Method for detecting baiCD gene and baiCD gene detection kit

Abstract

To provide a baiCD gene detection method that can detect the baiCD gene in Ruminococcaceae bacteria conveniently by PCR with accuracy comparable to that of metagenomics.SOLUTION: The present invention relates to a method for detecting the baiCD gene in Ruminococcaceae bacteria included in a sample. The method includes an amplification step for amplifying baiCD gene fragments by using a primer set, which is a combination of primers to hybridize with different two consensus regions, respectively. Each consensus region is a region in which at least 15 specific bases continue in a specific base sequence.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for detecting the baiCD gene and a baiCD gene detection kit used therefor. [Background technology]

[0002] Primary bile acids, which are steroid compounds biosynthesized in the liver, are secreted into bile in conjugated forms (e.g., cholic acid conjugated with taurine or glycine, chenodeoxycholic acid), some of which are converted in the large intestine to secondary bile acids (e.g., deoxycholic acid and lithocholic acid) by the 7-α-dehydroxylation activity (enzymatic activity that removes the 7-α hydroxyl group from the steroid nucleus) possessed by some enterobacteria. It is known that this 7-α-dehydroxylation activity is not carried out by a single enzyme, but by a group of enzymes expressed from the bai (bile acid inducible) operon gene group possessed by bacteria. The baiCD gene is one of the genes that constitute the bai operon gene group.

[0003] It has been reported that the secondary bile acids produced by enterobacteria in this way are toxic and are involved in various diseases and health conditions, such as colon cancer (Non-Patent Documents 1 and 2, etc.). Therefore, from the viewpoint of evaluating risk factors for diseases such as colon cancer, it is important to detect whether or not bacteria carrying genes included in the bai operon gene group, such as the baiCD gene, are present in the intestine.

[0004] On the other hand, it has also been reported that the presence of bacteria carrying the bai operon gene group in the intestine suppresses Clostridium difficile infection (CDI) (Non-Patent Documents 3, 4, etc.). For example, Patent Document 1 describes a method for reducing the risk of Clostridium infection by administering a bacterial strain expressing the bai operon gene group to a subject. Clostridium infection is an infection in which Clostridium produces toxins in the intestine, causing symptoms such as diarrhea, and may even lead to death. Therefore, from the perspective of evaluating the severity of Clostridium infection, it is important to detect whether or not bacteria carrying genes included in the bai operon gene group, such as the baiCD gene, are present in the intestine.

[0005] To determine what types and amounts of bacteria are contained in the intestinal environment, methods that do not rely on cell culture but mainly use metagenomic analysis such as shotgun metagenomic analysis and 16S metagenomic analysis to decipher and identify genetic information. For example, shotgun metagenomic analysis can be suitably used to analyze the extent to which bacteria carrying the bai operon gene group are contained in the intestinal flora. For example, a library is created by fragmenting the DNA (metagenomic DNA) of the entire intestinal flora by size, and the resulting sequence data is then compared with the sequence of the bai operon gene group in a database to analyze which bacterial gene group it is part of. However, metagenomic analysis is not suitable as a detection method for a large number of individual specimens because it requires complex operations, a large amount of information, and expensive equipment.

[0006] In addition, conventionally, a culture method for culturing bacteria from a specimen is used for bacterial testing of a specimen, but since many of the bacteria whose existence has been confirmed by such metagenomic analysis are strains that have never actually been cultured alone, it is difficult to apply such a culture method. Furthermore, among the bacteria that possess the bai operon gene group, specific bacteria such as Clostridium hiranonis and Clostridium scindens whose existence has been confirmed by culture can be detected by PCR using primers specific to those genes, as described in Non-Patent Document 4. However, with primers designed specifically for these bacteria, it has been difficult to comprehensively detect bacteria that possess the baiCD gene or the bai operon gene group, including bacteria that have never actually been cultured alone, such as some bacteria belonging to the Ruminococcaceae family described in Non-Patent Document 2. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] U.S. Patent Application No. 2019 / 0381113 [Non-patent literature]

[0008] [Non-Patent Document 1] Anna Dart,Nature Reviews,Microbiology,Vol.16,August 2018,p.453 [Non-Patent Document 2] Marius Vital et al.,Computational and Structural Biotechnology Journal,17,2019,p.1016-1019 [Non-Patent Document 3] Jenessa A. Winston et al., Anaerobe., 2016 October, 41, p. 44-50 [Non-Patent Document 4] Philipp Solbach et al., PLOS ONE, May 8, 2018, https: / / doi.org / 10.1371 / journal.pone.0196977 Summary of the Invention [Problem to be solved by the invention]

[0009] The present invention has been made in consideration of the above-mentioned problems, and aims to provide a baiCD gene detection method that can easily detect the baiCD gene by PCR with the same accuracy as metagenomic analysis, even for bacteria whose existence has previously only been confirmed by metagenomic analysis, such as some bacteria belonging to the Ruminococcaceae family, and a baiCD gene detection kit for use therein. [Means for solving the problem]

[0010] The present inventors first performed metagenomic analysis on DNA extracted from stool samples, and identified the baiCD gene region by combining the data of bacteria belonging to the family Ruminococcaceae and a phylogenetic group to which species containing a large amount of baiCD genes belong in the phylogenetic tree described in Figure 1 of Non-Patent Document 2, which is available from among the public metagenomic data; and the data of a strain (Firmicutes bacterium CAG:103 strain) for which genome information is known. Next, the sequences between the obtained baiCD gene regions were analyzed, and a common region in which the sequences were conserved between them was found. By designing a primer in a specific region within such a common region, it was found that it is possible to easily detect the baiCD gene by PCR with the same accuracy as metagenomic analysis, even for bacteria whose existence could only be confirmed by metagenomic analysis, such as some bacteria belonging to the family Ruminococcaceae, and thus the present invention was completed.

[0011] That is, the present invention relates to a baiCD gene detection method and a baiCD gene detection kit used therein, and more specifically, provides the following. [1] This is a method for detecting the baiCD gene of bacteria belonging to the Ruminococcaceae family in a sample, The method includes an amplification step of amplifying a baiCD gene fragment using a primer set, which is a combination of primers that hybridize to two mutually different consensus regions, The consensus regions are regions in which 15 or more consecutive bases other than n are present in the base sequence set forth in SEQ ID NO: 1. baiCD gene detection method. [2] The method for detecting the baiCD gene according to [1], wherein the consensus region is a region selected from the group consisting of a region represented by the base sequence set forth in SEQ ID NO: 2, a region represented by the base sequence set forth in SEQ ID NO: 3, a region represented by the base sequence set forth in SEQ ID NO: 4, a region represented by the base sequence set forth in SEQ ID NO: 5, a region represented by the base sequence set forth in SEQ ID NO: 6, a region represented by the base sequence set forth in SEQ ID NO: 7, a region represented by the base sequence set forth in SEQ ID NO: 8, and a region represented by the base sequence set forth in SEQ ID NO: 9. [3] The primer set is a primer set I which is a combination of a forward primer I having a total of 20 to 25 bases containing the base sequence set forth in SEQ ID NO: 10 and a reverse primer I having a total of 20 to 25 bases containing the base sequence set forth in SEQ ID NO: 11; a primer set II comprising a forward primer II having a total of 20 to 25 bases and containing the base sequence set forth in SEQ ID NO: 12, and a reverse primer I having a total of 20 to 25 bases and containing the base sequence set forth in SEQ ID NO: 11; a primer set III which is a combination of a forward primer III having a total of 20 to 25 bases including the base sequence set forth in SEQ ID NO: 13 and a reverse primer III having a total of 20 to 25 bases including the base sequence set forth in SEQ ID NO: 14; A primer set IV which is a combination of a forward primer IV having a total of 20 to 25 bases including the base sequence set forth in SEQ ID NO: 15 and a reverse primer III having a total of 20 to 25 bases including the base sequence set forth in SEQ ID NO: 14; and A primer set V is a combination of a forward primer V having a total of 20 to 25 bases including the base sequence set forth in SEQ ID NO: 16 and a reverse primer V having a total of 20 to 25 bases including the base sequence set forth in SEQ ID NO: 17. Any one selected from the group consisting of: A method for detecting the baiCD gene according to [1] or [2]. [4] detecting the amplification product obtained in the amplification step; detecting 16S rRNA in the sample; and a correction step of correcting the amount of the detected amplification product with the amount of the detected 16S rRNA; The method for detecting a baiCD gene according to any one of [1] to [3], further comprising: [5] A kit for use in the baiCD gene detection method according to any one of [1] to [4], A primer set is provided, which is a combination of primers that hybridize to two mutually different consensus regions, The consensus regions are regions in which 15 or more consecutive bases other than n are present in the base sequence set forth in SEQ ID NO: 1. baiCD gene detection kit. [6] The baiCD gene detection kit described in [5], wherein the consensus region is a region selected from the group consisting of a region represented by the base sequence set forth in SEQ ID NO: 2, a region represented by the base sequence set forth in SEQ ID NO: 3, a region represented by the base sequence set forth in SEQ ID NO: 4, a region represented by the base sequence set forth in SEQ ID NO: 5, a region represented by the base sequence set forth in SEQ ID NO: 6, a region represented by the base sequence set forth in SEQ ID NO: 7, a region represented by the base sequence set forth in SEQ ID NO: 8, and a region represented by the base sequence set forth in SEQ ID NO: 9. [7] The primer set is a primer set I which is a combination of a forward primer I having a total of 20 to 25 bases containing the base sequence set forth in SEQ ID NO: 10 and a reverse primer I having a total of 20 to 25 bases containing the base sequence set forth in SEQ ID NO: 11; a primer set II comprising a forward primer II having a total of 20 to 25 bases and containing the base sequence set forth in SEQ ID NO: 12, and a reverse primer I having a total of 20 to 25 bases and containing the base sequence set forth in SEQ ID NO: 11; a primer set III which is a combination of a forward primer III having a total of 20 to 25 bases including the base sequence set forth in SEQ ID NO: 13 and a reverse primer III having a total of 20 to 25 bases including the base sequence set forth in SEQ ID NO: 14; A primer set IV which is a combination of a forward primer IV having a total of 20 to 25 bases including the base sequence set forth in SEQ ID NO: 15 and a reverse primer III having a total of 20 to 25 bases including the base sequence set forth in SEQ ID NO: 14; and A primer set V is a combination of a forward primer V having a total of 20 to 25 bases including the base sequence set forth in SEQ ID NO: 16 and a reverse primer V having a total of 20 to 25 bases including the base sequence set forth in SEQ ID NO: 17. At least one selected from the group consisting of: The baiCD gene detection kit according to [5] or [6]. [8] A baiCD gene detection kit according to any one of [5] to [7], further comprising a primer set capable of detecting bacterial 16S rRNA. Effect of the Invention

[0012] According to the present invention, it is possible to provide a baiCD gene detection method that can easily detect the baiCD gene by PCR with the same accuracy as metagenomic analysis, even for bacteria belonging to the Ruminococcaceae family, whose existence has previously only been confirmed by metagenomic analysis, and a baiCD gene detection kit for use therein. [Brief description of the drawings]

[0013] [Figure 1A] FIG. 1 shows a common region and preferred consensus regions 1 to 8 set forth in SEQ ID NO: 1 (bases 1 to 960 of common region 1). [Figure 1B] FIG. 1 shows the common region and preferred consensus regions 1 to 8 set forth in SEQ ID NO: 1 (bases 961 to 1945 of common region 1). [Figure 2A] FIG. 1 shows the sequence contained in the common region among Nos. 1 to 8 in the baiCD gene region. [Figure 2B] FIG. 1 shows the sequence contained in the common region among Nos. 1 to 8 in the baiCD gene region. [Figure 2C] FIG. 1 shows the sequence contained in the common region among Nos. 1 to 8 in the baiCD gene region. [Figure 3A] This is an electrophoretic photograph obtained in the evaluation of primers for the baiCD gene detection evaluation (primer sets 1 to 3). [Figure 3B] This is an electrophoretic photograph obtained in the evaluation of primers for the baiCD gene detection evaluation (primer sets 4 to 5). [Figure 4] 1 is a graph showing the results of correcting the amount of baiCD gene obtained using primer sets 1, 2, 3, and 5 with the amount of total DNA. [Diagram 5] 1 is a graph showing the results of correcting the amount of baiCD gene obtained using primer sets 1, 2, 3, and 5 with the amount of 16S rRNA. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] Hereinafter, the present invention will be described in detail according to its preferred embodiments.

[0015] <baiCD gene detection method> The baiCD gene detection method of the present invention is a method for detecting the baiCD gene of bacteria belonging to the family Ruminococcaceae in a sample, and includes an amplification step of amplifying a baiCD gene fragment using a primer set which is a combination of primers that hybridize to two different consensus regions respectively, wherein each of the consensus regions is a region where 15 or more consecutive bases other than n in the nucleotide sequence set forth in SEQ ID NO: 1 are shown. In the present invention, the detection of the baiCD gene includes the quantification or semi-quantification of the baiCD gene.

[0016] [Sample] The "sample" targeted by the baiCD gene detection method of the present invention is not particularly limited as long as it is a sample in which bacteria that may possess the baiCD gene or its DNA may exist. Generally, samples such as urine, feces, oral mucosa, pharyngeal mucosa, intestinal mucosa, and various biopsy samples collected from a subject (preferably a human) for detecting the baiCD gene, such as a diagnostic target, can be mentioned. The sample according to the present invention is preferably feces. The sample may be appropriately diluted or suspended with a diluent, adjusted in pH, or subjected to treatments such as grinding or freezing. Examples of the diluent include buffers such as phosphate buffer, Tris buffer, Good buffer, and borate buffer.

[0017] [Common region] According to the present invention, by designing a primer within the common region found by the present inventors, in addition to bacteria that have been conventionally cultured alone, bacteria belonging to the family Ruminococcaceae whose existence has been conventionally confirmed only by metagenomic analysis can be easily detected by PCR with the same accuracy as metagenomic analysis. Specifically, the "bacteria belonging to the family Ruminococcaceae whose existence has been conventionally confirmed only by metagenomic analysis" are bacteria belonging to the family Ruminococcaceae in Figure 1 of Non-Patent Document 2, and belonging to a phylogenetic group (GeversD_2014_SKBSTL008_bin.71 to SRR5091468_bin.55 in Figure 1) to which species belong that have a number of MAGs (metagenome-assembled genomes) containing the baiCD gene (n_MAGs) of 100 or more in the data of Study A. Whether or not a target bacterium belongs to the phylogenetic group can be determined by obtaining the bin sequence of the bacterium, combining it with all the bacteria shown in Figure 1 of Non-Patent Document 2 to create a phylogenetic tree by the method described in the same document, and finding the following species: GeversD_2014_SKBSTL008_bin.71, CosteaPI_2017_SID713A002-11-0-0_bin.25, CosteaPI_2017_SID713B026-11-90-0_bin.59, and CosteaPI_2017_SID713A023-11-0-0_bin.14 and belong to a clade that includes at least one of the following species: XieH_2016_YSZC12003_37181R1_bin.102, Obregon-TitoAJ_2015_SM11_bin.2, KarlssonFH_2013_S54_bin.27, FengQ_2015_SID31866_bin.65, XieH_2016_YSZC12003_35390_bin.5, LiJ_2014_MH0358_bin.56, RaymondF_2016_P21E7_bin.22, FengQ_2015_SID531416_bin.49, VogtmannE_2016MMRS15137911ST-27-0-0_bin.3, and FengQ_2015_SID31866_bin.46 This can be confirmed by the fact that it is not in the same phylogenetic group as all of the above.

[0018] In the present invention, the common region for designing primers is a region on genomic DNA that is common to the base sequences of the baiCD genes of bacteria belonging to the family Ruminococcaceae, in particular, to the base sequences of the baiCD genes of bacteria belonging to the family Ruminococcaceae, the existence of which has previously only been confirmed by metagenomic analysis.

[0019] The base sequence of the common region is shown in SEQ ID NO: 1. In the base sequence shown in SEQ ID NO: 1 (common region 1), "n" represents a, c, g, or t, or a deletion of a base (one base). Details of SEQ ID NO: 1 are shown in Figs. 1A and 1B. In Figs. 1A and 1B, "n" represents A, C, G, or T, or a deletion of a base (one base), and "N" represents A, C, G, or T. The common region shown in SEQ ID NO: 1 is more preferably the sequence shown in SEQ ID NO: 20. In the base sequence shown in SEQ ID NO: 20 (common region 2), "n" represents a, c, g, or t.

[0020] [Consensus Area] In the present invention, primers are designed to hybridize to two mutually different consensus regions. The two consensus regions may partially overlap each other. The consensus region according to the present invention is a consensus region of the baiCD gene, and refers to a region in the base sequence of SEQ ID NO: 1 in which 15 or more consecutive bases other than n are present, more preferably a region in the base sequence of SEQ ID NO: 20 in which 15 or more consecutive bases other than n are present, preferably a region in which 15 to 100 consecutive bases other than n are present, and more preferably a region in which 17 to 30 consecutive bases other than n are present.

[0021] Such a consensus region is not particularly limited, but is preferably a region selected from the group consisting of, for example, the region represented by the base sequence set forth in SEQ ID NO:2 (sometimes referred to as "consensus region 1"), the region represented by the base sequence set forth in SEQ ID NO:3 (sometimes referred to as "consensus region 2"), the region represented by the base sequence set forth in SEQ ID NO:4 (sometimes referred to as "consensus region 3"), the region represented by the base sequence set forth in SEQ ID NO:5 (sometimes referred to as "consensus region 4"), the region represented by the base sequence set forth in SEQ ID NO:6 (sometimes referred to as "consensus region 5"), the region represented by the base sequence set forth in SEQ ID NO:7 (sometimes referred to as "consensus region 6"), the region represented by the base sequence set forth in SEQ ID NO:8 (sometimes referred to as "consensus region 7"), and the region represented by the base sequence set forth in SEQ ID NO:9 (sometimes referred to as "consensus region 8"). Consensus regions 1 to 8 are shown in Figures 1A to 1B as underlines, or as underlines and overlines when two regions overlap. In Figures 1A and 1B, the underlined or overlined numbers correspond to the numbers of the consensus regions.

[0022] As these consensus regions, two different regions can be appropriately selected, but a preferred combination, from the viewpoint of detection accuracy by PCR, is such that the number of bases in the PCR amplified product (fragment), i.e., the number of bases from the 5'-end base of one consensus region on the 5'-end side to the 3'-end base of the other consensus region on the 3'-end side, is a distance of 50 or more bases, more preferably 50 to 300 bases, and even more preferably 50 to 250 bases, from the first base in the 5'-end base of one consensus region on the 5'-end side.

[0023] Such preferred combinations include a combination of consensus region 1 and consensus region 2, a combination of consensus region 3 and consensus region 2, a combination of consensus region 4 and consensus region 5, a combination of consensus region 6 and consensus region 5, and a combination of consensus region 7 and consensus region 8. Among these, from the viewpoint of further improving the detection accuracy by PCR, the combination of consensus region 1 and consensus region 2, the combination of consensus region 3 and consensus region 2, and the combination of consensus region 4 and consensus region 5 are preferred, the combination of consensus region 1 and consensus region 2, and the combination of consensus region 3 and consensus region 2 are more preferred, and the combination of consensus region 1 and consensus region 2 is even more preferred.

[0024] [Primer set] The primer set according to the present invention is a combination of primers that hybridize to two mutually different consensus regions among the above consensus regions.

[0025] In the present invention, when a primer "hybridizes" to the consensus region, it includes not only hybridizing to the sense strand of the consensus region of the present invention (e.g., the base sequences shown in SEQ ID NOs: 2 to 9) but also hybridizing to its complementary strand.

[0026] In the present invention, the primer "hybridizes" to the consensus region means that at least a part of the primer and the consensus region can form a double strand, and the primer does not have to be completely complementary. In the present invention, the hybridization condition is preferably such that the sequence complementarity of the primer and the consensus region is 80% or more, 90% or more, 95% or more (e.g., 96% or more, 97% or more, 98% or more, 99% or more). The sequence complementarity can be calculated appropriately by a person skilled in the art using a known method (e.g., BLAST (NCBI)). In the present invention, the hybridization condition may be such that the primer has 1 to 5 consecutive bases (preferably 1 to 3 bases, e.g., 3 bases, 2 bases, or 1 base) inserted, deleted, or substituted at one or more positions of the base sequence completely complementary to the entire length of the base sequence of the consensus region.

[0027] The base sequence constituting such a primer can be appropriately designed to meet the above hybridization conditions, to match the base sequence of the consensus region, and more preferably to avoid hybridizing to other regions.

[0028] The length of the primer according to the present invention is preferably at least 15 bases, and is usually 15 to 100 bases, preferably 17 to 30 bases, and more preferably 20 to 25 bases. The primer can be synthesized, for example, by a commercially available oligonucleotide synthesizer. Furthermore, the primer according to the present invention does not have to be composed only of natural nucleotides (deoxyribonucleotides and / or ribonucleotides), and may be composed partly or entirely of non-natural nucleotides such as PNA (polyamide nucleic acid), LNA (registered trademark, locked nucleic acid), and ENA (registered trademark, 2'-O,4'-C-Ethylene-bridged nucleic acids).

[0029] More specifically, such a primer set includes: a primer set I which is a combination of a forward primer I having a total of 20 to 25 bases containing the base sequence set forth in SEQ ID NO: 10 and a reverse primer I having a total of 20 to 25 bases containing the base sequence set forth in SEQ ID NO: 11; a primer set II comprising a forward primer II having a total of 20 to 25 bases and containing the base sequence set forth in SEQ ID NO: 12, and a reverse primer I having a total of 20 to 25 bases and containing the base sequence set forth in SEQ ID NO: 11; a primer set III which is a combination of a forward primer III having a total of 20 to 25 bases including the base sequence set forth in SEQ ID NO: 13 and a reverse primer III having a total of 20 to 25 bases including the base sequence set forth in SEQ ID NO: 14; A primer set IV which is a combination of a forward primer IV having a total of 20 to 25 bases including the base sequence set forth in SEQ ID NO: 15 and a reverse primer III having a total of 20 to 25 bases including the base sequence set forth in SEQ ID NO: 14; and A primer set V is a combination of a forward primer V having a total of 20 to 25 bases including the base sequence set forth in SEQ ID NO: 16 and a reverse primer V having a total of 20 to 25 bases including the base sequence set forth in SEQ ID NO: 17. Among these, from the viewpoint of further improving the detection accuracy by PCR, primer set I, primer set II, or primer set III is preferred, primer set I or primer set II is more preferred, and primer set I is even more preferred.

[0030] [Amplification process (PCR process)] The baiCD gene detection method of the present invention includes an amplification step of amplifying a baiCD gene fragment using the primer set. By using the primer set set in the common region, it is possible to specifically amplify a baiCD gene, in particular, a nucleotide fragment (referred to as a "baiCD gene fragment" in this specification) contained in the baiCD gene of a bacterium belonging to the family Ruminococcaceae, the existence of which has been confirmed only by metagenomic analysis in the past.

[0031] In the amplification step, for example, first, DNA is extracted from the sample, and the primer set and complementary strand synthesis enzyme are added to the extracted DNA to perform a PCR reaction. If the desired consensus region, i.e., the baiCD gene, is present, each primer hybridizes to it, and a baiCD gene fragment is synthesized using the region sandwiched between the primer set as a template, and an amplification product (PCR product) in which the baiCD gene fragment is amplified can be obtained.

[0032] The method for extracting DNA from the sample can be appropriately selected and used without any particular limitation, and examples thereof include the SDS phenol method and a method using a commercially available nucleic acid extraction and purification kit. In addition, the reaction conditions such as the type of complementary strand synthesizing enzyme in the PCR, the composition of the reaction solution, the reaction temperature, time, and the number of cycles can also be appropriately selected without any particular limitation, and a commercially available PCR kit or the like can be used. In the present invention, general PCR conditions can be adopted by using the above-mentioned primer set according to the present invention, and for example, the temperature of complementary strand synthesis, more preferably DNA elongation by DNA polymerase, can be in the range of 50 to 75°C, more preferably 52 to 68°C. In addition, the PCR can be, for example, multiplex PCR in which a primer set for a standard nucleic acid and a primer set capable of detecting 16S rRNA described below are used simultaneously.

[0033] [Detection process] The amplification product obtained in the amplification step can be detected by a known method or a method based thereon, as appropriate, to detect the amplified baiCD gene fragment, which uses the common region common to the baiCD genes as a template, thereby making it possible to detect the baiCD gene, i.e., to detect the presence of the baiCD gene, and further indirectly to detect bacteria carrying the baiCD gene in the sample.

[0034] As a detection method, for example, a fluorescent dye (e.g., ethidium bromide, Syber Green (registered trademark), SYTO63, etc.) can be intercalated into the obtained amplification product and the fluorescence intensity is measured to quantify the amplification product and obtain the amount of the baiCD gene.

[0035] The amplification step and the detection step may be carried out simultaneously (in real time). Examples of such real-time methods (real-time PCR) include, but are not limited to, a method in which PCR is carried out in the presence of a fluorescent dye, such as the intercalation method (the so-called Syber Green method), and a method in which an oligonucleotide probe bound to a fluorescent dye (hereinafter sometimes simply referred to as a "probe"), such as the double-dye probe method (the so-called TaqMan (registered trademark) probe method).

[0036] Other examples of the detection method include a DNA array method in which measurements are performed using a substrate on which an oligonucleotide probe that hybridizes to the baiCD gene fragment is fixed; high performance liquid chromatography-isotope dilution mass spectrometry (LC-IDMS); inductively coupled plasma optical emission spectrometry (ICP-OES); inductively coupled plasma mass spectrometry (ICP-MS); and atomic absorption spectrometry (AAS), which are used alone or in combination with other methods, but are not limited to these, and a conventionally known method or a method similar thereto can be appropriately used.

[0037] The probe for the detection method is preferably one that specifically hybridizes to the baiCD gene fragment under normal hybridization conditions, preferably under stringent hybridization conditions, and preferably satisfies the above-mentioned hybridization conditions, for example, and can be designed based on the base sequence information of the baiCD gene fragment by a conventionally known method or a method similar thereto.

[0038] The length of the probe in the detection method is at least 15 bases. It is usually 15 to 100 bases, and preferably 17 to 30 bases. The probe can be synthesized, for example, by a commercially available oligonucleotide synthesizer. The probe does not have to be composed of only natural nucleotides (deoxyribonucleotides and / or ribonucleotides), and may be composed, for example, of a part or the whole of the non-natural nucleotides.

[0039] Moreover, the probe is preferably labeled with a labeling substance as appropriate. In this way, a signal corresponding to the labeling substance can be detected as an indicator of the baiCD gene fragment. The "signal" includes color development (coloring), reflected light, luminescence, quenching, fluorescence, radiation from a radioisotope, and the like, and includes signals that can be confirmed by the naked eye as well as signals that can be confirmed by a measuring method or device according to the type of signal. Examples of the labeling substance include fluorescent substances such as FITC, FAM, DEAC, R6G, TexRed, Cy5, and BODIPY FL; enzymes such as β-D-glucosidase, luciferase, and HRP; 3 H, 14 C. 32 P, 35 S, 123 Examples of such substances include radioisotopes such as I; affinity substances such as biotin and streptavidin; and luminescent substances such as luminol, luciferin, and lucigenin.

[0040] [Correction process] The amount of the amplification product (i.e., the amplified baiCD gene fragment) detected in the detection process may be quantified based on a calibration curve prepared using standard nucleic acid, and the quantitative value may be taken as the amount corresponding to the amount of the baiCD gene (hereinafter simply referred to as "baiCD gene amount"); however, since samples such as stool contain organisms other than bacteria belonging to the Ruminococcaceae family and DNA derived from the subject (patient, etc.) from which the sample was collected, it is preferable to correct for this.

[0041] The correction method may be, for example, a method of correcting based on the total amount of DNA in the sample (e.g., baiCD gene amount (correction value) = amount of amplified product / total amount of DNA), but from the viewpoint of improving accuracy, it is preferable to further include a step of detecting 16S rRNA in the sample and to correct based on the amount of 16S rRNA in the sample. In this case, the correction can be made, for example, by the following formula: baiCD gene amount (correction value) = amount of amplified product / amount of 16S rRNA.

[0042] The method for detecting 16S rRNA in a sample is not particularly limited, and may be, for example, a method in which DNA extracted from the sample is used as a template, PCR is performed using a primer set capable of detecting bacterial 16S rRNA, and the amplified product is detected. The method for extracting DNA from the sample, the PCR method, and the method for detecting the amplified product are the same as those described in the amplification step and detection step, respectively.

[0043] The primer set capable of detecting the bacterial 16S rRNA includes a primer set designed for a highly conserved sequence of bacterial 16S rRNA, and is not particularly limited, for example, a primer set designed for a conserved region present between variable regions 1 to 9. Examples of such primer sets include, but are not limited to, the 16S-primer set described in the Examples (forward primer: 16S-forward primer (SEQ ID NO: 18); reverse primer: 16S-reverse primer (SEQ ID NO: 19)).

[0044] According to the baiCD gene detection method of the present invention, for example, by using a specimen such as feces collected from a diagnostic subject such as a human as the sample, it is possible to easily detect the presence or absence of the baiCD gene in the intestine of the subject, that is, to detect whether bacteria carrying the baiCD gene are present in the intestine, and, if present, to quantify the amount of bacteria. Therefore, the present invention can be effectively used for predicting the risk of diseases associated with bacteria carrying the baiCD gene, such as evaluating risk factors for diseases such as colorectal cancer and evaluating the severity of Clostridium infection in the subject, and for assisting in diagnosis and treatment.

[0045] <Kit> The present invention also provides a baiCD gene detection kit for use in the baiCD gene detection method of the present invention. The baiCD gene detection kit of the present invention is a kit comprising a primer set that is a combination of primers that hybridize to two mutually different consensus regions, and each of the consensus regions is a region in which 15 or more consecutive bases other than n are present in the base sequence described in SEQ ID NO: 1. In addition, it is also preferable that the baiCD gene detection kit of the present invention further comprises a primer set capable of detecting bacterial 16S rRNA.

[0046] The primer set, which is a combination of primers hybridizing to the consensus region, and the primer set capable of detecting bacterial 16S rRNA, including their preferred embodiments, are as described in the baiCD gene detection method of the present invention. In the baiCD gene detection kit of the present invention, each primer may contain other components such as a buffer solution, a stabilizer, a preservative, and an antiseptic.

[0047] In addition, the baiCD gene detection kit of the present invention may further include a diluent for the sample; reagents for extracting and purifying DNA; reagents such as enzymes, buffers, pH adjusters, etc. necessary for PCR; reagents necessary for the detection process; standard nucleic acids; instructions for use, etc. EXAMPLES

[0048] Hereinafter, the present invention will be described more specifically based on examples, but the present invention is not limited to the following examples. In each example, the display of "% " indicates weight / volume (w / v: g / mL) percent unless otherwise specified.

[0049] <Obtaining the sequence of the baiCD gene region> (1) Obtaining metagenomic data from a sample and extracting the baiCD gene region from the metagenomic data As samples, feces collected from 5 humans (Samples 1 to 5: about 3 mg each) were used. First, DNA was extracted from each sample using the "PureLink Microbiome DNA Purification kit" manufactured by Thermo Fisher Scientific (hereinafter, the DNA extracted from Samples 1 to 5 will be referred to as Sample1 to Sample5, respectively). Next, for the extracted DNA (Sample1 to Sample5), a DNA library for sequencing analysis was prepared using the "Nextera DNA Flex library prep kit" manufactured by Illumina.

[0050] For the prepared DNA library, whole-genome sequence information was obtained by shotgun sequencing analysis using "NextSeq" manufactured by Illumina. For the obtained sequence information, assembly analysis was performed using a genome assembler (in this example, "SPAdes") to create a draft genome sequence. Next, binning software (in this example, "MetaBAT2") was used for binning division to construct a bin sequence. Specifically, contig sequences considered to be derived from the same organism were grouped based on information such as base composition and data coverage. In addition, gene prediction software (in this example, "Prokka") was used to predict gene regions from the bin sequence.

[0051] Next, a homology search program (in this example, "Blast+") was used to perform a homology search against the predicted gene regions above, using the amino acid sequence of the baiCD gene registered under entry number AAF22846 in the Kyoto Encyclopedia of Genes and Genomes (KEGG), an integrated database of biological systems information that integrates genome, protein, and compound information through intermolecular interactions, reactions, and relationship networks, as a query, to identify the region corresponding to the baiCD gene in each bin sequence (baiCD gene region).

[0052] In addition, the amount of the baiCD gene region in Samples 1 to 5 was quantified by mapping and compiling the metagenomic data obtained for the baiCD gene region in each bin sequence. As a result, it was confirmed that Samples 3 to 5 all contained a large amount of the baiCD gene region, and that the specimens from which these were extracted contained many bacteria carrying the baiCD gene. On the other hand, Samples 1 to 2 contained almost no baiCD gene region, and that the specimens from which these were extracted contained almost no bacteria carrying the baiCD gene.

[0053] (2) Acquisition of public metagenomic data and extraction of the baiCD gene region from the metagenomic data First, the following reference: (Reference 1) Pasolli et al., Cell 176, January 24, 2019, p. 649-662 The 4,930 bin sequences of "The SGBs' representatives" described in were obtained from the University of Trento, Segata Lab Computational Metagenomics (http: / / segatalab.cibio.unitn.it / data / Pasolli_et_al.html, search date: November 30, 2020), and gene regions were predicted from the bin sequences using gene prediction software (in this example, "Prokka"). Next, a homology search was performed on the predicted gene regions using the amino acid sequence of the baiCD gene registered in KEGG with entry number AAF22846 as a query, and the baiCD gene region in each bin sequence was identified.

[0054] Next, from the identified baiCD gene region, the following species were identified among those shown in FIG. 1 of Non-Patent Document 2: GeversD_2014_SKBSTL008_bin.71 (hereinafter referred to as "Gevers_bin.71"), CosteaPI_2017_SID713A002-11-0-0_bin.25 (hereinafter referred to as "Costea_bin.25"), CosteaPI_2017_SID713B026-11-90-0_bin.59 (hereinafter sometimes referred to as "Costea_bin.59"), and CosteaPI_2017_SID713A023-11-0-0_bin.14 (hereinafter referred to as "Costea_bin.14") (hereinafter, these are collectively referred to as "A species"), and XieH_2016_YSZC12003_37181R1_bin.102, Obregon-TitoAJ_2015_SM11_bin.2, KarlssonFH_2013_S54_bin.27, FengQ_2015_SID31866_bin.65, XieH_2016_YSZC12003_35390_bin.5, LiJ_2014_MH0358_bin.56, RaymondF_2016_P21E7_bin.22, FengQ_2015_SID531416_bin.49, VogtmannE_2016MMRS15137911ST-27-0-0_bin.3, and FengQ_2015_SID31866_bin.46 (Hereinafter, these are collectively referred to as "Type B" in some cases) The baiCD gene region possessed by bacteria belonging to a different taxonomic group was extracted. The taxonomic group is the taxonomic group to which the species belonging to the Ruminococcaceae family and containing the baiCD gene and having the number (n_MAGs) of MAGs (metagenome-assembled genomes) of 100 or more in the data of studyA belong in FIG. 1 of Non-Patent Document 2.

[0055] (3) Extraction of baiCD gene region from CAG Regarding the genomic sequence (NCBI accession number: CAWL010000000) of the Firmicutes bacterium CAG:103 strain (hereinafter sometimes referred to as "CAG") registered in NCBI, gene prediction software (in this example, "Prokka") was used to predict gene regions from the genomic sequence. Next, a homology search was performed using the amino acid sequence of the baiCD gene registered in KEGG with entry number: AAF22846 as a query against the predicted gene regions to identify the baiCD gene region. Note that CAG belongs to the same taxonomic group as the taxonomic group containing the above four species of Type A described in FIG. 1 of Non-Patent Document 2.

[0056] <baiCD gene detection evaluation> (1) Design of common region and primers Using the sequences of the baiCD gene regions identified in (1) to (3) of the above <Acquisition of the Sequence of the baiCD Gene Region>, a multiple alignment program (in this example, "MAFFT") and a consensus sequence creation program (in this example, "cons") were used to obtain the nucleotide sequences common to all baiCD genes. The obtained nucleotide sequences (referred to as "common region" or "common region 1" in this specification) are shown in SEQ ID NO: 1 and FIGS. 1A to 1B.

[0057] Also, among the sequences of the baiCD gene regions identified from Costea_bin.59 (No. 1), the sequences of the baiCD gene regions identified from CAG (No. 2), the sequences of the baiCD gene regions identified from Sample3 (No. 3), the sequences of the baiCD gene regions identified from Costea_bin.25 (No. 4), the sequences of the baiCD gene regions identified from Gevers_bin.71 (No. 5), the sequences of the baiCD gene regions identified from Sample5 (No. 6), the sequences of the baiCD gene regions identified from Costea_bin.14 (No. 7), and the sequences of the baiCD gene regions identified from Sample4 (No. 8), the sequences included in the common region are shown in FIGS. 2A to 2C. The nucleotide sequence common to the nucleotide sequences from positions 1 to 1683 of the No. 1 to 8 sequences shown in FIGS. 2A to 2C (common region 2) is represented by SEQ ID NO: 20. Note that there are portions where misalignments occur due to different alignment algorithms between the alignment of the No. 1 to 8 sequences shown in FIGS. 2A to 2C and the sequence shown in SEQ ID NO: 1 or FIGS. 1A to 1B.

[0058] Next, primer sets shown in Table 1 below were designed and synthesized for the nucleotide sequence shown in SEQ ID NO: 1. Table 1 also shows the regions (consensus regions) where the primers were designed and the SEQ ID numbers of the nucleotide sequences. Such consensus regions are underlined (or, in the overlapping portions of two consensus regions, underlined and overlined) in the sequences of FIGS. 1A to 1B and the No. 1 sequence in FIGS. 2A to 2C. The numbers attached to the underlines or overlines in FIGS. 1A to 1B correspond to the numbers of the consensus regions shown in Table 1.

[0059]

Table 1

[0060] (2) Primer Evaluation (qPCR) Using the DNA (Sample1 - 5) extracted from each sample in (1) of <Obtaining the Sequence of the <baiCD Gene Region>> as the template DNA, qPCR was performed to evaluate the primer set designed in (1) of <Evaluation of <baiCD Gene Detection>>. As the DNA polymerase, "NEBNext (R) Q5 (R) Hot Start HiFi PCR Master Mix" (manufactured by New England Biolabs, M0543S) was used. The composition of the reaction solution (total volume: 10 μL) and the PCR program were as follows, and PCR was performed using a "Veriti Thermal Cycler" (manufactured by Thermo fisher Scientific). (Composition of the Reaction Solution) DNA polymerase 5 μL (final concentration: 1×) 10 μM forward primer 0.5 μL (final concentration: 0.5 μM) 10 μM reverse primer 0.5 μL (final concentration: 0.5 μM) 1 ng / μL template DNA 1 μL (final concentration: 1 ng / reaction) Water 3 μL (PCR Program) 98℃ 30 sec 30 cycles: 98℃ 10 sec 68℃ 10 sec 72℃ 10 sec 4℃ ∞.

[0061] The reaction solution after PCR was subjected to electrophoresis using "4200 TapeStation" (ScreenTape: D1000 (5067-5582); manufactured by Agilent Technologies). The obtained electrophoretic photographs are shown in FIG. 3A (primer sets 1 to 3: set 1 to 3) and FIG. 3B (primer sets 4 to 5: set 4 to 5). As shown in FIG. 3A to FIG. 3B, primer dimers were observed when primer sets 1 and 2 were used, and extra bands were observed when primer set 4 was used. However, it was confirmed that, in any primer set, when Samples 1 to 2 (without baiCD gene region) were used, fragments of the target size were not amplified, whereas when Samples 3 to 5 (with baiCD gene region) were used, fragments of the target size (set 1: 241 bp, set 2: 242 bp, set 3: 205 bp, set 4: 207 bp, set 5: 156 bp) were amplified.

[0062] (3) Preparation of qPCR standards PCR was performed in the same manner as in (2) above, except that Sample 4 (containing the baiCD gene region, for baiCD gene quantification) or DNA extracted from Bacteroides ovatus ATCC8483 (for 16S rRNA quantification) was used as template DNA, primer sets 1, 2, 3, and 5 were used, and the reaction solution composition was as follows in a total volume of 100 μL. (Composition of reaction solution) DNA polymerase 50 μL (final concentration: 1x) 10μM forward primer 5μL (final concentration: 0.5μM) 10μM reverse primer 5μL (final concentration: 0.5μM) 1ng / μL template DNA 1μL (final concentration: 1ng / reaction) 39μL of water.

[0063] For the reaction solution after PCR, electrophoresis was performed using "Mupid-exU" (Advance), 1×TAE buffer, and 2% agarose gel. After electrophoresis, the band of the target size was cut out from the gel and purified to prepare a standard for qPCR. "100bp ladder" (manufactured by TOYOBO, DNA-035) was used as the DNA size marker, "Midori green direct" (manufactured by Nippon Genetics Co., Ltd., NE-MG06) was used for DNA staining, "ImageQuant LAS 500" (manufactured by GE healthcare) was used for photographing the gel after electrophoresis, and "NucleoSpin Gel and PCR Clean-up" (manufactured by Macherey-Nagel, 740609.10) was used for cutting out and purifying the band, respectively.

[0064] (4) qPCR Using primer sets 1, 2, 3, 5, and the 16S-primer set (forward primer: 16S-forward primer (SEQ ID NO: 18); reverse primer: 16S-reverse primer (SEQ ID NO: 19)) designed for the highly conserved sequence of bacterial 16S rRNA, qPCR was performed using the DNA (Sample1 - 5) extracted from each sample in (1) of the above <Acquisition of the baiCD gene region sequence> as the template DNA. The composition of the reaction solution (total volume: 10 μL) and the PCR program were as follows, and qPCR was performed using "Stratagene Mx3005P" (manufactured by Agilent technologies). (Composition of the reaction solution) KOD SYBR (R) qPCR Mix (manufactured by TOYOBO, QKD-201) 5 μL (final concentration: 1×) 10 μM forward primer 0.2 μL (final concentration: 0.2 μM) 10 μM reverse primer 0.2 μL (final concentration: 0.2 μM) 50×ROX Passive Reference (manufactured by TOYOBO, KOD SYBR (R) qPCR Mix accessory) 0.02 μL (final concentration: 1×) 1 μL of 1 ng / μL template DNA (final concentration: 1 ng / reaction) 3.58 μL of water (PCR program) 98°C for 2 min 40 cycles: 98°C for 10 sec 68°C for 30 sec (Data collection) Melting curve: 95°C for 15 sec 55°C for 60 sec (Data collection) 95°C for 15 sec.

[0065] Furthermore, qPCR was performed in the same manner as above, except that each concentration of qPCR standard prepared in (3) above was used as the template DNA, a calibration curve for the DNA amount (the amount of the baiCD gene region or the amount of 16S rRNA) was created, and for the results of each qPCR, the value obtained using the calibration curve was taken as the baiCD gene amount or 16S rRNA amount.

[0066] Using primer sets 1, 2, 3, 5, with Sample1 - 5 as template DNA, the baiCD gene amounts obtained by performing the above qPCR were corrected to the amount per each template DNA amount (total DNA amount) (baiCD gene amount / total DNA amount). For Sample1 - 5, the results of correcting the baiCD gene amounts obtained using primer sets 1, 2, 3, 5 (Set 1, 2, 3, 5) by the total DNA amount are shown in Figure 4. Figure 4 shows the relative values (vertical axis) with the values obtained when each primer set was used with Sample3 as the template DNA set to 1. Also shown is the amount of the baiCD gene region (Shotgun) quantified from the metagenomic data in (1) of the above <Acquisition of the sequence of the baiCD gene region>.

[0067] Also, using primer sets 1, 2, 3, and 5, with Samples 1 to 5 as template DNAs, the amount of baiCD gene obtained by performing the above qPCR was corrected to the amount per 16S rRNA amount obtained using the 16S - primer set (baiCD gene amount / 16S rRNA amount). For Samples 1 to 5, the results of correcting the baiCD gene amount obtained using primer sets 1, 2, 3, and 5 (Set 1, 2, 3, 5) with the 16S rRNA amount are shown in Fig. 5. Fig. 5 shows the relative values (vertical axis) with the value when each primer set was used with Sample 3 as the template DNA set to 1. Also shown is the amount of the baiCD gene region quantified from metagenomic data (Shotgun) in (1) of the above <Acquisition of the sequence of the baiCD gene region>.

[0068] As shown in Figs. 4 and 5, for any primer set, when Samples 1 to 2 (without the baiCD gene region) were used, almost no amplification of the baiCD gene region was confirmed, whereas when Samples 3 to 5 (with the baiCD gene region) were used, amplification of the baiCD gene region was confirmed, and it was confirmed that the baiCD gene region could be detected equivalently to the quantification result from metagenomic data. Furthermore, by correcting the baiCD gene amount to the amount per 16S rRNA amount obtained using the 16S - primer set, it was confirmed that a result closer to the quantification result from metagenomic data could be obtained.

Industrial Applicability

[0069] According to the present invention, it becomes possible to provide a method for detecting the baiCD gene, which can simply detect the baiCD gene with the same accuracy as metagenomic analysis by PCR even for bacteria belonging to the family Ruminococcaceae, whose existence has hitherto been confirmed only by metagenomic analysis, and a kit for detecting the baiCD gene used therefor.

Sequence Listing Free - Text

[0070] Sequence number: 1 <223> common area 1 <223> n is a, c, g, t, or deletion SEQ ID NO:2 <223> Consensus region 1 SEQ ID NO:3 <223> Consensus region 2 SEQ ID NO:4 <223> Consensus Region 3 SEQ ID NO:5 <223> Consensus region 4 SEQ ID NO:6 <223> Consensus Region 5 SEQ ID NO:7 <223> Consensus Region 6 SEQ ID NO:8 <223> Consensus Region 7 SEQ ID NO:9 <223> Consensus Region 8 SEQ ID NO:10 <223> Forward Primer I SEQ ID NO:11 <223> Reverse primer I SEQ ID NO:12 <223> Forward Primer II SEQ ID NO:13 <223> Forward Primer III SEQ ID NO:14 <223> Reverse primer III SEQ ID NO:15 <223> Forward Primer IV SEQ ID NO:16 <223> Forward Primer V SEQ ID NO:17 <223> Reverse primer V SEQ ID NO:18 <223> 16S-forward primer SEQ ID NO:19 <223> 16S-reverse primer SEQ ID NO:20 <223> Common area 2 <223> n is a, c, g, or t

Claims

1. A method for detecting the baiCD gene of bacteria belonging to the family Ruminococcus in a sample, The method includes an amplification step of amplifying a baiCD gene fragment using a primer set that is a combination of primers that hybridize to two mutually different consensus regions, the consensus region is a region in which 15 or more consecutive bases other than n are present in the base sequence set forth in SEQ ID NO: 1, The primer set is A primer set III which is a combination of a forward primer III consisting of the base sequence set forth in SEQ ID NO: 13 and a reverse primer III consisting of the base sequence set forth in SEQ ID NO: 14; and A primer set V is a combination of a forward primer V having a base sequence as set forth in SEQ ID NO: 16 and a reverse primer V having a base sequence as set forth in SEQ ID NO:

17. Any one selected from the group consisting of: baiCD gene detection method.

2. detecting the amplification product obtained in the amplification step; detecting 16S rRNA in the sample; and A correction step of correcting the amount of the detected amplification product with the amount of the detected 16S rRNA; The method for detecting the baiCD gene according to claim 1, further comprising:

3. A kit for use in the baiCD gene detection method according to claim 1 or 2, A primer set is provided which is a combination of primers that hybridize to two mutually different consensus regions, the consensus region is a region in which 15 or more consecutive bases other than n are present in the base sequence set forth in SEQ ID NO: 1, The primer set is A primer set III which is a combination of a forward primer III consisting of the base sequence set forth in SEQ ID NO: 13 and a reverse primer III consisting of the base sequence set forth in SEQ ID NO: 14; and A primer set V is a combination of a forward primer V having a base sequence as set forth in SEQ ID NO: 16 and a reverse primer V having a base sequence as set forth in SEQ ID NO:

17. Any one selected from the group consisting of: baiCD gene detection kit.

4. The baiCD gene detection kit according to claim 3, further comprising a primer set capable of detecting bacterial 16S rRNA.

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