Method for detecting bacteria involved in the onset of rheumatoid arthritis, reagent for detecting the bacteria, method for determining the presence or absence of predisposition to rheumatoid arthritis, and agent for determining the presence or absence of the predisposition

Abstract

The objective of the present invention is to provide a means for identifying a bacterium associated with the development of rheumatoid arthritis and determining the possibility of contracting rheumatoid arthritis earlier and more easily using the bacterium. The present invention relates to a method and reagent for detecting a bacterium involved with the development of rheumatoid arthritis, and a method and agent for determining the presence or absence of a predisposition to rheumatoid arthritis.

Description

Technical Field

[0001] The present invention relates to a method for detecting bacteria involved in the onset of rheumatoid arthritis, a reagent for detecting the bacteria, a method for determining the presence or absence of a predisposition to rheumatoid arthritis, and an agent for determining the presence or absence of the predisposition.

Background Art

[0002] Rheumatoid arthritis (rheumatoid-like arthritis, RA) is a chronic systemic autoimmune disease that occurs in about 1% of the total population and mainly affects joints. Rheumatoid arthritis causes damage through cytokines, chemokines, and metalloproteases.

[0003] The onset of rheumatoid arthritis is usually insidious and often starts with systemic and joint symptoms. As a characteristic of the pathological condition of rheumatoid arthritis, inflammation occurs symmetrically in peripheral joints (e.g., finger joints, middle finger joints), and as a result, the joint structure is progressively destroyed, usually accompanied by systemic symptoms. Rheumatoid arthritis occurs two to three times more frequently in women than in men and can occur at any age, with the highest incidence between 35 and 50 years old, but it can also occur in childhood or old age (Non-Patent Document 1).

[0004] When the symptoms of rheumatoid arthritis worsen, the physical and mental pain of the patient increases, and at the same time, the cost of drugs used for treatment soars. Therefore, a mechanism for providing appropriate treatment at an appropriate time is required.

[0005] Currently, the diagnosis of rheumatoid arthritis is comprehensively performed by scoring the number of swollen and painful joints and the presence or absence of rheumatoid factor and anti-CCP antibody in blood tests. However, the development of biomarkers that can determine the possibility of suffering from rheumatoid arthritis earlier is required.

[0006] On the other hand, Non-Patent Document 2 discloses that the prevalence rate of Prevotella copri in the intestine increases in the early stage of rheumatoid arthritis.

Prior Art Documents

Non-Patent Literature

[0007]

Non-Patent Literature 1

Non-Patent Literature 2

Summary of the Invention

Problems to be Solved by the Invention

[0008] Rheumatoid factors and anti-CCP antibodies that can be measured by the above blood tests and are used for the diagnosis of rheumatoid arthritis may be detected even in healthy individuals, while there are problems with their specificity, such as not being detected in patients with rheumatoid arthritis. In addition, Non-Patent Literature 2 discloses that the prevalence of Prevotella copri in the intestine increases at the early stage of rheumatoid arthritis, but the relationship with the onset of rheumatoid arthritis is not clear.

[0009] An object of the present invention is to identify bacteria related to the onset of rheumatoid arthritis and provide a means for more early and simply determining the possibility of suffering from rheumatoid arthritis by using the bacteria.

Means for Solving the Problems

[0010] The inventors isolated Prevotella copri from fecal samples of rheumatoid arthritis patients and healthy subjects, and found that Prevotella copri derived from rheumatoid arthritis patients (hereinafter abbreviated as RA-P.copri) has arthritis-inducing activity and causes exacerbation of arthritis compared with Prevotella copri derived from healthy subjects (hereinafter abbreviated as HC-P.copri). Furthermore, as a result of analyzing the genomic sequences of RA-P.copri and HC-P.copri, it was revealed that there are regions specific to the genome of RA-P.copri. Based on the above findings, the present invention was completed.

[0011] The present invention is as follows. 1. A method for detecting bacteria involved in the onset of rheumatoid arthritis, comprising a step of specifically detecting any one of the following nucleic acid sequences (1) to (3) or a partial sequence thereof. (1) The nucleic acid sequences represented by SEQ ID NOs: 1 to 3 (2) A nucleic acid sequence having 95% or more homology with the nucleic acid sequences represented by SEQ ID NOs: 1 to 3 (3) A nucleic acid sequence that hybridizes under stringent conditions with the nucleic acid sequence described in SEQ ID NOs: 1 to 3 or a nucleic acid sequence complementary to the nucleic acid sequence 2. The detection method according to 1 above, wherein the bacteria involved in the onset of rheumatoid arthritis belong to the genus Prevotella. 3. The detection method according to 2 above, wherein the bacteria belonging to the genus Prevotella are Prevotella copri. 4. The detection method according to any one of 1 to 3 above, wherein the step of specifically detecting any one of the nucleic acid sequences (1) to (3) or a partial sequence thereof is performed by polymerase chain reaction using DNA derived from the feces of a subject as a template. 5. The detection method according to 4 above, wherein at least two primers used in the polymerase chain reaction are oligonucleotides containing a nucleic acid sequence consisting of at least 15 consecutive bases among the nucleic acid sequences shown in any one of SEQ ID NOs: 1 to 3 or a nucleic acid sequence complementary to the sequence. 6. The detection method according to item 5 above, wherein the primer is one oligonucleotide selected from the nucleic acid sequences represented by SEQ ID NOs: 4 to 19. 7. The detection method according to any one of items 1 to 3 above, wherein the step of specifically detecting the nucleic acid sequence or a partial sequence thereof is performed by metagenomic analysis using a next-generation sequencer on DNA derived from the feces of a subject. 8. A method for determining the presence or absence of a predisposition to rheumatoid arthritis, comprising the step of detecting, by the detection method according to any one of items 1 to 7 above, bacteria involved in the onset of rheumatoid arthritis in the feces of a subject. 9. A reagent for detecting bacteria involved in the onset of rheumatoid arthritis, comprising a primer or a probe that specifically detects any one of the following nucleic acid sequences (1) to (3) or a partial sequence thereof. (1) Nucleic acid sequences represented by SEQ ID NOs: 1 to 3 (2) Nucleic acid sequences having 95% or more homology with the nucleic acid sequences represented by SEQ ID NOs: 1 to 3 (3) Nucleic acid sequences that hybridize under stringent conditions with the nucleic acid sequences described in SEQ ID NOs: 1 to 3 or nucleic acid sequences complementary to the nucleic acid sequences 10. The detection reagent according to item 9 above, wherein the primer or the probe is an oligonucleotide containing a nucleic acid sequence consisting of at least 15 consecutive bases among the nucleic acid sequences shown in any one of SEQ ID NOs: 1 to 3 or nucleic acid sequences complementary to the sequences. 11. The detection reagent according to item 10 above, wherein the primer or the probe is one oligonucleotide selected from the nucleic acid sequences represented by SEQ ID NOs: 4 to 19. 12. An agent for determining the presence or absence of a predisposition to rheumatoid arthritis, comprising a primer or a probe that specifically detects any one of the following nucleic acid sequences (1) to (3) or a partial sequence thereof. (1) Nucleic acid sequences represented by SEQ ID NOs: 1 to 3 (2) Nucleic acid sequences having 95% or more homology with the nucleic acid sequences represented by SEQ ID NOs: 1 to 3 (3) Nucleic acid sequences that hybridize under stringent conditions with the nucleic acid sequences described in SEQ ID NOs: 1 to 3 or nucleic acid sequences complementary to the nucleic acid sequences 13. The agent according to item 12, wherein the primer or the probe is an oligonucleotide containing a nucleic acid sequence consisting of at least 15 consecutive bases out of the nucleic acid sequences shown in any one of SEQ ID NOs: 1 to 3 or a nucleic acid sequence complementary to the sequence. 14. The agent according to item 13, wherein the primer or the probe is an oligonucleotide selected from the nucleic acid sequences represented by SEQ ID NOs: 4 to 19.

Advantages of the Invention

[0012] According to the method for detecting bacteria and the reagent for detecting bacteria of the present invention, it is possible to specifically and simply detect bacteria involved in the onset of rheumatoid arthritis from before the onset of rheumatoid arthritis. Further, according to the method for determining the presence or absence of a predisposition to rheumatoid arthritis and the agent for determining the presence or absence of the predisposition of the present invention, it is possible to determine the possibility of suffering from rheumatoid arthritis from before the onset of rheumatoid arthritis and to perform appropriate treatment at an appropriate time, which can be a new strategy from the perspective of preventive medicine.

Brief Description of the Drawings

[0013]

Figure 1

Figure 2

Figure 3

Figure 4

Modes for Carrying Out the Invention

[0014] Hereinafter, unless otherwise specified, the terms used in this specification have the meanings commonly used in the art.

[0015] <1. Method for detecting bacteria> The bacteria in the present invention are intestinal bacteria having the activity of inducing arthritis. The activity of inducing arthritis can be determined by an increase in the arthritis score. The method for determining the increase in the arthritis score is not particularly limited, but specifically, for example, the following methods can be mentioned.

[0016] In one aspect, the bacteria in the present invention have the following nucleic acid sequence of (1) or (2) or a partial sequence thereof. (1) The nucleic acid sequences represented by SEQ ID NOs: 1 to 3 (2) A nucleic acid sequence having 95% or more homology with the nucleic acid sequences represented by SEQ ID NOs: 1 to 3

[0017] Here, SEQ ID NOs: 1 to 3 represent the nucleic acid sequences of regions specifically present in the genome of RA-P. copri having arthritis-inducing activity. The homology between nucleic acid sequences is calculated using the homology search program BLAST. When the bacteria have a nucleic acid sequence having 95% or more homology with the nucleic acid sequences represented by SEQ ID NOs: 1 to 3, it can be said that, similar to RA-P. copri having the nucleic acid sequences represented by SEQ ID NOs: 1 to 3, there is a high possibility of having the activity of inducing arthritis.

[0018] As a method for determining the activity of inducing arthritis by an increase in the arthritis score, the method described by Tsuyoshi Kakitani et al. in New Biochemistry Experiment Course 12, Molecular Immunology II, pages 360 - 372, Tokyo Kagaku Dojin (1989) can be mentioned. Specifically, the following method can be mentioned. First, an antibiotic (for example, ampicillin, neomycin, metronidazole, and vancomycin) is administered to an arthritis model mouse (for example, DBA / 1j mouse) for several days to kill the intestinal bacteria. Subsequently, it is divided into a group to which the test bacteria are administered and a group to which bacteria derived from a healthy subject (control group), and each bacterium is orally administered continuously for several days (for example, 5 days). Three days after the final administration, feces are collected to confirm that each bacterium has colonized in the mouse, and an emulsion mixed with type II collagen and an adjuvant is intradermally administered to the mice in each group. Three weeks later, as a booster, an emulsion mixed with type II collagen and an adjuvant is readministered, and the arthritis score is evaluated over time. When the arthritis score is statistically significantly higher compared to the control group 5 to 8 weeks after the second administration of the emulsion, it can be determined that the test bacteria have the activity of inducing rheumatoid arthritis.

[0019] The bacterium in the present invention is Prevotella copri, which is a gram - negative anaerobic bacterium. Whether the bacterium isolated from the isolation source is Prevotella copri can be determined, for example, by performing phylogenetic analysis by comparing the nucleotide sequence data of the 16S ribosomal RNA gene with the sequence data of known species.

[0020] In this specification, "detection of bacteria" includes not only determining the presence or absence of bacterial cells but also quantifying their abundance.

[0021] For the detection of bacteria, total DNA is recovered from a sample. Examples of the types of the sample include, but are not limited to, feces or intestinal contents of a subject (a healthy subject, a rheumatoid arthritis patient, or an individual suspected of having rheumatoid arthritis), isolated / cultured bacteria, and the like. Methods for isolating / purifying DNA from a sample are known in the art and can be performed, for example, by extraction with phenol-chloroform, extraction using a commercially available DNA extraction reagent, or purification using a commercially available column kit.

[0022] The DNA recovered from the sample is dissolved in an appropriate buffer, such as TE (10 mM Tris-HCl, 1 mM EDTA, pH 8.0), etc., and subjected to the detection method of the present invention.

[0023] The method for detecting bacteria of the present invention is characterized by including a step of specifically detecting any one of the following nucleic acid sequences (1) to (3) or a partial sequence thereof. (1) The nucleic acid sequences represented by SEQ ID NOs: 1 to 3 (2) A nucleic acid sequence having 95% or more homology with the nucleic acid sequences represented by SEQ ID NOs: 1 to 3 (3) A nucleic acid sequence that hybridizes under stringent conditions with the nucleic acid sequences described in SEQ ID NOs: 1 to 3 or a nucleic acid sequence complementary to the nucleic acid sequences

[0024] Nucleic acid sequences that hybridize under stringent conditions include nucleic acid sequences having a certain degree or more of sequence identity with the nucleic acid sequence used as a probe. For example, nucleic acid sequences having at least 60% or more homology with the target nucleic acid sequence, preferably nucleic acid sequences having 80% or more homology, more preferably nucleic acid sequences having 90% or more homology, and most preferably genes having 95% or more homology can be mentioned. Further, for example, if 100 nucleic acids in the nucleic acid sequence are taken as one unit, in the nucleic acid sequence of the target gene, per unit, 1 to several, preferably 1 to 40, preferably 1 to 35, preferably 1 to 30, preferably 1 to 25, preferably 1 to 20, more preferably 1 to 15, still more preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, and even more preferably 1, 2, 3, 4 or 5 nucleic acid deletions, substitutions, additions, etc. can be mentioned.

[0025] "Deletion of nucleic acid" means that there is a deletion or disappearance of nucleic acid in the sequence, "substitution of nucleic acid" means that the nucleic acid in the sequence is replaced by another nucleic acid, and "addition of nucleic acid" means that a new nucleic acid is added so as to be inserted.

[0026] In one aspect, the detection method of the present invention preferably uses a primer or a probe that can specifically detect any one of the nucleic acid sequences of (1) to (3) above or a partial sequence thereof.

[0027] The primer may be any one as long as it is designed to specifically PCR amplify a partial region of the above nucleic acid sequence. Any one of the nucleic acid sequences of (1) to (3) above or a partial sequence thereof is a nucleic acid sequence present in the genome of RA-P.copri having arthritis-inducing activity in the present invention.

[0028] Here, "specifically" means that the primer PCR amplifies a partial region of any one of the nucleic acid sequences of (1) to (3) above, but does not PCR amplify a nucleic acid sequence that does not contain any one of the nucleic acid sequences of (1) to (3) above or a partial sequence thereof.

[0029] As the primer, for example, a combination of oligonucleotides containing a nucleic acid sequence represented by any one of SEQ ID NOs: 1 to 3 or a continuous nucleic acid sequence complementary to the sequence, preferably 15 to 50 bases, more preferably 18 to 30 bases, and the fragment length of the nucleic acid amplified by them is preferably 50 to 1,000 bases, more preferably 100 to 500 bases, and a pair of oligonucleotides can be mentioned.

[0030] Specific examples of preferred primers include combinations of oligonucleotides selected from the nucleic acid sequences represented by SEQ ID NOs: 4 to 19.

[0031] The temperature setting, reaction time, and number of cycles in PCR can be appropriately set according to the amount of template DNA used, the type of primer, etc. The annealing temperature in PCR can be appropriately set based on the GC content of the primer. For example, using genomic DNA of bacteria as a template and an oligonucleotide consisting of the nucleic acid sequence represented by SEQ ID NO: 4 and an oligonucleotide consisting of the nucleic acid sequence represented by SEQ ID NO: 5 as primers, the reaction can be carried out under the conditions of 95°C for 30 seconds, 55°C for 30 seconds, and 72°C for 45 seconds for 30 cycles.

[0032] The probe is an oligonucleotide that hybridizes under stringent conditions to a continuous nucleic acid sequence contained in the nucleic acid sequence represented by SEQ ID NOs: 1 to 3 or a nucleic acid sequence complementary to the nucleic acid sequence, preferably 15 bases or more, more preferably 18 to 500 bases, still more preferably 18 to 200 bases, and particularly preferably 18 to 50 bases.

[0033] Hybridization can be carried out according to a method known per se or a method analogous thereto, for example, the method described in Molecular Cloning, 2nd Edition (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989). Examples of stringent conditions include, for example, washing one or more times at 65° C. in 0.2×SSC / 0.1% SDS after a hybridization reaction at 45° C. in 6×SSC (sodium chloride / sodium citrate). Those skilled in the art can easily adjust the desired stringency by appropriately changing the salt concentration of the hybridization solution, the temperature of the hybridization reaction, the probe concentration, the length of the probe, the number of mismatches, the time of the hybridization reaction, the salt concentration of the washing solution, the temperature of the washing, etc.

[0034] The length of the probe is preferably 15 bases or more, more preferably 18 to 500 bases, still more preferably 18 to 200 bases, and particularly preferably 18 to 50 bases.

[0035] From the viewpoint of specificity, examples of the probe preferably include an oligonucleotide that hybridizes to a nucleic acid sequence represented by any one of SEQ ID NOs: 4 to 19 or its complementary sequence. More preferably, an oligonucleotide containing a consecutive 15- to 50-base subsequence of a nucleic acid sequence represented by any one of SEQ ID NOs: 4 to 19 or its complementary sequence is mentioned.

[0036] Specific examples of preferred probes include oligonucleotides selected from the nucleic acid sequences represented by SEQ ID NOs: 4 to 19.

[0037] The primer or probe may contain an additional sequence (a nucleic acid sequence not complementary to the polynucleotide to be detected) as long as it does not interfere with specific detection.

[0038] The oligonucleotide used as the primer or probe may be deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). In the case of ribonucleic acid, the thymidine residue (T) in the nucleotide sequence is appropriately read as the uridine residue (U). It may also be DNA containing a uridine residue synthesized by changing T at any position to U. Similarly, it may be RNA containing a thymidine residue synthesized by changing U at any position to T. Further, as long as the specificity of hybridization is not reduced, point mutations such as deletions, insertions or substitutions, or modified nucleotides may be present in the oligonucleotide.

[0039] Also, the primer or probe may be labeled with a suitable labeling agent, for example, a radioisotope (for example, 125 I, 131 I, 3 H, 14 C, 32 P, 33 P, 35 S, etc.), an enzyme (for example, β-galactosidase, β-glucosidase, alkaline phosphatase, peroxidase, malate dehydrogenase, etc.), a fluorescent substance (for example, fluorescamine, fluorescein isothiocyanate, etc.), a luminescent substance (for example, luminol, luminol derivative, luciferin, lucigenin, etc.), biotin, etc.

[0040] The oligonucleotide used as the primer or probe can be chemically synthesized, for example, using a general-purpose DNA synthesizer. The oligonucleotide may also be synthesized using any of the other methods well known in the art.

[0041] It includes performing PCR using the primer contained in the detection reagent of the present invention with the DNA recovered from the sample as a template. The obtained PCR product is separated by electrophoresis (for example, agarose gel electrophoresis, polyacrylamide gel electrophoresis, etc.). After electrophoresis, the gel is stained with a staining solution known per se such as an ethidium bromide solution, and the PCR product is detected using a transilluminator or the like. Then, based on the presence or absence and amount of the specific PCR product, the presence or absence and abundance of the bacteria of the present invention in the sample are determined.

[0042] The PCR used in the detection method of the present invention may be quantitative PCR. Quantitative PCR can be performed by known methods, and two analysis methods are known. The first one is a method that utilizes the characteristic that the reaction product increases exponentially to a certain amount in the PCR reaction and then reaches a plateau, analyzes the amount of the reaction product during the exponential growth phase, and calculates the initial template amount. The second one is a method of determining the number of PCR cycles (Ct) at which the amount of the reaction product exceeds a certain value (threshold) by monitoring the reaction product in real time. For both analysis methods, it is necessary to perform PCR by changing the amount of DNA with a known concentration, analyze the reaction product at each cycle number, and determine a quantitative PCR cycle number range from its kinetics. Based on the results, the abundance of the target gene in the unknown sample is estimated. Thereby, the abundance of the bacteria of the present invention in the sample can be quantified, and when it is estimated that the target gene is contained even in one copy in the test sample, it can be determined that the bacteria of the present invention are present.

[0043] In one aspect, the detection method of the present invention includes a step of bringing the probe contained in the detection reagent of the present invention into contact with the total DNA in the sample. The contact conditions are appropriately set so that the probe hybridizes with the nucleic acid sequence specifically present in the genome of RA - P.copri to form a nucleic acid complex. And the complex is detected as indicating the presence of the bacteria of the present invention.

[0044] When using a probe, it can be carried out by various known hybridization techniques [for example, fluorescence in situ hybridization method (abbreviated as Fluorescence In Situ Hybridization (hereinafter referred to as FISH)) and the like]. In the FISH method, the probe penetrates into the cytoplasm of bacteria and hybridizes under appropriate hybridization conditions to the nucleic acid sequence present in the genome of RA-P.copri present therein. At this time, by labeling the probe with a radioisotope, a fluorescent substance [for example, fluorescein isothiocyanate (FITC), TAMRA, Cy3, Cy5, etc.], a chemiluminescent substance, etc., the phenomenon of specific hybridization can be monitored by an appropriate method (for example, autoradiography, fluorescence microscopy, flow cytometry, etc.). For example, when the probe is labeled with a radioisotope, an assay can be carried out by a method such as autoradiography, when it is labeled with a fluorescent substance, an assay can be carried out with a fluorescence microscope, etc., and when it is labeled with a chemiluminescent substance, analysis using a photosensitive film or digital analysis using a CCD camera can be carried out. Thereby, bacteria in the sample can be detected.

[0045] In one aspect, the detection method of the present invention includes metagenomic analysis by a next-generation sequencer. A next-generation sequencer is a nucleotide sequence analysis (decoding) device that has dramatically improved the analysis speed by parallel processing, and is contrasted with a fluorescence capillary sequencer (referred to as the "first-generation sequencer") that uses the Sanger sequencing method. By using metagenomic analysis by a next-generation sequencer, it is possible to analyze all bacterial species constituting the gut microbiota and their abundance ratios. It is not particularly limited as long as it is a method for clarifying the diversity of the gut microbiota and the abundance ratio of specific bacteria constituting the gut microbiota.

[0046] Examples of commercially available next-generation sequencers include various next-generation sequencers sold by manufacturers such as Illumina (e.g., HiSeq 2500, HiSeq X Ten, NextSeq 500), Roche (454) (e.g., GS FLX+ system), Life Technologies (e.g., 5500xl SOLiD), and Ion Torrent (e.g., Proton Sequencer). It is also possible to use next-generation sequencers that will be developed and sold in the future, not limited to existing ultra-parallel next-generation sequencers.

[0047] <2. Reagent for detecting bacteria> The detection reagent of the present invention detects the presence of the bacteria of the present invention in a sample by detecting a nucleic acid sequence present in the genome of RA-P. copri in the present invention. The detection reagent of the present invention includes the primer or probe described above in <1. Method for detecting bacteria> that can specifically detect any one of the following nucleic acid sequences (1) to (3). (1) Nucleic acid sequences represented by SEQ ID NOs: 1 to 3 (2) Nucleic acid sequences having 95% or more homology with the nucleic acid sequences represented by SEQ ID NOs: 1 to 3 (3) Nucleic acid sequences that hybridize under stringent conditions with the nucleic acid sequences described in SEQ ID NOs: 1 to 3 or nucleic acid sequences complementary to the nucleic acid sequences

[0048] The detection reagent of the present invention may further contain nucleic acid synthesizing enzymes (e.g., DNA polymerase, RNA polymerase, reverse transcriptase, etc.), other enzymes, substrates corresponding to the enzymes (e.g., dNTP, rNTP, etc.), etc. as other components. It may also contain a labeled detection substance, a buffer solution, etc.

[0049] By using the detection reagent of the present invention, the presence or absence of bacteria that induce rheumatoid arthritis in a sample can be easily determined in a short time, which is useful for diagnosing the predisposing factors of rheumatoid arthritis.

[0050] <3. Method for determining the presence or absence of predisposing factors for rheumatoid arthritis> As shown in the examples described below, the bacteria of the present invention having the nucleic acid sequences represented by SEQ ID NOs: 1 to 3 were detected from the feces of patients with rheumatoid arthritis. Considering that the bacteria of the present invention have the activity of inducing arthritis, the presence of the bacteria of the present invention in the intestine of a subject indicates that the subject has a predisposition (predisposition to rheumatoid arthritis) to develop rheumatoid arthritis, regardless of whether the subject has already developed rheumatoid arthritis.

[0051] Therefore, by detecting the bacteria involved in the development of rheumatoid arthritis in the feces of a subject by the detection method described in <1. Bacterial detection method>, the presence or absence of a predisposition to rheumatoid arthritis can be determined. That is, the present invention provides a method for determining the presence or absence of a predisposition to rheumatoid arthritis, which includes detecting the bacteria of the present invention in the feces of a subject.

[0052] The bacteria of the present invention can be detected by the detection method described in <1. Bacterial detection method> using the feces of a subject as a sample. When the bacteria of the present invention are detected in the feces of a subject, it can be determined that the subject has a predisposition to rheumatoid arthritis and has a relatively high risk of developing rheumatoid arthritis. Conversely, when the bacteria of the present invention are not detected, it can be determined that the subject has a relatively low risk of developing rheumatoid arthritis.

[0053] <4. Agent for determining the presence or absence of a predisposition to rheumatoid arthritis> The present invention provides an agent for determining the presence or absence of a predisposition to rheumatoid arthritis. The agent of the present invention determines the presence or absence of a predisposition to rheumatoid arthritis by detecting the presence of the bacteria of the present invention in the feces of a subject. Therefore, the agent of the present invention preferably includes the detection reagent described in <2. Detection reagent for bacteria>, and more preferably includes a primer set of oligonucleotides selected from the nucleic acid sequences represented by SEQ ID NOs: 4 to 19. By using the agent of the present invention, the presence or absence of a predisposition to rheumatoid arthritis can be easily determined by the above method.

Examples

[0054] Examples are shown below, but the present invention is not limited to the following examples.

[0055] 1.Isolation of Prevotella copri Isolation of Prevotella copri (hereinafter abbreviated as P. copri) from the feces of rheumatoid arthritis patients and healthy subjects was performed by the following method. 1 g of feces stored at -80°C was thawed and suspended in 10 mL of PBS (1.37 mM NaCl, 10 mM Na2HPO4, 2.7 mM KCl, 1.76 mM KH2PO4, pH 7.4) that had been anaerobically replaced in an anaerobic chamber. Serial 10-fold dilutions of this suspension were prepared with PBS (10 4 ~10 8 -fold dilution). 50 μL of each dilution was spread on Columbia 5% sheep blood agar medium (manufactured by Becton Dickinson Japan) and cultured anaerobically at 37°C for 2 days. The colonies that appeared were picked with an inoculation loop, and PCR was performed using Prevotella primers (g-Prevo-F; SEQ ID NO: 20 and g-Prevo-R; SEQ ID NO: 21) with the bacterial cells of each colony as a template.

[0056] PCR was performed with the following reaction composition. In the following reaction composition, "one loopful" refers to the amount of bacterial cells adhering to the inoculation loop when a colony is picked once with an inoculation loop. Bacterial cells of colony One loopful 10xPCR buffer 5.0 μL 2 mM dNTP 5.0 μL rTaq (manufactured by Toyobo Life Science) 0.2 μL Primer g-Prevo-F (100 pM) 0.2 μL Primer g-Prevo-R (100 pM) 0.2 μL Deionized water 39.4 μL

[0057] The PCR reaction was performed under the condition of 35 cycles of 95°C for 30 seconds, 55°C for 30 seconds, and 72°C for 90 seconds. The reaction solution was subjected to electrophoresis using a 2% agarose gel to confirm the amplification of the fragment. The colonies from which amplification products were obtained were plated again on Columbia 5% sheep blood agar medium and anaerobically cultured at 37°C for 2 days. From the colonies that appeared, PCR was performed using the universal primers for the 16S rRNA gene (8F; SEQ ID NO: 22 and 15R; SEQ ID NO: 23) in the same manner as described above. It was confirmed by electrophoresis that the PCR products were amplified. The PCR products were purified by a conventional method, and using the purified DNA as a template, PCR was performed with sequencing primers (SEQ ID NOs: 24 to 28) under the following reaction composition.

[0058] Purified DNA 4.5 μL 5x Sequencing buffer 2.0 μL BigDye v3.1 (manufactured by ThermoFisher) 1.0 μL Sequencing primer (10 μM) 1.0 μL Deionized water 1.5 μL

[0059] The PCR reaction was carried out under the condition of performing 25 cycles of 96°C for 10 seconds, 50°C for 5 seconds, and 60°C for 4 minutes. After ethanol precipitation of the reaction solution, the nucleotide sequence was determined using a sequencer. The obtained 16S rRNA sequence was analyzed by BLAST search, and the bacteria that matched the sequence of Prevotella copri were identified as Prevotella copri.

[0060] For the strains identified as Prevotella copri, they were anaerobically cultured at 37°C for 1 to 2 days using GAM liquid medium (manufactured by Nissui Pharmaceutical Co., Ltd.). The culture solution was mixed with an equal volume of sterilized 80% glycerol solution and stored at -80°C.

[0061] 2. Evaluation of arthritis-inducing activity by P. copri An experiment was conducted to culture P. copri derived from arthritis patients (hereinafter abbreviated as RA-P.copri) and P. copri derived from healthy subjects (hereinafter abbreviated as HC-P.copri) isolated in 1. under anaerobic conditions, administer them to arthritis model mice, and evaluate the susceptibility.

[0062] DBA / 1j male mice (Charles River Laboratories Japan) at 6 - 7 weeks of age were administered four types of antibiotics (ampicillin 500 mg / L, neomycin 500 mg / L, metronidazole 500 mg / L, vancomycin 250 mg / L) in drinking water for 5 days to kill intestinal bacteria. From 2 days after the end of antibiotic administration, cultured RA - P. copri and HC - P. copri were orally administered continuously for 5 days. The administered bacterial cells were cultured in GAM liquid medium in an anaerobic chamber for 24 hours and then suspended in PBS so that the OD600 value became 1.5.

[0063] Three days after the final administration day, feces were collected to extract DNA, and the amount of P. copri bacteria contained in the feces was quantified by qPCR using P. copri - specific primers to confirm that each P. copri had colonized the mice. Two days after the end of bacterial cell administration, an emulsion (final collagen concentration 1 mg / mL) was prepared by mixing bovine type II collagen (Collagen Technical Training Association K41 TypeII) and adjuvant [Freund’s complete adjuvant (BD; DF0638 - 60 - 7)] at a ratio of 1:1, and 100 μL of it was administered intradermally to DBA / 1j mice. Three weeks later, as a booster, 100 μL of the emulsion prepared in the same manner was readministered.

[0064] Thereafter, the arthritis score of the mice was evaluated over time every week. The evaluation method for mouse arthritis was scored as 0 points; normal, 1 point; swelling or erythema in one joint, 2 points; swelling or erythema in two or more joints, 3 points; swelling or erythema in all joints, 4 points; deformation / rigidity, with a maximum of 16 points for each joint of the forelimbs and hindlimbs. The results are shown in Figure 1.

[0065] As shown in Figure 1, 5 - 8 weeks after the second emulsion administration, mice administered RA - P. copri showed severe arthritis compared to mice administered HC - P. copri. From this result, it was shown that RA - P. copri may act as a worsening factor for rheumatoid arthritis.

[0066] 3. Genome analysis of isolated strains 1. For the isolated RA-P. copri and HC-P. copri, DNA was extracted using the DNeasy PowerSoil Kit (manufactured by QIAGEN). Whole-genome sequencing of each strain was performed using long-read sequencing by MinION (manufactured by Oxford Nanopore) and short-read sequencing by MiSeq (manufactured by Illumina).

[0067] Using the obtained long reads and short reads, a hybrid assembly method by Unicycler was performed to construct a full-length genome. For the genomic sequences of each constructed strain, a homology search was performed using blastn with each other's sequences as references to obtain regions where there are overlapping regions between genomes. By visualizing the regions obtained by blastn search using Circos, regions where deletions were only observed in the strain were discovered.

[0068] In addition, the estimation of phylogenetic relationships based on genetic information was performed by creating a core genome. First, after performing gene annotation using RAST from the genomic sequences, a homology search using blastp was performed on the amino acid sequences of each coding region to search for the same gene. At that time, it was limited to those with single copy, amino acid homology of 95% or more, and a length ratio (shorter / longer) of 0.8 or more, and genes commonly found among the strains were extracted. The common genes were aligned by mafft for each gene, and then the amino acid sequences were combined in the same order for each strain to create a core genome sequence. The distance between core genomes was calculated by FastTree and used for the estimation of phylogenetic relationships.

[0069] As a result, the existence of regions specific to the genome of RA-P. copri containing regions 1 to 3 was revealed. The results are shown in Fig. 2(A). The sequences of regions 1 to 3 specific to the genome of RA-P. copri are shown as SEQ ID NOs: 1 to 3, respectively.

[0070] 4. Detection by PCR After thoroughly suspending 1 g of feces from healthy subjects and patients with rheumatoid arthritis in 10 mL of PBS, DNA was extracted from 200 μL of the suspension by the method described by Maeda et al. (Arthritis Rheumatol, 2016; 68: 2646-2661).

[0071] PCR was performed with the following reaction composition. Fecal-extracted DNA (50 ng / μL) 1.0 μL 2xHSGoTaq (manufactured by Promega) 7.5 μL Primer Fw (10 μM) 1.0 μL Primer Rv (10 μM) 1.0 μL Nuclease-Free Water 4.5 μL

[0072] The PCR reaction was carried out under the condition of performing 30 cycles of 95°C for 30 seconds, 55°C for 30 seconds, and 72°C for 45 seconds. The combinations of primers A to H used in each PCR reaction, the amplification length, and the positions in the genome of RA-P.copri are shown in Table 1. Figure 2(B) is a diagram showing the positions of the primers used in the examples in the genome of RA-P.copri. After the reaction, each reaction solution was subjected to electrophoresis using a 2% agarose gel to confirm the amplification of the fragment.

[0073]

Table 1

[0074] As shown in Table 1, amplification of the DNA fragment of the target size was confirmed only when DNA extracted from feces derived from patients with rheumatoid arthritis was used. From the above, it was found that a region specific to the genome of RA-P.copri was amplified by PCR.

[0075] Although the present invention has been described in detail using specific embodiments, it will be apparent to those skilled in the art that various changes and modifications can be made without departing from the spirit and scope of the present invention. This application is based on a Japanese Patent Application (Japanese Patent Application No. 2019-209926) filed on November 20, 2019, the entire contents of which are incorporated herein by reference.

Claims

1. An agent for determining the presence or absence of a predisposition to rheumatoid arthritis, comprising a primer or a probe that specifically detects any one of the following nucleic acid sequences (1) to (3). (1) Nucleic acid sequences represented by SEQ ID NOs: 1 to 3 (2) Nucleic acid sequences having 95% or more homology with the nucleic acid sequences represented by SEQ ID NOs: 1 to 3 (3) Nucleic acid sequences that hybridize under stringent conditions with the nucleic acid sequences described in SEQ ID NOs: 1 to 3 or nucleic acid sequences complementary to the nucleic acid sequences

2. The agent according to claim 1, wherein the primer or the probe is an oligonucleotide containing a nucleic acid sequence consisting of at least 15 consecutive bases among the nucleic acid sequences shown in any one of SEQ ID NOs: 1 to 3 or a nucleic acid sequence complementary to the sequence.

3. The agent according to claim 2, wherein the primer or the probe is one oligonucleotide selected from the nucleic acid sequences represented by SEQ ID NOs: 4 to 19.

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