JP7701052B2 - Method for detecting bacteria involved in the onset of rheumatoid arthritis, reagent for detecting said bacteria, method for determining the presence or absence of a predisposition to rheumatoid arthritis, and agent for determining the presence or absence of said predisposition - Google Patents

Description

The present invention relates to a method for detecting bacteria involved in the onset of rheumatoid arthritis, a reagent for detecting said 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 said predisposition.

Rheumatoid arthritis (RA) is a chronic, systemic autoimmune disease that affects approximately 1% of the population and primarily affects the joints. RA causes damage mediated by cytokines, chemokines, and metalloproteases.

The onset of rheumatoid arthritis is usually insidious, and often begins with systemic and joint symptoms. The pathology of rheumatoid arthritis is characterized by symmetric inflammation of peripheral joints (e.g., wrist joints, metacarpophalangeal joints), resulting in progressive destruction of joint structures, usually accompanied by systemic symptoms. Rheumatoid arthritis occurs two to three times more frequently in women than in men, and can develop at any age, most commonly between 35 and 50 years of age, but can also develop in childhood or old age (Non-Patent Document 1).

When symptoms of rheumatoid arthritis worsen, the patient experiences greater physical and mental pain and the cost of medication used in treatment rises, so there is a need for a system to provide appropriate treatment at the appropriate time.

Currently, rheumatoid arthritis is diagnosed based on a comprehensive assessment of factors such as the number of swollen and painful joints and the presence or absence of rheumatoid factor and anti-CCP antibodies in blood tests, but there is a need for the development of biomarkers that can determine the possibility of developing rheumatoid arthritis at an earlier stage.

On the other hand, non-patent literature 2 discloses that the presence rate of Prevotella copri in the intestines increases in the early stages of rheumatoid arthritis.

Apostolos Kontzias, MSD Manual Professional Edition, 06. Musculoskeletal Disorders and Connective Tissue, February 2017 (https://www.msdmanuals.com/ja-jp) Manimozhiyan Arumugam et al., NATURE,VOL 473, 2011, p. 174-180

The rheumatoid factor and anti-CCP antibodies that can be measured by the above-mentioned blood tests used to diagnose rheumatoid arthritis have specificity issues, such as not being detected in patients with rheumatoid arthritis, while being detected in healthy individuals. In addition, Non-Patent Document 2 discloses that the presence ratio of Prevotella copri in the intestine increases in the early stages of rheumatoid arthritis, but the relationship with the onset of rheumatoid arthritis is unclear.

The present invention aims to identify bacteria associated with the onset of rheumatoid arthritis and to provide a means for using said bacteria to more easily and earlier determine the possibility of contracting rheumatoid arthritis.

The present inventors isolated Prevotella copri from stool samples of rheumatoid arthritis patients and healthy individuals, and found that Prevotella copri derived from rheumatoid arthritis patients (hereinafter abbreviated as RA-P. copri) has arthritis-inducing activity and causes arthritis to become more severe than Prevotella copri derived from healthy individuals (hereinafter abbreviated as HC-P. copri). Furthermore, analysis of the genome sequences of RA-P. copri and HC-P. copri revealed the presence of a specific region in the genome of RA-P. copri. Based on the above findings, the present invention was completed.

The present invention is as follows.
1. A method for detecting a bacterium involved in the onset of rheumatoid arthritis, comprising the step of specifically detecting any one of the following nucleic acid sequences (1) to (3) or a partial sequence thereof:
(1) A nucleic acid sequence represented by SEQ ID NO: 1 to 3 (2) A nucleic acid sequence having 95% or more homology with a nucleic acid sequence represented by SEQ ID NO: 1 to 3 (3) A nucleic acid sequence that hybridizes under stringent conditions with a nucleic acid sequence described in SEQ ID NO: 1 to 3 or a nucleic acid sequence complementary to said nucleic acid sequence 2. The detection method according to 1 above, wherein the bacterium involved in the onset of rheumatoid arthritis belongs to the genus Prevotella.
3. The detection method according to 2 above, wherein the bacterium belonging to the genus Prevotella is Prevotella copri.
4. The detection method according to any one of 1 to 3 above, wherein the step of specifically detecting the nucleic acid sequence of any one of (1) to (3) above or a partial sequence thereof is carried out by polymerase chain reaction using DNA derived from feces of the subject as a template.
5. The detection method according to 4 above, wherein the at least two primers used in the polymerase chain reaction are oligonucleotides containing a nucleic acid sequence consisting of at least 15 consecutive bases selected from the nucleic acid sequences shown in any one of SEQ ID NOs: 1 to 3 or a nucleic acid sequence complementary to said sequence.
6. The detection method according to 5 above, wherein the primer is an oligonucleotide selected from the nucleic acid sequences represented by SEQ ID NOs: 4 to 19.
7. The detection method according to any one of 1 to 3 above, wherein the step of specifically detecting the nucleic acid sequence or a partial sequence thereof is carried out by metagenomic analysis using a next-generation sequencer targeting DNA derived from feces of the subject.
8. A method for determining the presence or absence of a predisposition to rheumatoid arthritis, comprising a step of detecting the bacterium involved in the onset of rheumatoid arthritis in feces of a subject by the detection method according to any one of 1 to 7 above.
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) A nucleic acid sequence represented by SEQ ID NO: 1 to 3 (2) A nucleic acid sequence having 95% or more homology with a nucleic acid sequence represented by SEQ ID NO: 1 to 3 (3) A nucleic acid sequence that hybridizes under stringent conditions with a nucleic acid sequence described in SEQ ID NO: 1 to 3 or a nucleic acid sequence complementary to said nucleic acid sequence 10. The detection reagent described in 9 above, wherein the primer or the probe is an oligonucleotide containing a nucleic acid sequence consisting of at least 15 consecutive bases from the nucleic acid sequence represented by any one of SEQ ID NO: 1 to 3 or a nucleic acid sequence complementary to said sequence.
11. The detection reagent according to 10 above, wherein the primer or the probe is an 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) A nucleic acid sequence represented by SEQ ID NO: 1 to 3 (2) A nucleic acid sequence having 95% or more homology with a nucleic acid sequence represented by SEQ ID NO: 1 to 3 (3) A nucleic acid sequence that hybridizes under stringent conditions with a nucleic acid sequence described in SEQ ID NO: 1 to 3 or a nucleic acid sequence complementary to said nucleic acid sequence 13. The agent described in 12 above, wherein the primer or the probe is an oligonucleotide containing a nucleic acid sequence consisting of at least 15 consecutive bases from the nucleic acid sequence represented by any one of SEQ ID NO: 1 to 3 or a nucleic acid sequence complementary to said sequence.
14. The agent according to 13 above, wherein the primer or the probe is an oligonucleotide selected from the nucleic acid sequences represented by SEQ ID NOs: 4 to 19.

The bacterial detection method and the bacterial detection reagent of the present invention enable specific and simple detection of bacteria involved in the onset of rheumatoid arthritis before the onset of rheumatoid arthritis. Furthermore, 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 make it possible to determine the possibility of contracting rheumatoid arthritis before the onset of rheumatoid arthritis and to administer appropriate treatment at the appropriate time, which can also be a new strategy from the perspective of preventive medicine.

1 shows the results of evaluating the arthritis-inducing activity of P. copri. Figure 2 (A) shows the results of analyzing the genome structures of isolated RA-P. copri and HC-P. copri, and Figure 2 (B) shows the positions of the primers used in the examples in the genome of RA-P. copri. FIG. 3 shows the results of PCR carried out using DNA extracted from the feces of a rheumatoid arthritis patient as a template. FIG. 4 shows the results of PCR carried out using DNA extracted from feces of healthy individuals as a template.

The terms used herein below have the meanings commonly used in the relevant field unless otherwise specified.

1. Bacteria detection method
The bacteria in the present invention are intestinal bacteria having arthritis-inducing activity. The arthritis-inducing activity can be determined by an increase in arthritis score. The method for determining the increase in arthritis score is not particularly limited, but specific examples thereof include the following methods.

In one embodiment, the bacterium of the present invention has the following nucleic acid sequence (1) or (2) or a partial sequence thereof:
(1) A nucleic acid sequence represented by SEQ ID NO: 1 to 3. (2) A nucleic acid sequence having 95% or more homology to the nucleic acid sequence represented by SEQ ID NO: 1 to 3.

Here, SEQ ID NOs: 1 to 3 show the nucleic acid sequences of regions that are specifically present in the genome of RA-P. copri and have arthritis-inducing activity. The homology between nucleic acid sequences is calculated using the homology search program BLAST. When a bacterium has a nucleic acid sequence that has 95% or more homology with the nucleic acid sequence represented by SEQ ID NOs: 1 to 3, it can be said that it is highly likely to have arthritis-inducing activity, similar to RA-P. copri having the nucleic acid sequence represented by SEQ ID NOs: 1 to 3.

Methods for assessing the activity of inducing arthritis based on an increase in arthritis score include the method described in Takekazu Kakimoto et al., New Biochemical Experiment Lecture Series 12, Molecular Immunology II, pp. 360-372, Tokyo Kagaku Dojin (1989). Specifically, the following method can be used. First, antibiotics (e.g., ampicillin, neomycin, metronidazole, and vancomycin) are administered to arthritis model mice (e.g., DBA/1j mice) for several days to kill the intestinal bacteria. After that, the mice are divided into a group administered with the test bacteria or a group administered with bacteria derived from healthy subjects (control group), and each bacterium is orally administered for several consecutive days (e.g., 5 days). Three days after the final administration, feces are collected, and it is confirmed that each bacterium has been established in the mice, and an emulsion of type II collagen and an adjuvant is intradermally administered to the mice in each group. Three weeks later, the emulsion containing type II collagen and an adjuvant is administered again as a booster, and the arthritis score is evaluated over time. If the arthritis score is statistically significantly higher than that of the control group 5 to 8 weeks after the second administration of the emulsion, the test bacterium can be determined to have the activity of inducing rheumatoid arthritis.

The bacterium in the present invention is Prevotella copri, a gram-negative anaerobic bacterium. Whether or not the bacterium isolated from the isolation source is Prevotella copri can be determined, for example, by comparing the base sequence data of the 16S ribosomal RNA gene with the sequence data of known species and performing phylogenetic analysis.

In this specification, "detection of bacteria" includes not only determining the presence or absence of bacterial bodies, but also quantifying the amount of bacteria present.

For bacterial detection, total DNA is recovered from a sample. Examples of the sample include, but are not limited to, feces or intestinal contents of subjects (healthy individuals, rheumatoid arthritis patients, or individuals suspected of having rheumatoid arthritis), isolated/cultured bacteria, etc. Methods for isolating/purifying DNA from samples 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.

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), and subjected to the detection method of the present invention.

The method for detecting bacteria of the present invention is characterized by comprising a step of specifically detecting any one of the following nucleic acid sequences (1) to (3) or a partial sequence thereof:
(1) A nucleic acid sequence represented by SEQ ID NO: 1 to 3. (2) A nucleic acid sequence having 95% or more homology with a nucleic acid sequence represented by SEQ ID NO: 1 to 3. (3) A nucleic acid sequence that hybridizes under stringent conditions with a nucleic acid sequence represented by SEQ ID NO: 1 to 3 or a nucleic acid sequence complementary to said nucleic acid sequence.

Examples of nucleic acid sequences that hybridize under stringent conditions include nucleic acid sequences that have a certain level of sequence identity with the nucleic acid sequence used as a probe. For example, a nucleic acid sequence that has at least 60% homology with the target nucleic acid sequence, preferably a nucleic acid sequence that has 80% homology or more, more preferably a nucleic acid sequence that has 90% homology or more, and most preferably a gene that has 95% homology or more. In addition, for example, if 100 nucleic acids in a nucleic acid sequence are considered as one unit, the nucleic acid sequence of the target gene may have one 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, even more preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, and even more preferably 1, 2, 3, 4, or 5 nucleic acids deleted, substituted, added, etc.

"Nucleic acid deletion" means that a nucleic acid is missing or lost in a sequence, "nucleic acid substitution" means that a nucleic acid in a sequence is replaced with another nucleic acid, and "nucleic acid addition" means that a new nucleic acid is added, such as by insertion.

In one aspect, the detection method of the present invention preferably uses a primer or probe capable of specifically detecting any one of the nucleic acid sequences (1) to (3) above or a partial sequence thereof.

The primers may be any primers designed to specifically amplify a partial region of the nucleic acid sequence by PCR. Any one of the nucleic acid sequences (1) to (3) above or a partial sequence thereof is a nucleic acid sequence present in the genome of RA-P. copri that has arthritis-inducing activity in the present invention.

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

The primers include, for example, a combination of oligonucleotides containing a consecutive nucleic acid sequence of preferably 15 to 50 bases, more preferably 18 to 30 bases, selected from the nucleic acid sequence represented by any one of SEQ ID NOs: 1 to 3 or a nucleic acid sequence complementary to said sequence, and the length of the nucleic acid fragment amplified by them is preferably 50 to 1,000 bases, more preferably 100 to 500 bases, and a pair of oligonucleotides can be mentioned.

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

The temperature setting, reaction time, and number of cycles in PCR can be set appropriately depending on the amount of template DNA used, the type of primer, etc. The annealing temperature in PCR can be set appropriately based on the GC content of the primers. For example, a reaction can be performed using bacterial genomic DNA as a template, 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, and a cycle of 30 cycles of 95°C for 30 seconds, 55°C for 30 seconds, and 72°C for 45 seconds.

The probe is an oligonucleotide that hybridizes under stringent conditions to a consecutive nucleic acid sequence of preferably 15 or more bases, more preferably 18 to 500 bases, even more preferably 18 to 200 bases, and particularly preferably 18 to 50 bases, contained in the nucleic acid sequence represented by SEQ ID NO: 1 to 3 or a nucleic acid sequence complementary to the nucleic acid sequence.

Hybridization can be performed according to a method known per se or a method similar thereto, for example, the method described in Molecular Cloning, 2nd Edition (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989). Stringent conditions include, for example, a hybridization reaction in 6xSSC (sodium chloride/sodium citrate) at 45°C, followed by one or more washes in 0.2xSSC/0.1% SDS at 65°C. Those skilled in the art can easily adjust the stringency to the desired level 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 hybridization reaction time, the salt concentration of the washing solution, the washing temperature, etc.

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

From the viewpoint of specificity, the probe is preferably an oligonucleotide that hybridizes to a nucleic acid sequence represented by any one of SEQ ID NOs: 4 to 19 or a complementary sequence thereof. More preferably, the probe is an oligonucleotide that includes a partial sequence of 15 to 50 consecutive bases of a nucleic acid sequence represented by any one of SEQ ID NOs: 4 to 19 or a complementary sequence thereof.

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

The primer or probe may contain additional sequences (nucleic acid sequences that are not complementary to the polynucleotide to be detected) as long as this does not interfere with specific detection.

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 may be appropriately read as a uridine residue (U). It may also be DNA containing a uridine residue synthesized by changing T to U at any position. Similarly, it may be RNA containing a thymidine residue synthesized by changing U to T at any position. In addition, point mutations such as deletions, insertions, or substitutions, or modified nucleotides may be present in the oligonucleotide as long as they do not reduce the specificity of hybridization.

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

The oligonucleotides used as primers or probes can be chemically synthesized, for example, using a conventional DNA synthesizer. Oligonucleotides may also be synthesized using any of the other methods well known in the art.

The method includes performing PCR using the primers contained in the detection reagent of the present invention and DNA recovered from the sample as a template. The obtained PCR products are separated by electrophoresis (e.g., agarose gel electrophoresis, polyacrylamide gel electrophoresis, etc.). After electrophoresis, the gel is stained with a known staining solution such as an ethidium bromide solution, and the PCR products are detected using a transilluminator or the like. Then, the presence or absence and amount of the bacteria of the present invention in the sample are determined using the presence or absence and amount of the specific PCR product as an indicator.

The PCR used in the detection method of the present invention may be quantitative PCR. Quantitative PCR can be performed by known methods, but two analytical methods are known. The first is a method that utilizes the characteristic that the reaction product in a PCR reaction increases exponentially up to a certain amount and then reaches a plateau, analyzes the amount of the reaction product during the exponential increase phase, and calculates the initial template amount. The second is a method that determines the PCR cycle number (Ct) at which the amount of the reaction product exceeds a certain value (threshold) by monitoring the reaction product in real time. In both analytical methods, it is necessary to change the amount of DNA of a known concentration, perform PCR, analyze the reaction product at each cycle number, and determine a quantitative PCR cycle number range from the kinetics. Based on the results, the amount of the target gene present in the unknown sample is estimated. As a result, the amount of the bacterium of the present invention present in the sample can be quantified, and if it is estimated that at least one copy of the target gene is contained in the test sample, it can be determined that the bacterium of the present invention is present.

In one embodiment, the detection method of the present invention includes a step of contacting the probe contained in the detection reagent of the present invention with the total DNA in a sample. The contact conditions are appropriately set so that the probe hybridizes with a nucleic acid sequence that is specifically present in the genome of RA-P. copri to form a nucleic acid complex. The complex is then detected as an indication of the presence of the bacterium of the present invention.

When a probe is used, it can be performed by various known hybridization techniques (e.g., Fluorescence In Situ Hybridization (hereinafter abbreviated as FISH)). In the FISH method, the probe penetrates into the cytoplasm of the bacteria and hybridizes under appropriate hybridization conditions to the nucleic acid sequence present in the genome of RA-P. copri present therein. In this case, the probe is labeled with a radioisotope, a fluorescent substance (e.g., fluorescein isothiocyanate (FITC), TAMRA, Cy3, Cy5, etc.), a chemiluminescent substance, etc., so that the phenomenon of specific hybridization can be monitored by an appropriate method (e.g., autoradiography, fluorescence microscopy, flow cytometry, etc.). For example, when the probe is labeled with a radioisotope, the assay can be performed by a method such as autoradiography, when it is labeled with a fluorescent substance, the assay can be performed with a fluorescent microscope, and when it is labeled with a chemiluminescent substance, analysis can be performed using a photosensitive film or digital analysis can be performed using a CCD camera, thereby allowing detection of bacteria in a sample.

In one embodiment, the detection method of the present invention includes metagenomic analysis using a next-generation sequencer. A next-generation sequencer is a base sequence analysis (decoding) device that dramatically improves the analysis speed by parallel processing, and is in contrast to a fluorescent capillary sequencer (called a "first-generation sequencer") that uses the Sanger sequencing method. By using metagenomic analysis using a next-generation sequencer, it is possible to analyze all bacterial species that make up the intestinal flora and their abundance ratios. There are no particular limitations on the method, so long as it reveals the diversity of the intestinal flora and the abundance ratios of specific bacteria that make up the intestinal flora.

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). Not limited to existing super-parallel next-generation sequencers, next-generation sequencers that will be developed and released in the future may also be used.

2. Reagents for detecting bacteria
The detection reagent of the present invention detects the presence of the bacterium of the present invention in a sample by detecting a nucleic acid sequence present in the genome of RA-P. copri of the present invention. The detection reagent of the present invention contains the primer or probe described above in <1. Bacteria detection method>, which can specifically detect any one of the following nucleic acid sequences (1) to (3).
(1) A nucleic acid sequence represented by SEQ ID NO: 1 to 3. (2) A nucleic acid sequence having 95% or more homology with a nucleic acid sequence represented by SEQ ID NO: 1 to 3. (3) A nucleic acid sequence that hybridizes under stringent conditions with a nucleic acid sequence represented by SEQ ID NO: 1 to 3 or a nucleic acid sequence complementary to said nucleic acid sequence.

The detection reagent of the present invention may further contain other components such as a nucleic acid synthesis enzyme (e.g., DNA polymerase, RNA polymerase, reverse transcriptase, etc.), other enzymes, and substrates corresponding to the enzymes (e.g., dNTPs, rNTPs, etc.). It may also contain a labeled detection substance and a buffer solution.

By using the detection reagent of the present invention, the presence or absence of rheumatoid arthritis-inducing bacteria in a sample can be easily determined in a short period of time, making it useful for diagnosing a predisposition to rheumatoid arthritis.

<3. Method for determining the presence or absence of a predisposition to rheumatoid arthritis>
As shown in the Examples below, the bacteria of the present invention having the nucleic acid sequences shown in SEQ ID NOs: 1 to 3 were detected in the feces of patients suffering from 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 to developing rheumatoid arthritis (a predisposing factor for rheumatoid arthritis), regardless of whether the subject has already developed rheumatoid arthritis.

Therefore, the presence or absence of a predisposition to rheumatoid arthritis can be determined by detecting bacteria involved in the onset of rheumatoid arthritis in the feces of a subject using the detection method described in <1. Bacterial detection method>. 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.

The bacteria of the present invention can be detected by the detection method described in <1. Bacterial detection method> using a subject's feces as a sample. When the bacteria of the present invention are detected in the subject's feces, it can be determined that the subject has a predisposition to rheumatoid arthritis and is at 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 is at a relatively low risk of developing rheumatoid arthritis.

<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 contains a detection reagent described in <2. Reagent for detecting bacteria>, and more preferably contains 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-mentioned method.

Examples are provided below, but the present invention is not limited to the following examples.

1. Isolation of Prevotella copri Isolation of Prevotella copri (hereinafter abbreviated as P. copri) from feces of rheumatoid arthritis patients and healthy subjects was performed as follows. 1 g of feces stored at -80°C was thawed and suspended in 10 mL of PBS (1.37 mM NaCl, 10 mM Na ₂ HPO ₄ , 2.7 mM KCl, 1.76 mM KH ₂ PO ₄ , pH 7.4) that had been anaerobically replaced in an anaerobic chamber. This suspension was serially diluted 10-fold with PBS (10 ⁴ to 10 ⁸ dilutions). 50 μL of each dilution was applied to Columbia 5% sheep blood agar medium (manufactured by Nippon Becton Dickinson) and anaerobically cultured at 37°C for 2 days. The emerged colonies were picked with a toothpick, and PCR was carried out using the bacterial cells of each colony as a template and Prevotella primers (g-Prevo-F; SEQ ID NO: 20 and g-Prevo-R; SEQ ID NO: 21).

PCR was performed with the following reaction composition: In the reaction composition below, "one pinch of toothpick" refers to the amount of bacterial cells that adhered to a toothpick when a colony was picked once with the toothpick.
A pinch of colony bacteria 10x PCR buffer 5.0 μL
2mM dNTP 5.0μL
rTaq (Toyobo Life Sciences) 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

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

Purified DNA 4.5μL
5x Sequencing Buffer 2.0 μL
BigDye v3.1
(ThermoFisher) 1.0 μL
Sequencing primer (10 μM) 1.0 μL
Deionized water 1.5 μL

The PCR reaction was carried out under the conditions of 96°C for 10 seconds, 50°C for 5 seconds, and 60°C for 4 minutes, with 25 cycles. The reaction solution was precipitated with ethanol, and the base sequence was determined using a sequencer. The obtained 16S rRNA sequence was analyzed by BLAST search, and the bacterium that matched the sequence of Prevotella copri was identified as Prevotella copri.

The strain identified as Prevotella copri was anaerobically cultured in GAM liquid medium (Nissui Pharmaceutical) at 37°C for 1-2 days. The culture was mixed with an equal volume of sterilized 80% glycerol solution and stored at -80°C.

2. Evaluation of arthritis-inducing activity of P. copri P. copri from arthritis patients (hereinafter abbreviated as RA-P. copri) and P. copri from healthy subjects (hereinafter abbreviated as HC-P. copri) isolated in 1 were cultured under anaerobic conditions and administered to arthritis model mice to evaluate sensitivity.

6-7 week old DBA/1j male mice (Charles River Japan) were given four types of antibiotics (ampicillin 500mg/L, neomycin 500mg/L, metronidazole 500mg/L, vancomycin 250mg/L) in drinking water for 5 days to kill intestinal bacteria. Two days after the antibiotic administration was completed, cultured RA-P. copri and HC-P. copri were orally administered for 5 consecutive days. The administered bacteria were cultured in GAM liquid medium in an anaerobic chamber for 24 hours and then suspended in PBS to an OD600 value of 1.5.

Three days after the final administration, feces were collected and DNA was extracted, and the amount of P. copri bacteria contained in the feces was quantified by qPCR using P. copri specific primers, confirming that each P. copri had colonized the mice. Two days after the end of the bacterial administration, an emulsion (final collagen concentration 1 mg/mL) was prepared by mixing bovine type II collagen (Collagen Technology Workshop K41 Type II) and adjuvant [Freund's complete adjuvant (BD Company; DF0638-60-7)] in a 1:1 ratio, and 100 μL of the emulsion was administered intradermally to DBA/1j mice. Three weeks later, 100 μL of the emulsion prepared in the same manner was administered again as a booster.

Thereafter, the arthritis scores of the mice were evaluated over time every other week. The arthritis of the mice was evaluated by scoring each joint of the front and back legs as follows: 0 points: normal, 1 point: swelling or redness in one joint, 2 points: swelling or redness in two or more joints, 3 points: swelling or redness in all joints, 4 points: deformation or rigidity, up to a maximum of 16 points. The results are shown in Figure 1.

As shown in Figure 1, 5-8 weeks after the second emulsion administration, mice administered with RA-P. copri showed more severe arthritis than mice administered with HC-P. copri. These results suggest that RA-P. copri may act as an aggravating factor for rheumatoid arthritis.

3. Genomic analysis of isolated strains DNA was extracted from the RA-P. copri and HC-P. copri isolated in 1. using DNeasy PowerSoil Kit (QIAGEN). The whole genome sequence of each strain was performed by long-read sequencing using MinION (Oxford Nanopore) and short-read sequencing using MiSeq (Illumina).

A hybrid assembly method was performed using Unicycler using the obtained long reads and short reads to construct a full-length genome. A homology search was performed using blastn for the constructed genome sequences of each strain, using each sequence as a reference, to obtain regions that were overlapping or not overlapping between the genomes. A region that was deleted only in that strain was discovered by visualizing the regions obtained by the blastn search using Circos.

In addition, the estimation of phylogenetic relationships based on genetic information was performed by creating a core genome. First, gene annotation was performed from the genome sequence using RAST, and then a homology search was performed using blastp on the amino acid sequence of each coding region to search for identical genes. In this case, genes that were single copies, had an amino acid homology of 95% or more, and a length ratio (shorter side/longer side) of 0.8 or more were limited, and genes commonly found between strains were extracted. Each common gene was aligned using mafft, and then the amino acid sequences were combined in the same order for each strain to create a core genome sequence. The distance between the core genomes was calculated using FastTree and used to estimate phylogenetic relationships.

As a result, the presence of regions specific to the RA-P. copri genome, including regions 1 to 3, was revealed. The results are shown in Figure 2 (A). The sequences of regions 1 to 3 specific to the RA-P. copri genome are shown in SEQ ID NOs: 1 to 3, respectively.

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

PCR was carried out using the following reaction composition.
Fecal extract DNA (50ng/μL) 1.0μL
2xHSGoTaq (Promega) 7.5 μL
Primer Fw (10 μM) 1.0 μL
Primer Rv (10 μM) 1.0 μL
Nuclease-Free Water 4.5μL

The PCR reaction was carried out under the conditions of 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 location in the genome of RA-P. copri are shown in Table 1. Figure 2 (B) shows the location in the genome of RA-P. copri of the primers used in the examples. After the reaction, each reaction solution was subjected to electrophoresis using a 2% agarose gel to confirm the amplification of the fragment.

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

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

Claims (3)

An agent for determining the presence or absence of a predisposition to rheumatoid arthritis, comprising a primer or probe that specifically detects any one of the following nucleic acid sequences (1) to (3):
(1) A nucleic acid sequence represented by SEQ ID NO: 1 to 3. (2) A nucleic acid sequence having 95% or more homology with a nucleic acid sequence represented by SEQ ID NO: 1 to 3. (3) A nucleic acid sequence that hybridizes under stringent conditions with a nucleic acid sequence represented by SEQ ID NO: 1 to 3 or a nucleic acid sequence complementary to said nucleic acid sequence. 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 selected from the nucleic acid sequence shown in any one of SEQ ID NOs: 1 to 3 or a nucleic acid sequence complementary to said sequence. The agent according to claim 2 , wherein the primer or the probe is an oligonucleotide selected from the nucleic acid sequences represented by SEQ ID NOs: 4 to 19.

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