JPWO2014142186A1 - Bifidobacterium breve strain specific gene - Google Patents

Abstract

A gene specific to Bifidobacterium breve YIT 12272 and a method for using the same. A gene for specifically detecting the Bifidobacterium breve YIT 12272 strain, which is DNA consisting of the base sequence represented by SEQ ID NOs: 1-145 or DNA complementary to the sequence.

Description

  The present invention relates to a gene specific for Bifidobacterium breve YIT12272 and a method for using the same.

  Bifidobacterium is a major bacterium in the intestinal flora of humans, and is beneficial to human health such as rectification such as constipation and diarrhea improvement, serum cholesterol elevation suppression, and immunostimulation. It is known to have an effect. For this reason, many commercially available products are sold in the form of various fermented foods and drinks and viable bacterial preparations, and in particular, fermented milk foods and drinks have excellent palatability. Suitable for continuous intake.

  Bifidobacterium is an obligate anaerobic bacterium that is weak against oxygen, low pH, and high acidity, and in fermented foods and drinks, there are many points that are difficult to handle, such as growth during production and survival during storage. . In order to obtain the physiological effects of Bifidobacterium, it is thought that as many bacteria as possible need to reach the intestines alive, especially the survival of bacteria in food and drink, ie after food and drink Increasing the rate of reaching the intestines is an important factor.

  In order to solve such a problem, a Bifidobacterium bacterium having a high survivability is produced even under aerobic conditions, low pH, and high acidity conditions. Examples of such strains include Bifidobacterium breve YIT 10001 strain (FERM BP-8205) (patent document 1), Bifidobacterium breve SBR 3212 strain (FERM P-11915) (patent document 2), Bifidobacterium bifidum YIT 4002 strain (FERM BP-1038) (patent document 3), Bifidobacterium breve YIT 12272 strain (FERM BP-11320) (patent document 4) and the like can be mentioned. The Umbreve YIT 12272 strain is highly versatile and has high survivability and has enhanced artificial gastric juice resistance and artificial bile / intestinal fluid resistance, and is a versatile strain applicable to various foods and drinks. Bifidobacterium breve YIT 12272 has very superior characteristics compared to other Bifidobacterium genus bacteria and Bifidobacterium breve, so other Bifidobacterium genus bacteria and It is extremely important to search for a gene specific to the strain that does not exist in Bifidobacterium breve and to clarify its function.

  Examples of useful genes present in Bifidobacterium genus and Bifidobacterium breve include BL0674 gene (Non-patent Document 1) considered to be related to host cell adhesion, and β-galactosidase related to β-galactosidase production. Although a gene (Non-patent Document 2) has been reported, a gene specific to Bifidobacterium breve YIT 12272 has not been reported. In addition, a primer specific to Bifidobacterium breve YIT 12272 and a method for specific detection of the strain characterized by using these primers have already been reported (Patent Document 4). Is a primer designed by extracting the DNA from the bacterial cells of the strain, performing the RAPD method by performing PCR, searching for a base sequence specific to the strain, and based on the sequence, It was not the target. For this reason, a gene specific to Bifidobacterium breve YIT 12272 has not yet been clarified.

International Publication No. 2003/040350 Japanese Patent No. 2922013 Japanese Examined Patent Publication No. 61-19220 International Publication No. 2011/105335 Schell et al., Proc Natl Acad Sci USA, 99 (22): 14422-14427,2002 Mφller, P.L. et al., Appl & Environ.Microbial., (2001), 62, (5), 2276-2283

  An object of the present invention is to provide a gene specific to the Bifidobacterium breve YIT 12272 strain and a method for using the same.

  When the present inventors examined in view of the said subject, 145 genes specific to the said strain were identified from all the genes 1966 of Bifidobacterium breve YIT 12272 strain, and the function was estimated. In addition, it was found that Bifidobacterium breve YIT 12272 strain can be specifically detected and quantified by targeting the gene.

The present invention relates to the following 1) to 9).
1) A gene for specifically detecting the Bifidobacterium breve YIT 12272 strain, which is a DNA consisting of the base sequence shown in SEQ ID NOs: 1-145 or a DNA consisting of a base sequence complementary to the sequence.
2) The gene according to 1), which is DNA consisting of the base sequence shown in SEQ ID NOs: 1 to 36 or DNA consisting of a base sequence complementary to the sequence.
3) The gene according to 1), which is DNA consisting of the base sequence shown in SEQ ID NOs: 37 to 55 or DNA consisting of a base sequence complementary to the sequence.
4) A method for specific detection of Bifidobacterium breve YIT 12272, which targets at least one of the genes described in 1).
5) A method for quantifying the Bifidobacterium breve YIT 12272 strain, which targets at least one of the genes described in 1).
6) A screening method for a microorganism having the gene, wherein the presence and / or expression level of any one or more of the gene according to 1) or an expression product thereof is used as an index.
7) A microorganism selected by the screening method described in 6).
8) Introduction of a genetically modified microorganism characterized by introducing any one or more of the genes described in 1) and / or modifying a microorganism gene targeting any one or more of the genes described in 1) Production method.
9) A genetically modified microorganism obtained by the production method described in 8).

  Since the gene of the present invention is a gene specific to the Bifidobacterium breve YIT 12272 strain, by targeting the gene, only the Bifidobacterium breve YIT 12272 strain is detected from the sample at the gene level. Can be detected and quantified. Further, by using the gene of the present invention, the excellent characteristics of Bifidobacterium breve YIT 12272 strain can be elucidated at the gene level. Furthermore, using the presence and / or expression level of the gene of the present invention or its expression product as an index, a microorganism having the same properties as those inherent to the Bifidobacterium breve YIT 12272 strain can be screened. In addition, by introducing the gene of the present invention into another microorganism, or when the other microorganism has a gene corresponding to the gene of the present invention, the gene is modified to produce a genetically modified microorganism having a useful function. can do.

The DNA consisting of the nucleotide sequence represented by SEQ ID NOs: 1 to 145 of the present invention is a gene specific to the Bifidobacterium breve YIT 12272 strain, and is identified by the method described in Example 1 below. .
That is, first, the entire amino acid sequence of the strain obtained from the genome sequence information of Bifidobacterium breve YIT 12272 strain (FERM BP-11320) (see Patent Document 4) and the Bifidobacterium whose genome information is disclosed. Comparing the amino acid sequences of all genes of 4 bacterial strains belonging to Umbreve, homologous sequences with an e-value (expected value that includes an accidentally erroneous search result) of less than 0.01 and a coverage of more than 60% 161 genes that were not found in all four control strains were selected as candidate genes specific to the strain. Then, the base sequence of the 161 gene is compared with the genome sequence of a bacterial strain belonging to Bifidobacterium breve whose base sequence of the full-length genome is published on a public database, and the e-value is less than 0.01 and Homologous sequences having a coverage of 100% and a gap number of 0 were selected as Bifidobacterium breve YIT 12272 strain-specific genes that were not found in the control strain (Table 1). ).
Here, the “cover rate” refers to the ratio of the region where sequence similarity is recognized with other strains to the total length of the Bifidobacterium breve YIT 12272 strain subjected to the search. . Specifically, the calculation is performed as follows using the values of the alignment length and the number of gaps obtained from the result of the homology search. “Cover ratio” = (“Alignment length” − “Gap number”) / “Searched amino acid sequence length of Bifidobacterium breve YIT 12272 strain” × 100
For example, in the sequence a of Bifidobacterium breve YIT 12272 consisting of 100 amino acids, when the sequence b consisting of 105 amino acids of strain B is hit with an alignment length of 90 and a gap number of 10 by homology search, the coverage is (90-10) / 100 × 100 = 80%.

  In addition, for each gene, a homology search (e-value <0.01) was performed with respect to the entire amino acid sequence database published by the National Center for Biotechnology Information (NCBI). Estimated its function.

Among the 145 genes in Table 1 above, the 36 DNAs shown in SEQ ID NOs: 1 to 36 correspond when the e-value is searched with less than 0.01 using the entire amino acid sequence database published by NCBI. This gene has no homologous sequence and encodes a novel amino acid sequence for all microorganisms. In addition, 19 DNAs shown in SEQ ID NOs: 37 to 55 in Table 1 are genes that have no corresponding homologous sequence when searched under conditions where e-value is less than 0.01 and the coverage rate is more than 60%. .
Therefore, these 55 genes are particularly specific for the Bifidobacterium breve YIT 12272 strain for specific detection of the strain and screening for microorganisms that retain the same properties as the inherent properties of the strain. Is more useful.

The specific detection method of the Bifidobacterium breve YIT 12272 strain of the present invention is either a DNA comprising the base sequence represented by SEQ ID NO: 1-145 or a gene comprising a DNA comprising a base sequence complementary to the sequence. It targets one or more.
Specific examples of the detection method include a DNA probe method for detecting using an oligonucleotide probe that specifically binds to these DNAs, and a nucleic acid amplification method for amplifying these DNAs using primers capable of amplifying these DNAs. .
As an example of a nucleic acid amplification method, for DNA extracted from a sample using a primer made based on DNA consisting of a base sequence represented by SEQ ID NOs: 1-145 or DNA consisting of a base sequence complementary to the sequence A method of detecting the amplified DNA fragment by performing a PCR reaction or the like can be mentioned.

The primer used here can be prepared as a primer that amplifies the full length of the gene based on the base sequences of the 5 ′ end and 3 ′ end of each DNA. In the PCR method, it is usually preferable to use two kinds of primers as one set, and it is necessary that both are a combination of a leading strand and a lagging strand.
Such a primer can be easily synthesized by, for example, an automatic DNA synthesizer. These primers can also be used as probes.

  The PCR reaction conditions can be the normal PCR conditions. For example, a heat denaturation reaction for converting double-stranded DNA into a single strand is 90 to 98 ° C, an annealing reaction for hybridizing a primer to a template cDNA is 37 to 72 ° C, and an extension reaction for allowing DNA polymerase to act is 50 to 75 ° C. It can be performed by performing one to several tens of cycles under the temperature condition. Examples of preferable reaction conditions include heat denaturation 98 ° C. for 10 seconds, annealing 60 ° C. for 30 seconds, and extension reaction 72 ° C. for 120 seconds × 30 cycles.

As described in Example 2 below, the primer amplifies only DNA of Bifidobacterium breve YIT 12272 and does not amplify DNA of other Bifidobacterium breve strains. By observing the presence or absence of the product, the presence or absence of the Bifidobacterium breve YIT 12272 strain can be determined from the Bifidobacterium breve in the sample, preferably the sample.
Thus, by utilizing the 145 gene of the present invention, the Bifidobacterium breve YIT 12272 strain can be specifically detected at the gene level.

  As a method for extracting DNA in the specimen, a heat extraction method, an alkali heat extraction method, a phenol / chloroform extraction method, or the like may be used. The medium used for culturing the specimen before extraction is not particularly limited as long as the specimen can be cultivated, and examples thereof include a GAM medium known as a medium for growing bifidobacteria. In addition, glucose and agar can be added to the medium. Although the culture time varies depending on the specimen, it can be performed, for example, 48 to 144 hours, preferably 72 hours, and the culture temperature is not particularly limited as long as Bifidobacterium breve can be suitably grown, For example, it can be carried out at 34 to 40 ° C, preferably 37 ° C.

  The specimen is not particularly limited as long as it may contain microorganisms. For example, conjunctival swab, tartar, plaque, sputum, pharyngeal swab, saliva, nasal discharge, alveolar lavage, pleural effusion, gastric juice , Gastric lavage fluid, urine, cervical mucus, vaginal secretions, skin lesions, feces, blood, ascites, tissue, spinal fluid, joint fluid, affected area wipes, foods, pharmaceuticals, cosmetics, foods / pharmaceuticals Examples include subjects that may contain microorganisms such as cosmetic intermediates, microbial cultures, plants, soil, activated sludge, and wastewater.

  The buffer used for suspending the emerged colonies is not particularly limited, and examples thereof include TE buffer. As an example of the DNA heat extraction method, the heat treatment is performed by treating the buffer suspension at 72 to 100 ° C. for 1 to 10 minutes, preferably at 98 ° C. for 2 minutes. Centrifugation is performed at 4 to 30 ° C. and 6,000 to 15,000 rpm for 1 to 10 minutes, preferably at 4 ° C. and 15,000 rpm for 5 minutes. The supernatant thus prepared may be used as a template DNA solution for PCR reaction.

  In addition, the Bifidobacterium breve YIT 12272 strain in the sample can be quantified by using quantitative PCR using the primer. As an example of the method of quantitative PCR, International Journal of Food Microbiology (2008) Vol. 126, p210-215.

  Further, by using the presence and / or expression level of the gene of the present invention or its expression product as an index, the gene is possessed and retains the same properties as the inherent properties of Bifidobacterium breve YIT 12272 strain. Microorganisms can be screened. That is, by measuring the presence and / or expression level of the gene of the present invention or its expression product, useful microorganisms having excellent characteristics possessed by the strain can be screened. Examples of expression products of these genes include mRNA and polypeptides. Here, the presence and / or expression level of a gene or its expression product is measured by using a probe or primer that can detect the gene or its expression product, the presence or absence of the target gene in the microorganism, the copy number or the expression level. Can be performed by Southern hybridization method, Northern hybridization method, DNA microarray or RT-PCR method. Then, the target microorganism may be selected based on the presence and / or expression level of the gene or its expression product.

  Furthermore, by introducing the gene alone or in combination into another microorganism that does not originally have the gene of the present invention, a genetically modified microorganism having the same function as that of the strain or an enhanced function can be produced. . As a method for introducing these genes into other microorganisms, a competence method using DNA uptake ability, a protoplast PEG method using protoplasts, an electroporation method using high voltage pulses, or the like may be used. In addition, when a gene is incorporated into a microbial chromosome, a method using homologous recombination or a site-specific integration method can be used.

  In addition, when another microorganism has a gene corresponding to the gene of the present invention, it is possible to produce a genetically modified microorganism in which the function of the gene is enhanced or suppressed by modifying the gene. Examples of gene modification include inhibition or suppression of expression of these genes, or enhancement of expression.

  In order to inhibit the expression of genes, these genes can be destroyed or deleted. This method can be achieved by an inactivation method in which a completely different DNA fragment is inserted into the gene or by stepwise homologous recombination. A stepwise double crossover method in which part or all of the above are deleted may be used, and a stepwise double crossover method is particularly preferable. Specifically, when part or all of the target gene is deleted, the regions on both sides sandwiching the deletion region are separated from the chromosomal DNA or amplified by PCR and separated, such as pYSSE3. These DNA fragments are cloned in the same orientation as the original orientation in a plasmid vector that can grow in Escherichia coli but cannot replicate in the target microorganism. Next, the resulting recombinant plasmid DNA is introduced into the microorganism to be deleted by electroporation, etc., and the desired deletion region is cloned from the resulting antibiotic-resistant clone by PCR or the like. A clone in which recombination has occurred in the region homologous to the upstream or downstream cloned region and the plasmid has been inserted on the chromosome is selected. The clones obtained in this way are repeatedly subcultured in a medium that does not contain antibiotics, so that the plasmid is detached from the chromosome by recombination between the homologous regions where the plasmids are in close proximity, and disappears as the bacteria grow. Select clones that have lost antibiotic resistance. Clones lacking the target gene region can be isolated from the thus obtained clones by PCR or the like.

  In order to suppress gene expression, a method using so-called RNA interference by synthesizing a short RNA complementary to the 5′-terminal region of the target gene mRNA, or a region or regulatory gene that controls the expression of these genes is destroyed. Alternatively, a method of modification by deletion or the like can be used, and modification of a region controlling the expression of these genes is particularly preferable. Specifically, the amount of transcription of these genes into mRNA can be reduced by modifying the sequence of the promoter that controls the transcription of the genes.

  In order to enhance the expression of genes, these genes in the microorganism are introduced by introducing recombinant plasmids carrying these genes into the target microorganism, or by integrating these genes elsewhere in the chromosome by site-specific recombination. May be performed by a method such as increasing the copy number, modifying the control region controlling the expression of these genes, or modifying the control gene and increasing the amount of transcription to mRNA, etc. In particular, a method of increasing the copy number of these genes is preferable. Specifically, the gene of interest is cloned into a plasmid having a plurality of copies per microbial cell, including the original promoter sequence and ribosome binding site of the gene, or separated from another gene. Or, create a recombinant plasmid by cloning only the region encoding the polypeptide of the gene downstream of the promoter and the ribosome binding site created by chemical synthesis, and introduce it into microbial cells by electroporation etc. Can increase the copy number of the gene in microbial cells.

  Here, the microorganism that is the target of gene transfer or modification is not particularly limited, and Gram-positive bacteria, Gram-negative bacteria, yeasts, and the like can be used.

  The microorganism selected by the screening method and the genetically modified microorganism obtained by the gene introduction or modification can be used as a food or drink or a medicine. In this case, the microbial cell may be either a live cell or a heated cell (dead cell), may be freeze-dried, or a culture containing these microorganisms may be used. In addition, as long as the excellent characteristics of Bifidobacterium breve YIT 12272 are preserved, it is also possible to use treated cells.

  Such a pharmaceutical can be administered in the form of a conventional pharmaceutical preparation by mixing the microorganism with a solid or liquid non-toxic pharmaceutical carrier. Examples of such preparations include solid preparations such as tablets, granules, powders and capsules, liquid preparations such as solutions, suspensions and emulsions, and freeze-dried preparations. These preparations can be prepared by conventional means on the preparation. Examples of the non-toxic pharmaceutical carrier include glucose, lactose, sucrose, starch, mannitol, dextrin, fatty acid glyceride, polyethylene glycol, hydroxyethyl starch, ethylene glycol, polyoxyethylene sorbitan fatty acid ester, amino acid, gelatin, albumin , Water, physiological saline and the like. In addition, conventional additives such as stabilizers, wetting agents, emulsifiers, binders, isotonic agents, excipients and the like can be appropriately added as necessary.

  Moreover, the microorganisms can be used not only in the above-mentioned preparations but also in foods and drinks. What is necessary is just to add the said microorganisms as it is or with a various nutrient component when mix | blending with food-drinks. This food and drink can be used as a health food or a food material that exhibits various pharmacological actions according to the excellent properties of Bifidobacterium breve YIT 12272. Such foods and drinks may use additives that can be used as foods and drinks as appropriate, and may be formed into edible forms using conventional means, that is, granules, granules, tablets, capsules, pastes, etc. In addition, various foods such as processed foods such as ham and sausage, fishery processed foods such as kamaboko and chikuwa, bread, confectionery, butter, powdered milk, water, fruit juice, milk, soft drinks, You may add and use for drinks, such as a tea drink. The food and drink includes animal feed.

  Furthermore, examples of the food and drink include fermented food and drink such as fermented milk, lactic acid bacteria beverage, fermented soy milk, fermented fruit juice, and fermented plant liquid using the microorganism. Manufacture of these fermented food / beverage products should just follow a conventional method. For example, when producing fermented milk, first, the microorganism is inoculated and cultured in a sterilized milk medium alone or simultaneously with other microorganisms, and this is homogenized to obtain a fermented milk base. Then, a separately prepared syrup solution is added and mixed, homogenized with a homogenizer or the like, and further added with flavor to finish the final product. The fermented milk thus obtained can be a product of any form such as a plain type, a soft type, a fruit flavor type, a solid form, and a liquid form.

Hereinafter, although an example is given and the present invention is explained still in detail, the present invention is not limited to these. Example 1 Identification of a gene specific to Bifidobacterium breve YIT 12272 strain According to the procedure shown in the following 1) to 3), the Bifidobacterium breve YIT 12272 strain was identified from the genome sequence. 145 genes were identified.
1) For all gene regions on the Bifidobacterium breve YIT 12272 strain genome sequence determined using the gene prediction program GeneHackerPlus (Yada, T. et al. 2001, DNA Research, 8, 97-106.) Was converted into an amino acid sequence by the sequence conversion program EMBOSS-Transeq (version 6.4.0.0), and the entire amino acid sequence of the strain was obtained.

2) About these amino acid sequences, 4 bacterial strains belonging to Bifidobacterium breve (Bifidobacterium breve DSM 20213 ^T strain, whose genome information is disclosed on public databases (Genbank, version 192.0), Bifidobacterium breve ACS-071-V-Sch8b strain, Bifidobacterium breve UCC2003 strain, Bifidobacterium breve CECT 7263 strain) all genes amino acid sequences, search program NCBI-blastp (version The homology search according to 2.2.25+) was performed. Among all 1966 genes of Bifidobacterium breve YIT 12272 strain, there are homologous sequences whose e-value (expected value including accidental erroneous search result) is less than 0.01 and the coverage is more than 60% 161 genes that were not found in all four control strains were used as candidate genes specific to the strain.
The “cover ratio” means the ratio of the region where sequence similarity is recognized with other strains to the total length of the amino acid sequence of Bifidobacterium breve YIT 12272 strain subjected to the search. It is calculated as follows. “Cover ratio” = (“Alignment length” − “Gap number”) / “Searched amino acid sequence length of Bifidobacterium breve YIT 12272 strain” × 100

  3) Next, regarding the base sequence of the above 161 genes, two bacterial strains belonging to Bifidobacterium breve (bibibacterium breve) whose full-length base sequences are publicly disclosed on public databases (Genbank, version 192.0). Homology search was carried out with the search program NCBI-blastn (version 2.2.25+) against the genome sequences of Fidobacterium breve ACS-071-V-Sch8b strain and Bifidobacterium breve UCC2003 strain). Of these, 145 genes whose homologous sequences with an e-value of less than 0.01, a coverage of 100%, and a gap number of 0 were not found in any of the two strains as controls (Table 1 above) Was extracted as a gene for specifically detecting the YIT 12272 strain among the bacteria belonging to Bifidobacterium breve.

Among the 145 genes in Table 1 above, 36 genes shown in SEQ ID NOs: 1 to 36 are expressed using the whole amino acid sequence database published by the National Center for Biotechnology Information (NCBI). -It is a gene that encodes a novel amino acid sequence for all microorganisms when the value is less than 0.01 and no corresponding homologous sequence exists.
In addition, 19 genes shown in SEQ ID NOs: 37 to 55 in Table 1 are genes for which there is no corresponding homologous sequence when searching under conditions where e-value is less than 0.01 and the coverage rate exceeds 60%. .

Example 2 Preparation of specific primers based on specific gene sequences (1) Preparation of primers Targeting 18 genes shown in Table 2 randomly selected from the genes of the present invention of SEQ ID NOs: 1-145, Primers that amplify the full length of the gene were prepared using a DNA synthesizer based on the base sequences of the 5 ′ end and 3 ′ end (Table 2, SEQ ID NOS: 146 to 181).
In addition, as a positive control, a common sequence region of 16S rRNA gene and a dnaA gene having extremely high sequence commonality in Bifidobacterium breve species were used, and primers for amplifying these regions were similarly prepared (Table). 2, SEQ ID NOS: 182-185).

(2) Extraction of template DNA Plates of 13 strains belonging to Bifidobacterium breve having different origins shown in Table 3 with 1% glucose and 1.5% agar added to GAM medium (manufactured by Nissui Pharmaceutical) The culture was anaerobically cultured at 37 ° C. for 72 hours. One of the appearing colonies was suspended in 50 μL of TE buffer (10 mM Tris, 1 mM EDTA, pH 8.0) and treated at 98 ° C. for 2 minutes to extract DNA. Immediately after the heat treatment, the mixture was ice-cooled and centrifuged at 4 ° C. and 15,000 rpm for 5 minutes, and the supernatant was used as a template DNA solution for the following PCR reaction.

(3) PCR reaction 7.5 μL of EmeraldAmp PCR Master Mix (manufactured by Takara), 0.2 μM primer, 0.5 μL of the template DNA solution with water to make a total volume of 15 μL, iCycler (manufactured by Bio-Rad) ), 30 cycles of PCR reaction were performed at 98 ° C. for 10 seconds, 60 ° C. for 30 seconds, and 72 ° C. for 120 seconds.

(4) Specific detection of YIT 12272 strain By observing the binding ability between each primer and each strain DNA based on the presence or absence of the amplification product by the PCR reaction, 18 genes shown in Table 2 were transformed into Bifidobacterium breve YIT. It was verified whether these genes are specific to the 12272 strain and whether these genes can be used to specifically detect the strain. The reaction solution after the PCR reaction was subjected to electrophoresis at 100 V for 25 minutes with 1% agarose gel using Mupid-exU (manufactured by Advance), and stained with 0.5 mg / ml ethidium bromide. Amplification products were observed under UV irradiation. The presence or absence of amplification products when using each primer was as shown in Table 4. This revealed that the 18 genes shown in Table 2 are specific to the Bifidobacterium breve YIT 12272 strain and that these genes can be used to specifically detect the strain.

Claims (9)

  A gene for specifically detecting the Bifidobacterium breve YIT 12272 strain, which is DNA consisting of the base sequence represented by SEQ ID NOs: 1-145 or DNA complementary to the sequence.   The gene according to claim 1, which is DNA consisting of the base sequence shown in SEQ ID NOs: 1 to 36 or DNA consisting of a base sequence complementary to the sequence.   The gene according to claim 1, which is a DNA consisting of the base sequence shown in SEQ ID NOs: 37 to 55 or a DNA consisting of a base sequence complementary to the sequence.   A method for specific detection of Bifidobacterium breve YIT 12272, which targets at least one of the genes according to claim 1.   A method for quantifying the Bifidobacterium breve YIT 12272 strain, which targets at least one of the genes according to claim 1.   The screening method of the microorganisms which have the said gene using the presence and / or expression level of any one or more of the gene of Claim 1, or its expression product as a parameter | index.   A microorganism selected by the screening method according to claim 6.   A genetically modified microorganism characterized by introducing any one or more of the genes according to claim 1 and / or modifying a microbial gene targeting at least one of the genes according to claim 1. Production method.   A genetically modified microorganism obtained by the production method according to claim 8.