Detection method for Streptococcus mobilis

Abstract

To provide a fast detection method for Streptococcus mobilis bacteria.SOLUTION: The present invention provides a method for detecting Streptococcus mobilis bacteria using a protein consisting of a specific amino acid sequence as an index. For example, oligonucleotides consisting of specific base sequences are used as a primer pair, subjecting DNA extracted from a sample under test to PCR.SELECTED DRAWING: Figure 9

Description

[Technical Field]

[0001] The present invention relates to a method for detecting Streptococcus mobilis (formerly known as Okadaella gastrococcus). [Background technology]

[0002] Patent Document 1 discloses the bacterium Okadaella gastrococcus (newly named Streptococcus mobilis), which is believed to cause gastrointestinal diseases. This bacterium is resistant to triple antibiotic therapy, which uses tetracycline, metronidazole, and an H2 antagonist. Triple antibiotic therapy is a method used to eliminate Helicobacter pylori, which is believed to cause gastritis and gastric ulcers. However, in recent years, it has been discovered that some patients who have been treated with triple antibiotic therapy have developed gastritis and gastric ulcers, and the presence of Streptococcus mobilis has been speculated to be one of the causes of these diseases. In fact, it has been reported that Streptococcus mobilis (hereinafter sometimes referred to as S. mobilis) is present in the stomachs of patients with gastritis and gastric ulcers (Patent Document 1). Furthermore, there have been cases of gastric cancer occurring in patients chronically infected with S. mobilis, suggesting a possible link between the two. (Non-Patent Document 1). A molecular phylogenetic analysis of Okadaella gastrococcus (hereinafter sometimes referred to as Og bacteria) has revealed that it is included in the genus Mitis of Streptococcus, and a new name, Streptococcus mobilis, has been proposed. In this paper, Og bacteria and S. mobilis are the same bacterium, and the names can be used interchangeably.

[0003] However, although the gene sequence of S. mobilis described in Patent Document 1 is publicly known, no method for specifically detecting this S. mobilis has been known to date. It is also known that the base sequences of genes possessed by microorganisms can result in so-called SNPs (Single Nucleotide Polymorphisms), in which some bases are replaced with other bases. However, it is unknown whether these S. mobilis strains also have SNPs, and it has been necessary to detect S. mobilis strains that have SNPs. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Special Publication No. 2001-518288 [Non-patent literature]

[0005] [Non-Patent Document 1] Takayuki Okada et.al., Am. J. Gastroenterol., Abstract S61, Vol 100, 2005 Summary of the Invention [Problem to be solved by the invention]

[0006] An object of the present invention is to provide a method for rapid detection of, for example, Streptococcus mobilis bacteria. [Means for solving the problem]

[0007] The detection method according to the present invention is characterized in that it uses, as an indicator, a protein having a specific amino acid sequence contained in Streptococcus mobilis or a base sequence encoding the amino acid sequence. [Effects of the Invention]

[0008] The method according to the present invention allows rapid detection of Streptococcus mobilis bacteria. [Brief explanation of the drawings]

[0009] [Figure 1] Figure 1 is a sequence map showing the region where Og bacteria may be specifically detected. [Figure 2] Figure 2 is a phylogenetic tree based on the 16S-23S region of ribosomal RNA. [Figure 3] 3 shows the nucleotide sequences of the orf25 gene of three species of Og bacteria. A dot in the nucleotide sequence indicates that the base at that position is the same as that of the Og1 strain. [Figure 4] FIG. 4 shows the primer set used to amplify the orf25 gene of Og bacteria. [Figure 5] FIG. 5 shows the primer set used to amplify the orf25 homologue of the S. parasanguinis FW213 strain. [Figure 6] FIG. 6 is a diagram comparing the amplified region using the primer set shown in FIG. 4 with the amplified region using the primer set shown in FIG. [Figure 7] FIG. 7 is an image showing the specificity of the amplified products obtained by dot blot analysis using the primer sets shown in FIGS. [Figure 8] FIG. 8 is an image showing the detection results of three types of Og bacteria and S. parasanguinis F213 by dot blotting of the amplified products using the primer sets shown in FIGS. [Figure 9] Figure 9 is an image showing the results of colony hybridization using DNA probes (for Og-orf25, Og-orf25_5', and Og-orf25_3') capable of detecting the entire or partial region of the orf25 gene, and the Og1 strain and its related bacteria. [Figure 10]Figure 10 shows the results of colony hybridization using DNA probes (for Og-orf25, Og-orf25_5', and Og-orf25_3') capable of detecting the entire or partial region of the orf25 gene, and 20 species of Streptococcus and Streptococcus isolated from Russian patients with gastroesophageal junction inflammation, gastritis, or gastric ulcer. [Figure 11] FIG. 11 is an electrophoretic image showing the results of detection of amplification products by PCR specific to the orf25 gene. [Figure 12] Figure 12 shows an alignment of the amino acid sequences of ORF25 of three Og bacteria (Og1, Og2, and Og3). A dot in the amino acid sequence indicates an amino acid that is identical to the amino acid in the Og1 strain at that position. The amino acid sequence of Og1 is shown as SEQ ID NO: 1, the amino acid sequence of Og2 is shown as SEQ ID NO: 2, and the amino acid sequence of Og3 is shown as SEQ ID NO: 3. The amino acid sequence shown as SEQ ID NO: 1 corresponds to the nucleotide sequence shown as SEQ ID NO: 4 in Figure 3, the amino acid sequence shown as SEQ ID NO: 2 corresponds to the nucleotide sequence shown as SEQ ID NO: 5 in Figure 3, and the amino acid sequence shown as SEQ ID NO: 3 corresponds to the nucleotide sequence shown as SEQ ID NO: 6 in Figure 3. [Figure 13] Figure 13 shows the ORF of the S. parasanguinis FW213 strain, which has a base sequence similar to part of the base sequence of the ORF25 gene of three Og bacteria, and the base sequences of other ORFs of three Og bacteria (Og1-ORF25-homologue, Og2-ORF25-homologue, Og3-ORF25-homologue) that have a base sequence similar to part of the base sequence (FW213-ORF25-homolog). [Figure 14]Figure 14 shows the regions amplified by primers used to amplify the entire and partial regions of the orf of the S. parasanguinis FW213 strain, which contains a base sequence similar to part of the base sequence of the orf25 gene of three Og bacteria, and the base sequences of other orfs of three Og bacteria (Og1-ORF25-homologue, Og2-ORF25-homologue, Og3-ORF25-homologue) which contain a base sequence similar to part of the base sequence (FW213-ORF25-homolog). DETAILED DESCRIPTION OF THE INVENTION

[0010] The method of the present invention uses the ORF25 protein (hereinafter referred to as "ORF25 protein") contained in Og bacteria as an indicator. It also uses the nucleotide sequence encoding the ORF25 protein (including a stop codon; hereinafter referred to as "orf25 gene") as an indicator. The orf25 gene is one of the ORFs (Open Reading Frames) that can be used to specifically detect Og bacteria, among those discovered by analyzing the entire nucleotide sequence contained in Og bacteria.

[0011] The ORF25 protein consists of the amino acid sequence shown in any one of SEQ ID NOS: 1 to 3. Furthermore, proteins having the amino acid sequence shown in SEQ ID NOS: 1 to 3 in which one or more amino acid residues have been deleted, substituted, or added can also be used as an indicator for detecting Og bacteria according to the present invention. In one embodiment of the present invention, the term "one or more" refers to one to nine, preferably one to five, amino acid residues. It is known that polypeptides that have one or more amino acid residues deleted, added, or inserted, or substituted with other amino acids, retain their biological activity (Mark et al., Proc Natl Acad Sci U S A. 1984 Sep;81(18):5662-5666; Zoller et al., Nucleic Acids Res. 1982 Oct 25;10(20):6487-6500; Wang et al., Science. 1984 Jun 29;224(4656):1431-1433).

[0012] The method for detecting ORF25 protein may be any general method capable of detecting proteins used in testing for infectious diseases, etc., such as immunochromatography or fluorescent antibody techniques using anti-ORF25 protein antibodies, enzyme immunoassays (e.g., EIA and ELISA) using immobilized anti-ORF25 antibodies, Western blotting, etc. Methods using ORF25 protein as an indicator also include, for example, methods for detecting anti-ORF25 antibodies expressed in various animals, including humans.

[0013] As used herein, the terms "homology" or "sequence homology" refer to similarities (in the primary structure (i.e., sequence) of polynucleotides, polypeptides, etc.) resulting from divergence from a common ancestor. Furthermore, "homology search" refers to a method for detecting and analyzing "homology" by comparing the sequences of different molecules (e.g., polypeptide sequences, nucleotide sequences) based on their similarity. When detecting and analyzing homology, "sequence similarity" can be used as an indicator. "Sequence similarity" is defined as the percentage of identical residues ("percent identity," hereinafter referred to as "sequence identity (%)") between multiple sequences (e.g., two polypeptide sequences) being analyzed, or the percentage of residues ("similar residues") that conserve similar physicochemical properties between the sequences ("percent similarity," hereinafter referred to as "similar sequence identity (%)"). In other words, based on the observed sequence similarity, it is possible to discuss whether there is "homology."

[0014] As used herein, "sequence identity (%)" and "sequence similarity (%)" are calculated by comparing two optimally aligned sequences within the analysis range of the two sequences (hereinafter referred to as the "comparison window"). Specifically, the portion of the polynucleotide or polypeptide sequence within the comparison window may contain additions or deletions (i.e., gaps) when compared with the reference sequence for optimal alignment of the two sequences (the reference sequence here is assumed to have no additions or deletions, although gaps may occur if the other sequence contains additions). Positions where identical residues (nucleotide residues or amino acid residues) are found in both aligned sequences are identified, and the number of identified positions (the "number of identical residues") is divided by the "total number of positions" (within the comparison window) and then multiplied by 100 to calculate the "sequence identity." Similarly, the "sequence similarity" can be calculated by identifying the number of residues with conserved physicochemical properties (the "number of similar residues") instead of the "number of identical residues."

[0015] Homology searches can be performed using appropriate sequence comparison algorithms and programs known in the art. Examples of such algorithms and programs include the Basic Local Alignment Search Tool (BLAST), which can be used to evaluate homology based on the sequence similarity of polypeptides or polynucleotides. For BLAST, see, for example, Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87:2267-2268; Altschul et al., 1990, J. Mol. Biol. 215:403-410; Altschul et al., 1993, Nature Genetics 3:266-272; and Altschul et al., 1997, Nuc. Acids Res. 25:3389-3402. Search results can be quantified, for example, as positives or identities.

[0016] The ORF25 gene encodes the ORF25 protein. Nucleotide sequences that can be used as detection indicators include those encoding proteins consisting of the amino acid sequence set forth in any one of SEQ ID NOS: 1 to 3, or those encoding proteins having an amino acid sequence in which one or more amino acid residues have been deleted, substituted, or added in the amino acid sequence set forth in SEQ ID NOS: 1 to 3, or those having a sequence identity of 90% or more, preferably 94% or more, and more preferably 97% or more with these sequences. Examples of detection indicators include a polynucleotide consisting of the nucleotide sequence set forth in SEQ ID NOS: 4 as a nucleotide sequence encoding a protein having the amino acid sequence set forth in SEQ ID NOS: 1, a polynucleotide consisting of the nucleotide sequence set forth in SEQ ID NOS: 5 encoding a protein having the amino acid sequence set forth in SEQ ID NOS: 2, and a polynucleotide consisting of the nucleotide sequence set forth in SEQ ID NOS: 6 encoding a protein having the amino acid sequence set forth in SEQ ID NOS: 3. In addition to polynucleotides having the nucleotide sequences set forth in SEQ ID NOS: 4 to 6, polynucleotides having a sequence identity of 90% or more, preferably 94% or more, and more preferably 97% or more with a polynucleotide consisting of the nucleotide sequence set forth in any one of SEQ ID NOS: 4 to 6 can also be used as detection indicators for Og bacteria. In the present invention, the degree of sequence identity is determined by the value obtained by BLAST (NCBI BLAST http: / / www.ncbi.nlm.nih.gov / BLAST / ). In addition, in the method of using the nucleotide sequence encoding the ORF25 protein of the present invention as an index, not only the polynucleotide encoding the ORF25 protein but also the messenger RNA (mRNA) replicated during protein synthesis from the polynucleotide can be used as an index.

[0017] Any common method capable of detecting polynucleotides can be used to detect the orf25 gene. Examples include a method using a detection probe (e.g., a fluorescent probe) capable of complementarily binding to the orf25 gene, and a method in which polynucleotides extracted from a test sample are separated by electrophoresis or other methods and detected by luminescence from a color-changing or fluorescent substance. Usable detection probes are probes capable of complementarily binding to the entire orf25 gene or a partial region. Examples of probes capable of binding to the entire region include probes having nucleotide sequences capable of complementarily binding to the nucleotide sequences shown in SEQ ID NOS: 4 to 6. Examples of probes capable of complementarily binding to a partial region include probes having nucleotide sequences complementary to the nucleotide sequences shown in SEQ ID NOS: 7 to 9, which are the regions from 50 to 176 from the 5' end of the orf25 gene having the nucleotide sequences shown in SEQ ID NOS: 4 to 6, and probes having nucleotide sequences complementary to the nucleotide sequences shown in SEQ ID NOS: 10 to 12, which are the regions from 177 to 432 from the 5' end of the gene. The complementary probe may be DNA or RNA, and may be a probe having a base sequence capable of binding complementarily to a base sequence having 60% or more, preferably 75% or more, and more preferably 90% or more sequence identity with the base sequences shown in SEQ ID NOS: 4 to 6 or the base sequences shown in SEQ ID NOS: 7 to 12. The probe may be, for example, a complementary strand of a polynucleotide having a base sequence shown in SEQ ID NOS: 4 to 6 or a base sequence having 60% or more, preferably 75% or more, and more preferably 90% or more sequence identity therewith, or a complementary strand of a polynucleotide having a base sequence shown in SEQ ID NOS: 7 to 12 or a base sequence having 60% or more, preferably 75% or more, and more preferably 90% or more sequence identity therewith. The probe may contain a fluorescent substance or 32 The primers can be labeled with a radioisotope such as P. Labeled primers can be prepared by, for example, nick translation or random primer DNA labeling (random prime method). The probes can consist of a single probe having a specific base length, or they can contain multiple probes having different base lengths.

[0018] As used herein, the term "polynucleotide capable of complementarily binding" is used interchangeably with "polynucleotide capable of hybridizing under stringent conditions." These "stringent conditions" refer to well-known conditions commonly used in the art. Specifically, "stringent conditions" refer to conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed, for example, conditions under which polynucleotide pairs with a sequence identity higher than a certain standard hybridize and polynucleotide pairs with a lower sequence identity do not hybridize.

[0019] "Stringent conditions" are sequence-dependent and vary depending on various environmental parameters. General guidelines for polynucleotide (nucleic acid) hybridization can be found, for example, in Tijssen (1993), Laboratory Techniques in Biochemistry and Molecular Biology—Hybridization With Nucleic Acid Probes Part I, Chapter 2, "Overview of Principles of Hybridization and the Strategy of Nucleic Acid Probe Assay," Elsevier, New York. Several factors, such as temperature and salt concentration, can affect the stringency of a hybridization reaction. For details, see Ausubo et al., Current Protocols in Molecular Biology, Wiley Interscience Publishers, (1995). Examples of so-called "highly stringent conditions" include 0.0015 M sodium chloride, 0.0015 M sodium citrate, and conditions at 65 to 68° C., or 0.015 M sodium chloride, 0.0015 M sodium citrate, and 50% formamide at 42° C. Such conditions can be achieved according to the methods described in experimental texts such as Molecular Cloning 2nd ed., Current Protocols in Molecular Biology, Supplement 1-38, and DNA Cloning 1: Core Techniques, A Practical Approach, Second Edition, Oxford University Press (1995).Furthermore, "moderately stringent conditions" can be easily determined by those skilled in the art based on, for example, the length of DNA, and are shown, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed. Vol. 1, 7.42-7.45, Cold Spring Harbor Laboratory Press, 2001.

[0020] When detecting the orf25 gene, it is preferable to obtain an amplification product of the target gene (which may be the entire region of the gene or a partial region). The so-called polymerase chain reaction (PCR) method can be used to obtain the amplification product. The method for detecting the amplification product is not particularly limited, and examples include a method in which the amplification product is separated by electrophoresis after amplification and the target amplification product is confirmed using a stain such as ethidium bromide; a method in which a reagent that emits fluorescence when bound to the double-stranded DNA of the amplification product (intercalator: TB Green, SYBR Green, etc.) is added to the PCR reaction system; and a detection method using real-time PCR in which the amplification product is detected while amplification is occurring, such as the so-called TaqMan method, in which a fluorescently labeled probe bound to the amplified DNA is used.

[0021] Examples of primer sets required for PCR include: (1) a primer set consisting of a polynucleotide (primer) consisting of the nucleotide sequence shown in SEQ ID NO: 13 and a polynucleotide (primer) consisting of the nucleotide sequence shown in SEQ ID NO: 14 (which amplifies the entire region of the orf25 gene); (2) a primer set consisting of a polynucleotide (primer) consisting of the nucleotide sequence shown in SEQ ID NO: 15 and a polynucleotide (primer) consisting of the nucleotide sequence shown in SEQ ID NO: 16 (which amplifies the region from bases 50 to 176 from the 5' end of the orf25 gene); and (3) a primer set consisting of a polynucleotide (primer) consisting of the nucleotide sequence shown in SEQ ID NO: 17 and a polynucleotide (primer) consisting of the nucleotide sequence shown in SEQ ID NO: 14 (which amplifies the region from bases 177 to 432 from the 5' end of the orf25 gene). Other examples include (4) a primer set consisting of a polynucleotide (primer) consisting of the nucleotide sequence shown in SEQ ID NO: 18 and a polynucleotide (primer) consisting of the nucleotide sequence shown in SEQ ID NO: 19 (which amplifies the region from positions 94 to 301 from the 5' end of the orf25 gene), and (5) a primer set consisting of a polynucleotide (primer) consisting of the nucleotide sequence shown in SEQ ID NO: 20 and a polynucleotide (primer) consisting of the nucleotide sequence shown in SEQ ID NO: 21 (which amplifies the region from positions 93 to 194 from the 5' end of the orf25 gene). However, any polynucleotide capable of binding complementarily to the nucleotide sequences shown in SEQ ID NOs: 4 to 6 may be used as long as it is capable of amplifying the entire region or a portion thereof of the desired orf25 gene, and primers consisting of nucleotide sequences in which approximately 1 to 5 nucleotides have been substituted, deleted, or inserted from the nucleotide sequences of these primers may also be used.For example, (6) a primer set consisting of a polynucleotide (primer) consisting of the nucleotide sequence shown in SEQ ID NO: 23 and a polynucleotide (primer) consisting of the nucleotide sequence shown in SEQ ID NO: 14 (for example, amplifying the entire region of the orf25 gene shown in SEQ ID NO: 5); (7) a primer set consisting of a polynucleotide (primer) consisting of the nucleotide sequence shown in SEQ ID NO: 24 and a polynucleotide (primer) consisting of the nucleotide sequence shown in SEQ ID NO: 25 (for example, amplifying the region from bases 177 to 432 from the 5' end of the orf25 gene shown in SEQ ID NO: 5); and (8) a primer set consisting of a polynucleotide (primer) consisting of the nucleotide sequence shown in SEQ ID NO: 26 and a polynucleotide (primer) consisting of the nucleotide sequence shown in SEQ ID NO: 14 (for example, amplifying the region from bases 177 to 432 from the 5' end of the orf25 gene shown in SEQ ID NO: 5).

[0022] The composition of the reaction solution when performing PCR, and the reaction conditions such as the reaction temperature, number of reaction cycles, and reaction time are items that can be appropriately determined by a person skilled in the art, and examples of these are the composition of the reaction solution and reaction conditions shown in the Examples.

[0023] The detection kit of the present invention includes a primer set capable of specifically amplifying the orf25 gene. The primer set capable of amplifying the orf25 gene may include, for example, the primer sets (1) to (8) described above. The detection kit may further include PCR reaction reagents, such as a buffer constituting a reaction composition for amplification, reaction nucleic acids, and the detection probe, as well as an intercalator for detection in real-time PCR and a TaqMan probe used in the TaqMan method. Alternatively, the kit may include the full-length orf25 gene or a partial DNA capable of reacting with the target DNA.

[0024] The detection method of the present invention uses the ORF25 protein or the ORF25 gene as an indicator, as described above. It is desirable that, prior to detection, a sample suspected of containing the ORF25 protein or the ORF25 gene be prepared from a test sample. Examples of test samples include cells from a subject suspected of being infected with Og bacteria (e.g., cells obtained by puncturing the stomach), body fluids from the subject (e.g., gastric juice, saliva, blood such as serum, feces, and urine), and vomit. Since using the test sample as is may result in failure to detect the indicator protein or gene, unnecessary components are removed from the test sample to facilitate detection of the ORF25 protein or the ORF25 gene. Sample preparation methods may be any commonly used method for testing infectious diseases, etc., such as culturing Og bacteria under conditions suitable for culturing and recovering the resulting cells, or further preparing a fraction containing the ORF25 protein or the ORF25 gene by lysing, centrifuging, extracting, or other methods from the recovered cells. Furthermore, when the orf25 gene is used as an indicator, it is desirable to amplify the gene using the above-mentioned primer set using a sample prepared from the test sample or the test sample itself.

[0025] The present invention will be described in more detail below based on the following examples, but it goes without saying that the present invention is not limited to the following examples. [Example]

[0026] [Search by RAST server] Strains with the same properties as the Og bacteria described in Patent Document 1 were newly isolated from clinical sites. Genetic analysis of these strains revealed two new strains with different nucleotide sequences. For convenience, the strain described in Patent Document 1 will be referred to as Og1, and the two newly isolated strains will be referred to as Og2 and Og3, respectively. First, based on the genome data of the Og1 strain, all ORFs were estimated using the RAST server (Rapid Annotation using Subsystem Technology) to search for ORFs (open reading frames) that could potentially specifically detect Og bacteria. Of the 2,020 ORFs, 49 were predicted to be ORFs that could potentially specifically detect Og bacteria (see Figure 1). Figure 1 also shows the binding positions of primers used to amplify each ORF region.

[0027] [Classification of Og bacteria] A search for the ORFs of Og bacteria revealed that the S. parasanguinis type strain (S. parasanguinis ATCC 1591) and S. parasanguinis FW213 (Taiwan strain) possessed similar base counts, GC content, CDS, and RNA counts to those of Og bacteria. Therefore, a phylogenetic tree was constructed using several strains, including these two strains. The phylogenetic tree was constructed using software that applied the proximity joining method based on the base sequence of the 16S-23S region of ribosomal RNA possessed by each bacterium. The resulting phylogenetic tree is shown in Figure 2.

[0028] Furthermore, the Og1 strain and S. parasanguinis FW213 strain were analyzed using ANI (Average Nucleotide Ionization) analysis. Using an identity calculator from Kostas Lab, the identity rate was 94.3%, confirming that the Og1 strain and S. parasanguinis FW213 strain belong to different species, and that the newly detected Og2 and Og3 strains belong to the same species as Og1.

[0029] [Search for a region that can specifically detect Og bacteria] The 49 candidate ORFs identified above were searched for homology with S. parasanguinis strain FW213, the S. parasanguinis type strain, and several other strains of the genus Streptococcus that should be distinguished from Og strains in gene information databases. Homology searches were performed using BLAST (NCBI BLAST http: / / www.ncbi.nlm.nih.gov / BLAST / ). The results are shown in Table 1. As a result, the 432-bp ORF region at the 25th position from the top of Table 1 was deemed to be a target capable of specifically detecting Og strains. This target, designated orf25, was further investigated.

[0030] [Table 1]

[0031] An alignment of the nucleotide sequences of the orf25 gene, which is the ORF of the three Og bacteria, is shown in Figure 3. The nucleotide sequence of the Og1 strain (Og1-orf25) shown in Figure 3 is SEQ ID NO: 4, the nucleotide sequence of the Og2 strain (Og2-orf25) is SEQ ID NO: 5, and the nucleotide sequence of the Og3 strain (Og3-orf25) is SEQ ID NO: 6.

[0032] [Detection of orf25 gene] Based on the nucleotide sequence of the Og1 strain shown in Figure 3, a primer set for amplifying the orf25 gene was designed. The entire orf25 gene and a partial region were amplified, and the amplification products were detected. Three forward primers (Og25-Hyb-startF, Og25-Hyb-50F, and Og25-Hyb-177F; the nucleotide sequences are shown in SEQ ID NOs: 13, 15, and 17, respectively) were designed, and two reverse primers (Og25-Hyb-176R and Og25-Hyb-endR; the nucleotide sequences are shown in SEQ ID NOs: 16 and 14, respectively) were designed. Amplification was performed using the three primer combinations shown in Figure 4 (primer set orf25, primer set orf25_5', and primer set orf25_3'). The primer set orf25 (a combination of Og25-Hyb-startF and Og25-Hyb-endR) amplifies the entire region of orf25, the primer set orf25_5' (a combination of Og25-Hyb-50F and Og25-Hyb-176R) amplifies the region from the 50th base to the 176th base from the 5' end, and the primer set orf25_3' (Og25-Hyb-177F and Og25-Hyb-endR) amplifies the region from the 177th base to the 3' end.

[0033] The composition of the PCR reaction solution is shown in Table 2, and the PCR amplification conditions are shown in Table 3. The resulting amplified products were separated by agarose gel electrophoresis, and the amplified products were confirmed by blotting using three DNA probes (orf25, orf25_5', orf25_3') prepared corresponding to each amplified region.

[0034] Furthermore, a partial region of the nucleotide sequence (shown as FW213-orf25-homolog in Figure 13 , and its nucleotide sequence shown as SEQ ID NO: 22) of one ORF (referred to as the "orf-another2 gene") contained in the S. parasanguinis FW213 strain discovered during the search was similar to a partial region of the orf25 gene of Og bacteria, confirming that the three primer sets did not detect the S. parasanguinis FW213 strain. The nucleotide sequences of the orf-another2 genes of the three Og strains are shown as SEQ ID NO: 32 (Og1 strain: shown as Og1-orf25-homolog in Figure 13 ), SEQ ID NO: 33 (Og2 strain: shown as Og2-orf25-homolog in Figure 13 ), and SEQ ID NO: 34 (Og3 strain: shown as Og3-orf25-homolog in Figure 13 ). At this time, three forward primers and two reverse primers were prepared based on the base sequence of the S. parasanguinis FW213 strain shown for reference in Figure 11, and attempts were made to detect the Og1 strain and the S. parasanguinis FW213 strain. For confirmation, a primer set (orf-homolog-FW213) that amplifies the entire region of the orf-another2 gene, a primer set (orf-homolog-FW213) that amplifies the region from the 56th base from the 5' end of the orf gene to the 182nd base from the same end, a primer set (orf-homolog-FW213_5') that amplifies the region from the 183rd base from the 5' end to the 3' end of the orf gene, and primer sets (orf-homolog-FW213_3') that amplify the region that binds to each amplified region. 32 A P-labeled DNA probe was used. 32 The P-labeled DNA probe was prepared by the random primer method. Figure 4 shows the primer set used to amplify the orf25 gene of Og bacteria, Figure 5 shows the primer set used to amplify the orf25 homologue of S. parasanguinis strain FW213, and Figure 6 shows a comparison of the amplified regions of both primers.

[0035] The results are shown in Figure 7. As shown in Figure 7, the amplification products obtained with the three primer sets constructed based on the nucleotide sequence of the orf25 gene of the Og1 strain reacted with the PCR product derived from the Og1 strain (probe shown in Figure 7) but did not react with the PCR product derived from the orf-another2 gene of the S. parasanguinis FW213 strain (same probe). Furthermore, the amplification products obtained with the three primer sets constructed based on the nucleotide sequence of the orf-another2 gene of the S. parasanguinis FW213 strain reacted with the PCR product derived from the S. parasanguinis FW213 strain (same probe) but did not react with the PCR product derived from the Og1 strain. The DNA template was prepared using a standard method for preparing DNA samples from bacteria. Figure 14 shows the amplified regions of the primer sets used in PCR.

[0036] [Table 2]

[0037] [Table 3]

[0038] [Amplification of Og2 and Og3 strains] Next, we attempted to detect the target genes of the Og1 and S. parasanguinis FW213 strains by blotting using the DNA probes prepared above and bacterial DNA from Og1, Og2, Og3, S. parasanguinis FW213, and the S. parasanguinis type strain. The results are shown in Figure 8. Three DNA probes prepared based on the orf25 sequence of Og1 strain reacted with Og1, Og2, and Og3, respectively. Furthermore, three DNA probes prepared based on the S. parasanguinis FW213 sequence reacted with all strains except the type strain. Therefore, we confirmed that the orf25 gene in the three Og strains is a unique gene found in Og strains, and that detection of this region can specifically detect Og strains. Furthermore, it was concluded that a region having a sequence similar to that of the orf-another2 gene of the S. parasanguinis FW213 strain is also present in the Og bacterium, and that this region is located in a different position from the orf25 gene (see Figure 13). [Example]

[0039] Three probes (orf25, orf25_5', orf25_3') were prepared based on the base sequence of the orf25 gene of the Og1 strain, and various strains were analyzed. 32 Hybridization was performed using the above DNA probes labeled with P. Each strain was inoculated onto a nitrocellulose membrane placed on a medium. After incubation, the bacterial DNA was treated with alkali and then subjected to colony hybridization with the three probes. The results are shown in Figures 9 and 10. The three probes reacted with Og1, Og2, and Og3 strains, respectively, but not with other strains. Furthermore, they reacted well with strains thought to be Og bacteria (No. 21 and No. 22 in Figure 10) cultured from gastric puncture specimens of Russian patients with gastroesophageal junction inflammation, gastritis, or gastric ulcer. Therefore, it is presumed that the bacteria collected from these patients were Og bacteria. [Example]

[0040] A new primer set capable of amplifying a partial region of the orf25 gene was created, and an attempt was made to detect Og bacteria from the PCR amplification products. The newly created primer set (Og-Hypo25F: SEQ ID NO: 18 and Og-Hypo25R: SEQ ID NO: 19) is shown in Table 4.

[0041] [Table 4]

[0042] The amplification products amplified under the conditions shown in Tables 2 and 3 were subjected to agarose gel electrophoresis and confirmed with a fluorescently labeled probe. The results are shown in Figure 11. As shown in lane 4, a specific amplification product was also obtained from a bacterium (PI-1) thought to be Og bacteria cultured from a gastric puncture specimen from a Russian patient with gastroesophageal junction inflammation, gastritis, or gastric ulcer.

[0043] In addition, when the Og1 strain was detected by real-time PCR using the primer set shown in Table 5 (Og-ORF25-RT-F: sequence number 20 and Og-ORF25-RF-R: sequence number 21), a good peak was observed, whereas when applied to the S. parasanguinis FW213 strain as a reference example, no peak was observed (results not shown).

[0044] [Table 5] [Industrial Applicability]

[0045] The present invention can be used, for example, to rapidly detect S. mobilis bacteria, which are thought to be involved in gastric ulcers, gastric cancer, and other human pathologies.

Claims

1. A method for detecting Streptococcus mobilis bacteria using, as an indicator, a protein consisting of the amino acid sequence shown in any one of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, or an amino acid sequence in which 1 to 9 amino acid residues have been substituted, deleted, or inserted from these amino acid sequences.

2. A method for detecting Streptococcus mobilis bacteria using, as an indicator, a polynucleotide encoding the protein of claim 1 or a polynucleotide consisting of a base sequence that has 90% or more sequence identity with the base sequence of said polynucleotide.

3. A method for detecting Streptococcus mobilis bacteria using, as an indicator, a polynucleotide consisting of a base sequence shown in any of SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6, or a base sequence that has a sequence identity of 90% or more with any of these base sequences.

4. A PCR primer set used to detect Streptococcus mobilis, the primer set being any one of the following primer sets (1) to (8): (1) a polynucleotide consisting of the base sequence shown in SEQ ID NO: 13; or A polynucleotide consisting of a base sequence in which 1 to 2 nucleotide residues are substituted, deleted or inserted from the base sequence of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 13, and capable of complementarily binding to the base sequences shown in SEQ ID NOs: 4 to 6; A polynucleotide consisting of the base sequence shown in SEQ ID NO: 14, or A polynucleotide consisting of a base sequence in which 1 to 2 nucleotide residues are substituted, deleted or inserted from the base sequence of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 14, and capable of binding complementarily to the base sequences shown in SEQ ID NOs: 4 to 6. (2) a polynucleotide consisting of the base sequence shown in SEQ ID NO: 15; or A polynucleotide consisting of a base sequence in which 1 to 2 nucleotide residues are substituted, deleted or inserted from the base sequence of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 15, and capable of complementarily binding to the base sequences shown in SEQ ID NOs: 4 to 6; A polynucleotide consisting of the base sequence shown in SEQ ID NO: 16, or A polynucleotide consisting of a base sequence in which 1 to 2 nucleotide residues are substituted, deleted or inserted from the base sequence of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 16, and capable of binding complementarily to the base sequences shown in SEQ ID NOs: 4 to 6. (3) a polynucleotide consisting of the base sequence shown in SEQ ID NO: 17; or A polynucleotide having a base sequence in which one nucleotide residue is substituted, deleted or inserted from the base sequence of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 17, and capable of binding complementarily to the base sequences shown in SEQ ID NOs: 4 to 6; A polynucleotide consisting of the base sequence shown in SEQ ID NO: 14, or A polynucleotide consisting of a base sequence in which 1 to 2 nucleotide residues are substituted, deleted or inserted from the base sequence of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 14, and capable of binding complementarily to the base sequences shown in SEQ ID NOs: 4 to 6. (4) a polynucleotide consisting of the base sequence shown in SEQ ID NO: 18; or A polynucleotide having a base sequence in which one nucleotide residue is substituted, deleted or inserted from the base sequence of a polynucleotide having the base sequence shown in SEQ ID NO: 18, and capable of binding complementarily to the base sequences shown in SEQ ID NOs: 4 to 6; A polynucleotide consisting of the base sequence shown in SEQ ID NO: 19, or A polynucleotide having a base sequence in which 1 to 2 nucleotide residues are substituted, deleted or inserted from the base sequence of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 19, and capable of binding complementarily to the base sequences shown in SEQ ID NOs: 4 to 6. (5) A polynucleotide consisting of the base sequence shown in SEQ ID NO: 20, or A polynucleotide having the base sequence shown in SEQ ID NO: 20, in which 1 to 2 nucleotide residues have been substituted, deleted or inserted from the base sequence of the polynucleotide, and capable of binding complementarily to the base sequences shown in SEQ ID NOs: 4 to 6; A polynucleotide consisting of the base sequence shown in SEQ ID NO: 21, or A polynucleotide consisting of a base sequence in which 1 to 2 nucleotide residues are substituted, deleted or inserted from the base sequence of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 21, and capable of binding complementarily to the base sequences shown in SEQ ID NOs: 4 to 6. (6) a polynucleotide consisting of the base sequence shown in SEQ ID NO: 23; or A polynucleotide consisting of a base sequence in which 1 to 2 nucleotide residues are substituted, deleted or inserted from the base sequence of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 23, and capable of complementarily binding to the base sequences shown in SEQ ID NOs: 4 to 6. A polynucleotide consisting of the base sequence shown in SEQ ID NO: 14, or A polynucleotide consisting of a base sequence in which 1 to 2 nucleotide residues are substituted, deleted or inserted from the base sequence of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 14, and capable of binding complementarily to the base sequences shown in SEQ ID NOs: 4 to 6. (7) a polynucleotide consisting of the base sequence shown in SEQ ID NO: 24; or A polynucleotide consisting of a base sequence in which 1 to 2 nucleotide residues are substituted, deleted or inserted from the base sequence of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 24, and capable of complementarily binding to the base sequences shown in SEQ ID NOs: 4 to 6; A polynucleotide consisting of the base sequence shown in SEQ ID NO: 25, or A polynucleotide having a base sequence in which 1 to 2 nucleotide residues are substituted, deleted or inserted from the base sequence of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 25, and capable of binding complementarily to the base sequences shown in SEQ ID NOs: 4 to 6. (8) a polynucleotide consisting of the base sequence shown in SEQ ID NO: 26; or A polynucleotide having a base sequence in which one nucleotide residue is substituted, deleted or inserted from the base sequence of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 26, and capable of binding complementarily to the base sequences shown in SEQ ID NOs: 4 to 6; A polynucleotide consisting of the base sequence shown in SEQ ID NO: 14, or A polynucleotide consisting of a base sequence in which 1 to 2 nucleotide residues are substituted, deleted or inserted from the base sequence of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 14, and capable of binding complementarily to the base sequences shown in SEQ ID NOs: 4 to 6.

5. A detection probe having a base sequence capable of binding complementarily to a polynucleotide consisting of the base sequence shown in any one of SEQ ID NOs: 4 to 6 or 7 to 12, or a polynucleotide having a sequence identity of 90% or more with any of these polynucleotides.

6. A kit for detecting Streptococcus mobilis, comprising a primer set capable of amplifying a partial region or the entire region of the orf25 gene.

7. 7. The kit for detecting Streptococcus mobilis according to claim 6, wherein the primer set is any one of the primer sets according to claim 4.

8. A kit for detecting Streptococcus mobilis, comprising a primer set capable of amplifying a partial region or the entire region of the orf25 gene and any of the detection probes according to claim 5.

9. A method for detecting Streptococcus mobilis bacteria, comprising the steps of amplifying a polynucleotide in a test sample using a primer set capable of amplifying a partial region or the entire region of the orf25 gene, and detecting the amplification product amplified by the step.

10. 10. The method for detecting Streptococcus mobilis according to claim 9, wherein the primer set is any one of the primer sets according to claim 4.

11. An isolated polynucleotide consisting of the base sequence set forth in any one of SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, or a base sequence having a sequence identity of 90% or more with any one of these base sequences.

12. An isolated protein consisting of an amino acid sequence set forth in any one of SEQ ID NOs: 1 to 3, or said amino acid sequence, or an amino acid sequence in which 1 to 9 amino acids have been substituted, deleted or inserted from said amino acid sequence.

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