JP7637523B2 - baiCD gene detection method and baiCD gene detection kit - Google Patents

Description

The present invention relates to a method for detecting the baiCD gene and a baiCD gene detection kit used therein.

Primary bile acids, which are steroid compounds biosynthesized in the liver, are secreted into bile in a conjugated form (e.g., cholic acid conjugated with taurine or glycine, chenodeoxycholic acid), but some of them are converted in the large intestine to secondary bile acids (e.g., deoxycholic acid and lithocholic acid) by the 7-α-dehydroxylation activity (enzyme 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 the bacteria. The baiCD gene is one of the genes that make up this bai operon gene group.

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 colorectal cancer (Non-Patent Documents 1 and 2, etc.). Therefore, from the perspective of evaluating risk factors for diseases such as colorectal 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.

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 infection (CDI: Clostridium difficile infection) (Non-Patent Documents 3, 4, etc.). For example, Patent Document 1 describes a method of administering a bacterial strain expressing the bai operon gene group to a subject to reduce the risk of Clostridium infection. 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.

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. To analyze the extent to which bacteria carrying the bai operon gene group are contained in the intestinal flora, shotgun metagenomic analysis can be suitably used. 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, large amounts of information, and expensive equipment.

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 family Ruminococcus (Ruminococcaceae) described in Non-Patent Document 2.

US Patent Application No. 2019/0381113

Anna Dart, Nature Reviews, Microbiology, Vol. 16, August 2018, p. 453 Marius Vital et al., Computational and Structural Biotechnology Journal, 17, 2019, p. 1016-1019 Jenessa A. Winston et al., Anaerobe. , 2016 October, 41, p. 44-50 Philipp Solbach et al., PLOS ONE, May 8, 2018, https://doi. org/10.1371/journal. bone. 0196977

The present invention has been made in consideration of the above 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 could previously only be confirmed by metagenomic analysis, such as some bacteria belonging to the family Ruminococcaceae, and a baiCD gene detection kit used therefor.

The present inventors first performed metagenomic analysis on DNA extracted from stool samples, and identified the baiCD gene region by combining public metagenomic data with data on bacteria belonging to the family of Ruminococci and a phylogenetic group containing species with a large amount of baiCD genes in the phylogenetic tree described in Figure 1 of Non-Patent Document 2; and data on a strain with known genome information (Firmicutes bacterium CAG: 103 strain). Next, the sequences between the obtained baiCD gene regions were analyzed, and a common region with conserved sequences between them was found. By designing a primer for a specific region within the 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 of Ruminococci (Ruminococcaceae), and thus completed the present invention.

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]
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 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 a 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 is a combination of a forward primer I having a total of 20 to 25 bases including the base sequence set forth in SEQ ID NO: 10 and a reverse primer I having a total of 20 to 25 bases including the base sequence set forth in SEQ ID NO: 11;
A primer set II is a combination of a forward primer II having a total of 20 to 25 bases including the base sequence set forth in SEQ ID NO: 12 and a reverse primer I having a total of 20 to 25 bases including 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;
Primer set IV is a combination of forward primer IV having a total of 20 to 25 bases including the base sequence set forth in SEQ ID NO: 15 and reverse primer III having a total of 20 to 25 bases including the base sequence set forth in SEQ ID NO: 14; and primer set V is a combination of forward primer V having a total of 20 to 25 bases including the base sequence set forth in SEQ ID NO: 16 and 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:
The method for detecting the baiCD gene according to [1] or [2].
[4]
detecting the amplification product obtained in the amplification step;
A step of detecting 16S rRNA in the sample; and a correction step of correcting the amount of the detected amplification product by 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 according to [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 is a combination of a forward primer I having a total of 20 to 25 bases including the base sequence set forth in SEQ ID NO: 10 and a reverse primer I having a total of 20 to 25 bases including the base sequence set forth in SEQ ID NO: 11;
A primer set II is a combination of a forward primer II having a total of 20 to 25 bases including the base sequence set forth in SEQ ID NO: 12 and a reverse primer I having a total of 20 to 25 bases including 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;
Primer set IV is a combination of forward primer IV having a total of 20 to 25 bases including the base sequence set forth in SEQ ID NO: 15 and reverse primer III having a total of 20 to 25 bases including the base sequence set forth in SEQ ID NO: 14; and primer set V is a combination of forward primer V having a total of 20 to 25 bases including the base sequence set forth in SEQ ID NO: 16 and 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]
The baiCD gene detection kit according to any one of [5] to [7], further comprising a primer set capable of detecting bacterial 16S rRNA.

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 family Ruminococcaceae, whose existence could previously only be confirmed by metagenomic analysis, and a baiCD gene detection kit used therefor.

FIG. 1 shows the common region and preferred consensus regions 1 to 8 set forth in SEQ ID NO: 1 (bases 1 to 960 of common region 1). 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). FIG. 1 shows sequences contained in the common region among Nos. 1 to 8 of the baiCD gene region. FIG. 1 shows sequences contained in the common region among Nos. 1 to 8 of the baiCD gene region. FIG. 1 shows sequences contained in the common region among Nos. 1 to 8 of the baiCD gene region. Electrophoretic photographs obtained in the evaluation of primers in the baiCD gene detection evaluation (primer sets 1 to 3). Electrophoretic photographs obtained in the evaluation of primers for baiCD gene detection evaluation (primer sets 4 to 5). 1 is a graph showing the results of the baiCD gene amounts obtained using primer sets 1, 2, 3, and 5 corrected by the total DNA amount. 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.

The present invention will be described in detail below with reference to preferred embodiments.

<Method for detecting baiCD gene>
The baiCD gene detection method of the present invention is a method for detecting the baiCD gene of a bacterium belonging to the family Ruminococcus (Ruminococcaceae) in a sample, and 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, 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 the present invention, the detection of the baiCD gene also includes quantification or semi-quantification of the baiCD gene.

[sample]
The "sample" to be subjected to the baiCD gene detection method of the present invention is not particularly limited as long as it is a sample in which bacteria capable of carrying the baiCD gene or its DNA may be present. In general, examples of the sample include urine, stool, oral mucosa, pharyngeal mucosa, intestinal mucosa, and various biopsy samples collected from a subject (preferably a human) for which the baiCD gene is to be detected, such as a diagnostic subject. The sample according to the present invention is preferably stool. The sample may be appropriately diluted or suspended in a diluent, pH adjusted, or treated by pulverization, freezing, or the like. Examples of the diluent include buffer solutions such as phosphate buffer, Tris buffer, Good's buffer, and borate buffer.

[Common Area]
According to the present invention, by designing a primer within the common region discovered by the present inventors, in addition to bacteria that have traditionally been cultured alone, the baiCD gene of bacteria belonging to the family Ruminococcaeae, the presence of which has traditionally only been confirmed by metagenomic analysis, can be easily detected by PCR with the same accuracy as metagenomic analysis. Specifically, in Non-Patent Document 2, "bacteria belonging to the family Ruminococcus whose existence has been confirmed only by metagenomic analysis" are bacteria belonging to the family Ruminococcus (Ruminococcaceae) in Figure 1 of the same document, and 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 (metagome-assembled genomes) containing baiCD genes (n_MAGs) of 100 or more in the data of Study A. Whether or not a target bacterium belongs to the above phylogenetic group can be determined by obtaining the bin sequence of the bacterium, combining it with all the bacteria listed in Figure 1 of Non-Patent Document 2 to create a phylogenetic tree by the method described in the same document, and finding that 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 not being in the same phylogenetic group as all of the above.

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, and in particular, is common 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.

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" indicates a, c, g, or t, or a deletion of a base (1 base). Details of SEQ ID NO: 1 are shown in Figures 1A to 1B. In Figures 1A to 1B, "n" indicates A, C, G, or T, or a deletion of a base (1 base), and "N" indicates 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" indicates a, c, g, or t.

[Consensus Areas]
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 which 15 or more consecutive bases other than n are present in the base sequence described in SEQ ID NO: 1, more preferably a region in which 15 or more consecutive bases other than n are present in the base sequence described in SEQ ID NO: 20, 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.

Such a consensus region is not particularly limited, but is preferably a region selected from the group consisting of 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 and overlined numbers correspond to the numbers of the consensus regions.

As these consensus regions, two mutually different regions can be appropriately selected, but a preferred combination, from the viewpoint of detection accuracy by PCR, is one in which the number of bases in the PCR amplification 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 at a distance of 50 bases or more, more preferably 50 to 300 bases, and even more preferably 50 to 250 bases, from the viewpoint of detection accuracy by PCR.

Such preferred combinations include the combination of consensus region 1 and consensus region 2, the combination of consensus region 3 and consensus region 2, the combination of consensus region 4 and consensus region 5, the combination of consensus region 6 and consensus region 5, and the 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.

[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 consensus regions.

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.

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 condition for such hybridization is preferably that the sequence complementarity between 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). Note that 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 condition for hybridization may be that the primer has a base sequence in which 1 to 5 consecutive bases (preferably 1 to 3 bases, e.g., 3 bases, 2 bases, or 1 base) are inserted, deleted, or substituted at one or more positions of a base sequence that is completely complementary to the entire length of the base sequence of the consensus region.

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.

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 in part or in whole 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).

More specifically, such a primer set includes:
A primer set I is a combination of a forward primer I having a total of 20 to 25 bases including the base sequence set forth in SEQ ID NO: 10 and a reverse primer I having a total of 20 to 25 bases including the base sequence set forth in SEQ ID NO: 11;
A primer set II is a combination of a forward primer II having a total of 20 to 25 bases including the base sequence set forth in SEQ ID NO: 12 and a reverse primer I having a total of 20 to 25 bases including 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;
Primer set IV is a combination of forward primer IV having a total of 20 to 25 bases including the base sequence set forth in SEQ ID NO: 15 and reverse primer III having a total of 20 to 25 bases including the base sequence set forth in SEQ ID NO: 14; and primer set V is a combination of forward primer V having a total of 20 to 25 bases including the base sequence set forth in SEQ ID NO: 16 and 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.

[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 Ruminococcaceae family, the existence of which has only been confirmed by metagenomic analysis in the past.

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.

The method for extracting DNA from the sample can be appropriately selected from known methods without particular limitations, and examples of such methods 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 synthesis enzyme in the PCR, the composition of the reaction solution, the reaction temperature, time, and number of cycles, can also be appropriately selected without particular limitations, 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 for 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.

[Detection step]
The amplification product obtained in the amplification step can be detected by a known method or a method similar thereto, whereby the amplified baiCD gene fragment is detected, and since the baiCD gene fragment uses the common region common to the baiCD genes as a template, it is 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.

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, which can be used as the amount of the baiCD gene.

The amplification step and the detection step may be performed 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 performed in the presence of a fluorescent dye, such as the intercalation method (also known as the 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 (also known as the TaqMan (registered trademark) probe method).

Other examples of the detection method include a DNA array method using a substrate on which an oligonucleotide probe that hybridizes to the baiCD gene fragment is immobilized; 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 may be used alone or in combination with other methods. However, the detection method is not limited to these, and a conventionally known method or a method similar thereto may be appropriately used.

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 hybridization conditions, and can be designed by a conventionally known method or a method similar thereto, based on the base sequence information of the baiCD gene fragment.

The length of the probe used 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 only of natural nucleotides (deoxyribonucleotides and/or ribonucleotides), and may be composed, for example, in part or in whole of the non-natural nucleotides.

In addition, the probe is preferably labeled with a labeling substance as appropriate. This allows a signal corresponding to the labeling substance to be detected as an indicator of the baiCD gene fragment. The "signal" includes coloration (color development), reflected light, luminescence, quenching, fluorescence, radiation from a radioisotope, and the like, and includes signals that can be confirmed by a measurement method or device according to the type of signal as well as signals that can be confirmed by the naked eye. 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; radioisotopes such as ³ H, ¹⁴ C, ³² P, ³⁵ S, and ¹²³ I; affinity substances such as biotin and streptavidin; and luminescent substances such as luminol, luciferin, and lucigenin.

[Correction process]
The amount of the amplification product (i.e., the amplified baiCD gene fragment) detected in the detection step may be quantified based on a calibration curve prepared using a standard nucleic acid, and the quantified value may be taken as the amount corresponding to the amount of the baiCD gene (hereinafter simply referred to as the "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.

The correction method may be, for example, a method in which correction is made 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 make correction based on the amount of 16S rRNA in the same sample. In this case, correction can be made, for example, by the following formula: baiCD gene amount (correction value) = amount of amplified product/amount of 16S rRNA.

The method for detecting 16S rRNA in a sample is not particularly limited, but an example of such a method is to use DNA extracted from the sample as a template, perform PCR using a primer set capable of detecting bacterial 16S rRNA, and detect the amplified product. 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.

The primer set capable of detecting the bacterial 16S rRNA includes a primer set designed for a highly conserved sequence of the bacterial 16S rRNA, and is not particularly limited to 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)).

According to the baiCD gene detection method of the present invention, by using a specimen such as stool 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 to predict 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 to assist in diagnosis and treatment.

<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 which is a combination of primers hybridizing to two different consensus regions, each of which is a region in which 15 or more bases other than n are consecutive 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.

The primer set, which is a combination of primers that hybridize 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.

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, and pH adjusters required for PCR; reagents required for the detection process; standard nucleic acids; and instructions for use.

The present invention will be described in more detail below with reference to examples, but the present invention is not limited to the following examples. In each example, "%" indicates weight/volume (w/v: g/mL) percentage unless otherwise specified.

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

The prepared DNA library was subjected to shotgun sequencing analysis using Illumina's "NextSeq" to obtain whole genome sequence information. The obtained sequence information was subjected to assembly analysis using a genome assembler (in this example, "SPAdes") to create a draft genome sequence. Next, binning division was performed using binning software (in this example, "MetaBAT2") to construct bin sequences. Specifically, contig sequences thought to be derived from the same organism were grouped based on base composition and data coverage information. In addition, gene prediction software (in this example, "Prokka") was used to predict gene regions from the bin sequences.

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

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 a large amount of bacteria carrying the baiCD gene. On the other hand, Samples 1 and 2 contained almost no baiCD gene region, and that the specimens from which these were extracted contained almost no bacteria carrying the baiCD gene.

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

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 sometimes referred to as "Gevers_bin.71"),
CosteaPI_2017_SID713A002-11-0-0_bin.25 (hereinafter sometimes 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, sometimes 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 sometimes collectively referred to as "Type B"), the baiCD gene regions of bacteria belonging to a different phylogenetic group were extracted. The phylogenetic group is a phylogenetic group including species that belong to the family Ruminococcus (Ruminococcaceae) and have a number of MAGs (metagome-assembled genomes) (n_MAGs) containing the baiCD gene of 100 or more in the data of Study A in FIG. 1 of Non-Patent Document 2.

(3) Extraction of the baiCD gene region from CAG The genome sequence (NCBI accession number: CAWL010000000) of Firmicutes bacterium CAG:103 strain (hereinafter sometimes referred to as "CAG") registered in NCBI was used to predict the gene region from the genome sequence using gene prediction software (in this example, "Prokka"). Next, a homology search was performed on the predicted gene region using the amino acid sequence of the baiCD gene registered in KEGG under entry number: AAF22846 as a query, and the baiCD gene region was identified. Note that CAG belongs to the same phylogenetic group as the phylogenetic group containing the above four species of species A described in Figure 1 of Non-Patent Document 2.

<BaiCD gene detection evaluation>
(1) Design of common region and primers Using the sequence of the baiCD gene region identified in (1) to (3) of <Obtaining the sequence of the baiCD gene region> above, a base sequence common to all baiCD genes was obtained using a multiple alignment program (in this example, "MAFFT") and a consensus sequence creation program (in this example, "cons"). The obtained base sequence (referred to as the "common region" or "common region 1" in this specification) is shown in SEQ ID NO: 1 and Figures 1A to 1B.

In addition, among the sequence of the baiCD gene region identified from Costea_bin. 59 (No. 1), the sequence of the baiCD gene region identified from CAG (No. 2), the sequence of the baiCD gene region identified from Sample 3 (No. 3), the sequence of the baiCD gene region identified from Costea_bin. 25 (No. 4), the sequence of the baiCD gene region identified from Gevers_bin. 71 (No. 5), the sequence of the baiCD gene region identified from Sample 5 (No. 6), the sequence of the baiCD gene region identified from Costea_bin. 14 (No. 7), and the sequence of the baiCD gene region identified from Sample 4 (No. 8), the sequences contained in the common region are shown in Figures 2A to 2C. No. The base sequence common to the base sequences from base 1 to 1683 of sequences No. 1 to 8 (common region 2) is shown in SEQ ID NO: 20. Note that there are some discrepancies between the alignment of sequences No. 1 to 8 shown in Figures 2A to 2C and the sequences shown in SEQ ID NO: 1 or Figures 1A to 1B due to different alignment algorithms.

Next, a primer set shown in Table 1 below was designed and synthesized for the base sequence shown in SEQ ID NO:1. Table 1 also shows the region for which the primers were designed (consensus region) and the sequence number showing the base sequence. Such consensus regions are underlined (or underlined and overlined where two consensus regions overlap) in the sequences of Figures 1A to 1B and No. 1 in Figures 2A to 2C. The underlined or overlined numbers in Figures 1A to 1B correspond to the numbers of the consensus regions shown in Table 1.

(2) Primer Evaluation (qPCR)
qPCR was performed using the DNA (Samples 1 to 5) extracted from each sample in (1) of <Acquisition of the sequence of the baiCD gene region> as template DNA, and the primer set designed in (1) of <Evaluation of baiCD gene detection> was evaluated. For DNA polymerase, "NEBNext ^(R) Q5 ^(R) Hot Start HiFi PCR Master Mix" (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 "Veriti Thermal Cycler" (Thermo Fisher Scientific).
(Composition of reaction solution)
DNA polymerase 5 μL (final concentration: 1x)
10 μM forward primer 0.5 μL (final concentration: 0.5 μM)
10 μM reverse primer 0.5 μL (final concentration: 0.5 μM)
1ng/μL template DNA 1μL (final concentration: 1ng/reaction)
Water 3 μL
(PCR program)
98℃ 30sec
30 cycles:
98℃ 10sec
68℃ 10sec
72℃ 10sec
4℃∞.

The reaction solution after PCR was subjected to electrophoresis using a "4200 TapeStation" (ScreenTape: D1000 (5067-5582); Agilent Technologies). The electrophoretic images obtained are shown in Figure 3A (primer sets 1-3: set 1-3) and Figure 3B (primer sets 4-5: set 4-5). As shown in Figures 3A and 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, with either primer set, fragments of the desired size were not amplified when Samples 1 and 2 (without the baiCD gene region) were used, whereas fragments of the desired size (set 1: 241 bp, set 2: 242 bp, set 3: 205 bp, set 4: 207 bp, set 5: 156 bp) were amplified when Samples 3 to 5 (with the baiCD gene region) were used.

(3) Preparation of qPCR standard 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, and primer sets 1, 2, 3, and 5 were used. PCR was performed in the same manner as in (2) above, except that the reaction solution had the following composition in a total volume of 100 μL.
(Composition of reaction solution)
DNA polymerase 50 μL (final concentration: 1x)
10 μM forward primer 5 μL (final concentration: 0.5 μM)
10 μM reverse primer 5 μL (final concentration: 0.5 μM)
1ng/μL template DNA 1μL (final concentration: 1ng/reaction)
Water 39 μL.

The reaction solution after PCR was subjected to electrophoresis using "Mupid-exU" (Advance), 1x TAE buffer, and 2% agarose gel, and bands of the desired size were excised and purified from the gel after electrophoresis to prepare standards for qPCR. The DNA size marker used was the 100 bp ladder (TOYOBO, DNA-035), the DNA was stained with Midori green direct (Nippon Genetics, NE-MG06), the gel was photographed after electrophoresis with ImageQuant LAS 500 (GE healthcare), and the bands were excised and purified with NucleoSpin Gel and PCR Clean-up (Macherey-Nagel, 740609.10).

(4) qPCR
Using primer sets 1, 2, 3, and 5, as well as a 16S-primer set (forward primer: 16S-forward primer (SEQ ID NO: 18); reverse primer: 16S-reverse primer (SEQ ID NO: 19)) designed for a highly conserved sequence of bacterial 16S rRNA, qPCR was performed using the DNA (Samples 1 to 5) extracted from each specimen in (1) of <Acquisition of the sequence of the baiCD gene region> as 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 reaction solution)
KOD SYBR ^(R) qPCR Mix (manufactured by TOYOBO, QKD-201)
5μL (final concentration: 1x)
10 μM forward primer 0.2 μL (final concentration: 0.2 μM)
10 μM reverse primer 0.2 μL (final concentration: 0.2 μM)
50x ROX Passive Reference (TOYOBO, KOD ^SYBR® qPCR Mix accessory)
0.02μL (final concentration: 1x)
1ng/μL template DNA 1μL (final concentration: 1ng/reaction)
Water 3.58 μL
(PCR program)
98℃ 2min
40 cycles:
98℃ 10sec
68℃ 30sec
(Data collection)
Melting curve:
95℃ 15sec
55℃ 60sec
(Data collection)
95°C 15sec.

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

The baiCD gene amount obtained by performing the above qPCR using primer sets 1, 2, 3, and 5 and Samples 1 to 5 as template DNA was corrected to the amount (baiCD gene amount/total DNA amount) per amount of template DNA (total DNA amount). Figure 4 shows the results of correcting the baiCD gene amount obtained using primer sets 1, 2, 3, and 5 (Sets 1, 2, 3, and 5) for Samples 1 to 5 by the total DNA amount. Figure 4 shows the relative values (vertical axis) with the value when Sample 3 was used as template DNA and each primer set was set to 1. The amount (Shotgun) of the baiCD gene region quantified from the metagenomic data in (1) of <Acquisition of the sequence of the baiCD gene region> above is also shown.

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

As shown in Figures 4 and 5, for both primer sets, 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 at a level equivalent to the quantitative results from the metagenomic data. Furthermore, it was confirmed that by correcting the amount of the baiCD gene to the amount per 16S rRNA amount obtained using the 16S-primer set, results closer to the quantitative results from the metagenomic data could be obtained.

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 family Ruminococcaceae, whose existence could previously only be confirmed by metagenomic analysis, and a baiCD gene detection kit used therefor.

SEQ ID NO: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 (4)

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. detecting the amplification product obtained in the amplification step;
A step of detecting 16S rRNA in the sample; and a correction step of correcting the amount of the detected amplification product by the amount of the detected 16S rRNA.
The method for detecting the baiCD gene according to claim 1 , further comprising: 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. The baiCD gene detection kit according to claim 3 , further comprising a primer set capable of detecting bacterial 16S rRNA.