invention, the value of the indicator is the amount or concentration (control value) of the corresponding biomarker in a sample containing intestinal bacteria collected from an animal not treated with the test substance. determining that the test substance is provoking or exacerbating systemic lupus erythematosus if higher than .

A corresponding biomarker means the same bacterium as the target biomarker used as an indicator.

"High" means, for example, that the index value is 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold higher than the control value.

EXAMPLES The present invention will be described in detail below based on examples, but the present invention is not limited by these examples.

Test example 1. Biomarker screening of systemic lupus erythematosus I built my own pipeline that does In the metagenomic analysis pipeline, first, stool samples from 47 systemic lupus erythematosus patients and 203 healthy subjects were collected, lysed by the bead method, and DNA was extracted. Paired-end sequencing of 150 bp using Hiseq3000 yielded an average of 6.3 Gb of sequence reads per sample.

Quality control of sequence reads was performed as follows. A series of quality control steps were performed to maximize the quality of the dataset. The main steps in the quality control process are: (i) trimming of poor quality bases, (ii) identification and masking of human reads, and (iii) elimination of duplicate reads. Marking of duplicate reads was performed using PRINSEQ-lite (version 0.20.4, parameter: -derep 1). Trim raw reads using Trimmomatic (version 0.39; parameters: ILLUMINACLIP:TruSeq3-PE-2.fa:2:30:10:8:true LEADING:20 TRAILING:20 SLIDINGWINDOW:3:15 MINLEN:60) and clipped the Illumina adapter and cut the low quality bases on both ends. Reads less than 60 bp in length after trimming were discarded. Next, among the duplicated reads with the same sequence, only the longest read was retained and the duplicated portion was removed. Finally, quality-filtered reads were aligned to the human reference genome (hg38) using the default parameters of bowtie2 (version 2.3.5) and BMTagger (version 3.101). Only reads where both paired ends were not aligned with either tool were retained.

We used curated reference metagenomes to improve the efficiency and accuracy of the taxonomic annotation of metagenomes. A Japanese population reference metagenome constructed in a previous report was combined with a metagenome identified from the Human Gut Bacteria Project. Filtering genomes annotated to species with more than 50 reference genomes yielded a taxonomic reference genome dataset of 7,881 genomes. Filtered paired-end reads were aligned to the reference genome dataset using bowtie2 with default parameters. For multiple-mapped reads, only the best possible alignment was selected by alignment score. The number of reads mapped to each genome was divided by the length of the genome. The values for each genome were summed for each sample, and the relative compositional proportions of each clade at six levels (L2: Phylum, L3: Order, L4: Order, L5: Family, L6: Genus, L7: Species). Calculated. Then, outlier samples were detected by PCA. On the other hand, for gene and pathway analysis, each read was subjected to de novo assembly to create the longest nucleotide sequence (contig) possible. Regions encoding genes in the contigs were identified and clustering of similar regions between samples was performed. Comprehensively calculated the relative amounts of gene sequences contained in each sample by referring to amino acid sequence information and pathway information for genes and proteins registered in public databases. A case-control analysis was performed on the composition ratio of the microorganisms obtained above and the relative amount of genes, and two strains were identified as biomarkers (Streptococcus anginosus and Streptococcus intermedius).

Using these biomarkers, linear regression was performed with a regression formula adjusted for age and sex (bacteria ~ SLE + age + sex + Dataset + bacterial principal component (PC1 to PC25)), and Wald statistics for the systemic lupus erythematosus term were obtained. When tested for dose, these biomarkers were found to contribute significantly to the diagnosis of systemic lupus erythematosus.

FIG. 1 shows a volcano plot comparing the amounts of bacterial species.

Table 1 shows the effect size, standard error (SE), and P-value (P).

Even when the male samples and the drug user samples were excluded, the association between the two types of bacteria shown in Table 1 and systemic lupus erythematosus could be confirmed.

For both Streptococcus anginosus and Streptococcus intermedius, the composition ratio in systemic lupus erythematosus was higher than that in healthy subjects (Streptococcus anginosus: 2.9 times, Streptococcus intermedius: 2.2 times).

A Receiver Operating Characteristic (ROC) curve for the combination of Streptococcus anginosus and Streptococcus intermedius for diagnosing systemic lupus erythematosus showed an AUC of 0.79 (95% confidence interval 0.73 to 0.86).

Claims (9)

A method of testing for systemic lupus erythematosus, comprising:
(1) detecting at least one biomarker selected from the group consisting of Streptococcus anginosus and Streptococcus intermedius in an intestinal bacterium-containing sample collected from a subject;
inspection methods, including; Furthermore, (2) determining that the subject is suffering from systemic lupus erythematosus when the amount or concentration of the biomarker detected in step (1) is equal to or greater than the cutoff value;
The inspection method according to claim 1, comprising: The test method according to claim 1 or 2, wherein the biomarkers are Streptococcus anginosus and Streptococcus intermedius. The test method according to any one of claims 1 to 3, wherein the sample containing intestinal bacteria is stool. The test method according to any one of claims 1 to 4, wherein the subject is a human. A diagnostic agent for systemic lupus erythematosus, comprising at least one biomarker-detecting agent selected from the group consisting of Streptococcus anginosus and Streptococcus intermedius. A test kit for systemic lupus erythematosus, comprising at least one biomarker-detecting agent selected from the group consisting of Streptococcus anginosus and Streptococcus intermedius. The amount or concentration of at least one biomarker selected from the group consisting of Streptococcus anginosus and Streptococcus intermedius in a sample containing intestinal bacteria collected from an animal treated with the test substance as an indicator A method for screening an active ingredient of a prophylactic or therapeutic agent for systemic lupus erythematosus. The amount or concentration of at least one biomarker selected from the group consisting of Streptococcus anginosus and Streptococcus intermedius in a sample containing intestinal bacteria collected from an animal treated with the test substance as an indicator A method for evaluating induction or exacerbation of systemic lupus erythematosus.

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