JP2017216979A - Oligonucleotide - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide, for the purpose of evaluating the efficacy against humans and other animals, a method for detecting Bifidobacterium longum subspecies infantis, which can be detected by distinguishing four subspecies including a new group C subspecies, and a quantitative method thereof.SOLUTION: There are provided an oligonucleotide consisting of a specific nucleotide sequence, an oligonucleotide pair comprising a combination of the oligonucleotide, a nucleic acid amplification primer consisting of the oligonucleotide and the oligonucleotide pair, and a nucleic acid amplification kit comprising the nucleic acid amplification primer.SELECTED DRAWING: None

Description

  The present technology relates to oligonucleotides. More specifically, the present technology relates to an oligonucleotide having a specific sequence and a nucleic acid amplification primer comprising the oligonucleotide.

  Bifidobacterium genus bacteria are abundant in the intestinal tract of infants before weaning, and are thought to contribute to maintaining the intestinal environment of infants well. Physiological functions of Bifidobacterium include, for example, an intestinal infection-protecting action on the host, an immune function-enhancing action, nutrition, and intestinal decay-inhibiting action. Focusing on the usefulness of Bifidobacterium as described above, adding Bifidobacterium to infant formula, and administering the formulated Bifidobacterium to humans It has been devised to prevent infections or to promote treatment of allergic diseases. Bifidobacterium bacteria to be administered to humans are desirably those that inhabit the human intestinal tract from a physiological viewpoint. Examples of Bifidobacterium bacteria that inhabit the human intestinal tract include Bifidobacterium longum (B. longum).

  For Bifidobacterium longum, it was previously known that there were three subspecies. The three subspecies are B. longum subsp. Longum, B. longum subsp. Infantis, and B. longum subsp. Bifidobacterium longum subspices Switzerland (hereinafter also referred to as “Longham subspecies”, “Infantis subspecies”, and “Swiss subspecies”), respectively. However, based on genome information that has been rapidly accumulated in recent years, a group C subspecies has been proposed as a new subspecies (the following Non-Patent Document 1, pages 6-7). As a result, Bifidobacterium longum can be classified into four subspecies: Longum subspecies, Infantis subspecies, Swiss subspecies, and Group C subspecies. Group C subspecies include Bifidobacterium longum subspecies longum JDM301, Bifidobacterium longum BXY01 and Bifidobacterium longum subspecies longum CMCCP0001 (see the following non-patent document 1 figure). 3).

  In order to evaluate the effectiveness of Bifidobacterium on humans and other animals, the Bifidobacterium used must be detected separately from other Bifidobacterium. . In order to detect a specific Bifidobacterium genus, for example, there are a method of culturing a test bacterium and specifying the bacterium by morphological and biochemical properties and a DNA-DNA homology test. There is a problem that it is time-consuming and time-consuming and complicated, and the latter is time-consuming and also has a problem with accuracy. Thus, methods for analyzing Bifidobacterium bacteria using PCR primers that detect 16S rRNA gene and the like have been performed (Non-Patent Documents 2 to 8 below).

Pangenome analysis of Bifidobacterium longum and site-directed mutagenesis through by-pass of restriction-modification systems.BMC Genomics. 2015, Vol.16, No.1, p.832-850. Differentiation of bifidobacteria by use of pulsed-field gel electrophoresis and polymerase chain reaction.Int J Food Microbiol. 1996, Vol.29, No.1, p.11-29. Distribution of bifidobacterial species in human intestinal microflora examined with 16S rRNA-gene-targeted species-specific primers.Appl Environ Microbiol. 1999, Vol. 65, No. 10, p.4506-4512. Molecular differentiation of Bifidobacterium species with amplified ribosomal DNA restriction analysis and alignment of short regions of the ldh gene.FEMS Microbiol Lett. 2000, Vol.191, No.1, p.17-24. Evaluation of the PCR method for identification of Bifidobacterium species.Lett Appl Microbiol. 2008, Vol.46, No.1, p.7-13. Use of tuf gene-based primers for the PCR detection of probiotic Bifidobacterium species and enumeration of bifidobacteria in fermented milk by cultural and quantitative real-time PCR methods.J Food Sci. 2010, Vol.75, No.8, p.M521- 527. Establishment of a sensitive system for analysis of human vaginal microbiota on the basis of rRNA-targeted reverse transcription-quantitative PCR.J Microbiol Methods. 2015, Vol.111, p.93-104. Species specific identification of nine human Bifidobacterium spp. In feces. Syst Appl Microbiol. 2002, Vol. 25, No. 4, p.536-43. Comparative Genomic Analysis of 45 Type Strains of the Genus Bifidobacterium: A Snapshot of Its Genetic Diversity and Evolution.PloS ONE.2015, Vol.10, No.2, e0117912.doi: 10.1371 / journal.pone.0117912

  Bifidobacterium longum contains four subspecies, including a new group C subspecies. Differentiating and detecting these subspecies is necessary to assess the effectiveness of bacteria.

In addition, Bifidobacterium breve, which is closely related to Bifidobacterium longum, is present in the infant intestinal tract.
For example, in order to evaluate the effectiveness of adding Bifidobacterium longum to infant formula, it is necessary to detect Bifidobacterium breve and Bifidobacterium longum separately. In order to evaluate the effectiveness of Bifidobacterium longum subspecies infants in the intestinal tract of infants, Bifidobacterium breve and Bifidobacterium longum subspecies infants It is necessary to detect them separately.

  Non-Patent Documents 2 to 8 disclose methods for analyzing Bifidobacterium bacteria using PCR primers targeting a specific region such as a 16S rRNA gene. However, the PCR primers described in these non-patent documents cannot detect Bifidobacterium longum sub-species Infantis from the other three subspecies or detect Bifidobacterium breve. There is a problem. Therefore, there is a need for a technique capable of distinguishing and detecting a specific Bifidobacterium longum subspecies from other subspecies and also from Bifidobacterium breve.

  An object of the present technology is to provide a technology related to the detection of Bifidobacterium longum sub-species Infantis.

  Most of the conventional primers for detecting Bifidobacterium longum subspecies infantitis targeted sequences on the 16S rRNA gene. The present inventors investigated the sequence of clpC (proteolytic enzyme) gene, which has not been focused on in bacterial detection, and found a specific sequence that can solve the above-mentioned problems, and completed the present technology.

That is, the present technology provides an oligonucleotide having the base sequence of SEQ ID NO: 1.
Moreover, this technique provides the oligonucleotide which consists of a base sequence of sequence number 2.
The present technology also provides an oligonucleotide pair that is a combination of an oligonucleotide consisting of the base sequence of SEQ ID NO: 1 and an oligonucleotide consisting of the base sequence of SEQ ID NO: 2.

The present technology also provides a nucleic acid amplification primer comprising an oligonucleotide pair that is a combination of an oligonucleotide having the base sequence of SEQ ID NO: 1 and an oligonucleotide having the base sequence of SEQ ID NO: 2.
The primer for nucleic acid amplification of the present technology may be for detecting Bifidobacterium longum subspecies infantitis.
The primer for nucleic acid amplification of the present technology may be one that does not show a cross-reaction with Bifidobacterium longum subspecies other than Bifidobacterium longum subspecies Infantis.
The primer for nucleic acid amplification of the present technology may be one that does not show a cross-reaction with Bifidobacterium breve.

The present technology also provides a nucleic acid amplification kit containing the nucleic acid amplification primer.
The kit for nucleic acid amplification according to the present technology may be for detecting Bifidobacterium longum subspecies infantis.

The present technology also provides a method for detecting Bifidobacterium longum subspecies Infantis. In this detection method, a sample is subjected to a nucleic acid amplification process using an oligonucleotide pair that is a combination of an oligonucleotide consisting of the base sequence of SEQ ID NO: 1 and an oligonucleotide consisting of the base sequence of SEQ ID NO: 2. And determining the presence or absence of Bifidobacterium longum subspecies infantis or nucleic acid derived from Bifidobacterium longum subspecies infantis in the sample depending on the presence or absence of an amplification product.
The present technology also provides a method for quantifying Bifidobacterium longum subspecies Infantis. The quantification method uses a pair of oligonucleotides, which is a combination of an oligonucleotide consisting of the base sequence of SEQ ID NO: 1 and an oligonucleotide consisting of the base sequence of SEQ ID NO: 2, to perform a nucleic acid amplification process on the sample. Quantifying the amount of Bifidobacterium longum subspecies infantis or nucleic acid derived from Bifidobacterium longum subspecies infantis in the sample based on the Ct value of the sample after the treatment Including that.

  By this technique, Bifidobacterium longum subspecies infantis can be specifically detected. By this technique, Bifidobacterium longum subspecies infantis can be detected separately from other subspecies and can also be detected separately from Bifidobacterium breve. In addition, the effect show | played by this technique is not necessarily limited to the effect described here, and may be the any effect described in this specification.

It is a figure which shows the design area | region of primer 1 and 2. FIG.

  Hereinafter, preferred embodiments for carrying out the present technology will be described. In addition, embodiment described below shows an example of typical embodiment of this technique, and, thereby, the scope of this technique is not interpreted narrowly.

これらオリゴヌクレオチドによって、他方のオリゴヌクレオチドと組み合わせて核酸増幅に用いた場合にビフィドバクテリウム・ロンガム・サブスピーシーズ・インファンティスの検出ができるという効果が奏される。なお、本明細書内に記載の塩基配列は、他に言及されない限り、左端が５’端であり、右端が３’端である。 <1. Oligonucleotide>
The present technology provides an oligonucleotide having the following base sequence of SEQ ID NO: 1. The present technology also provides an oligonucleotide consisting of the following base sequence of SEQ ID NO: 2.

When these oligonucleotides are used for nucleic acid amplification in combination with the other oligonucleotide, there is an effect that Bifidobacterium longum subspices infantis can be detected. In addition, unless otherwise indicated, the left end is the 5 ′ end and the right end is the 3 ′ end in the base sequences described in the present specification.

  The oligonucleotides of the present technology correspond to a specific sequence in the clpC (proteolytic enzyme) gene region of Bifidobacterium longum subspecies Infantis. As a result, a specific region in the clpC gene region is amplified by performing nucleic acid amplification using the oligonucleotide of the present technology.

  Bifidobacterium longum is classified into four subspecies. In nucleic acid amplification using the above oligonucleotides, Bifidobacterium longum subspecies Infantis nucleic acid, especially chromosomal DNA is specifically Amplified. That is, in the nucleic acid amplification, the other three sub-species nucleic acids are not amplified, or even if they are amplified, they are below the detection limit and are not detected. Therefore, there is an effect that it is possible to detect the bacteria of this subspecies separately from the other three subspecies (that is, the longum subspecies, the Swiss subspecies, and the group C subspecies).

  Bifidobacterium longum is very closely related to Bifidobacterium breve (see FIG. 3 of Non-Patent Document 9), but in nucleic acid amplification using the above oligonucleotides, the nucleic acid of Bifidobacterium breve is Even if it is not amplified or even if it is amplified, it will be below the detection limit and will not be detected. Therefore, there is an effect that Bifidobacterium longum subspecies infantis can be detected separately from Bifidobacterium breve.

  The present technology also provides an oligonucleotide pair that is a combination of an oligonucleotide consisting of the base sequence of SEQ ID NO: 1 and an oligonucleotide consisting of the base sequence of SEQ ID NO: 2. By using the oligonucleotide pair for nucleic acid amplification, the effects described above can be achieved.

  Further, the present technology provides an oligonucleotide consisting of a base sequence complementary to the base sequence of SEQ ID NO: 1, an oligonucleotide consisting of a base sequence complementary to the base sequence of SEQ ID NO: 2, and a combination of these oligonucleotides. Certain oligonucleotide pairs are also provided. These oligonucleotides can also have the effects described above.

  Oligonucleotides of the present technology can be produced by synthetic methods and equipment known to those skilled in the art. Examples of the synthesis method include a solid phase method, and examples of the apparatus include, but are not limited to, an automatic oligonucleotide synthesizer.

<2. Primer for nucleic acid amplification>
The present technology provides a primer for nucleic acid amplification consisting of an oligonucleotide pair which is a combination of an oligonucleotide consisting of the base sequence of SEQ ID NO: 1 and an oligonucleotide consisting of the base sequence of SEQ ID NO: 2. By performing nucleic acid amplification using the nucleic acid amplification primer of the present technology, a specific region in Bifidobacterium longum subspecies Infantis nucleic acid, particularly DNA, and more particularly chromosomal DNA is specific. Therefore, it is possible to detect Bifidobacterium longum subspecies infantis. Therefore, the nucleic acid amplification primer of the present technology can be used for detecting Bifidobacterium longum subspecies infantitis.

  The primer for nucleic acid amplification of the present technology may be one that does not show a cross-reaction with Bifidobacterium longum subspecies other than Bifidobacterium longum subspecies Infantis. That is, in the nucleic acid amplification using the nucleic acid amplification primer of the present technology, the Bifidobacterium longum subspecies Infantis nucleic acid is specific among the four subspecies contained in Bifidobacterium longum. The nucleic acids of the other three subspecies (i.e., Longham subspecies, Swiss subspecies, and Group C subspecies) are not amplified or, if amplified, are below the detection limit and are not detected. As described above, the nucleic acid of Bifidobacterium longum sub-species Infantis among these four subspecies is specifically amplified by the primer for nucleic acid amplification of the present technology. More specifically, only the nucleic acid of Bifidobacterium longum subspecies Infantis among these four subspecies can be amplified by the nucleic acid amplification primer of the present technology.

  The primer for nucleic acid amplification of the present technology may be one that does not show a cross-reaction with Bifidobacterium breve. That is, in the nucleic acid amplification using the nucleic acid amplification primer of the present technology, the nucleic acid of Bifidobacterium breve that is closely related to Bifidobacterium longum is not amplified, or even if it is amplified, it is below the detection limit. ,Not detected. In this way, the nucleic acid amplification primer of the present technology specifically amplifies the nucleic acid of Bifidobacterium longum subspecies infantis without amplifying the Bifidobacterium breve nucleic acid. .

  In the present technology, nucleic acid amplification refers to amplification of a nucleic acid to be amplified (target nucleic acid) among nucleic acids in a sample by utilizing complementarity of the base sequence of the target nucleic acid. Specific examples of the nucleic acid amplification method include, but are not limited to, PCR, LAMP, NASBA, SDA, TMA, and 3SR. In the present technology, the nucleic acid amplification is preferably PCR.

  In the present technology, PCR may be performed by the same method as PCR that is generally performed. Typically, PCR prepares a reaction solution containing a template DNA, a primer, a DNA polymerase, and other necessary components, and the reaction solution is applied at temperatures suitable for the denaturation step, annealing step, and extension reaction step, respectively. It can be performed by repeating a series of steps of keeping the temperature for a predetermined time 20 to 40 cycles. The temperature, the heat retention time, and the number of cycles in each step can be appropriately determined by those skilled in the art.

The reaction conditions for PCR can be appropriately set by those skilled in the art depending on the enzyme, apparatus, etc. used. Specifically, for example, after heating at 95 ° C. for 3 minutes, denaturation step: 90 to 98 ° C. for 10 to 40 seconds, annealing step: 50 to 65 ° C. for 20 to 40 seconds, and extension reaction step: 70 to 75 ° C. A reaction condition of repeating a series of steps of 20 to 40 seconds for 20 to 40 cycles and finally heating at 72 ° C. for 10 minutes can be mentioned. The amount ratio of the template DNA to the primer is preferably 0.2 to ² μmol of primer with respect to 10 ² to 10 ⁸ copies of chromosomal DNA.

  The DNA polymerase is not particularly limited as long as it can be used for PCR. Examples of the DNA polymerase include commercially available AmpliTaq Gold, Platinum Taq DNA polymerase (Thermo Fisher SCIENTIFIC), Takara Taq and Takara Ex Taq (Takara Bio), and Plameh Platinum Platum. Examples thereof include, but are not limited to, DNA polymerases such as SCIENTIFIC) and Pyrobest DNA polymerase (Takara Bio). The DNA polymerase used in the PCR may be a composition comprising these enzymes and a buffer solution.

  Bacteria detection using the primer for nucleic acid amplification according to the present technology is performed by amplifying bacterial nucleic acid, so that bacterial culture does not have to be performed. As a result, according to the present technology, bacteria can be detected quickly, simply, at low cost and with high accuracy.

  Further, prior to detection, bacteria may be cultured. When culturing bacteria, for example, bacteria obtained from stool, food, or soil can be anaerobically cultured in the presence of carbon dioxide on a suitable medium, such as a GAM agar medium supplemented with 3% glucose. Moreover, you may extract bacteria from a culture medium with water, a buffer solution, an organic solvent, etc. as needed.

  The nucleic acid amplified using the nucleic acid amplification primer of the present technology is DNA or RNA, and in particular may be chromosomal DNA or a fragment thereof. The nucleic acid to be amplified can be extracted from bacteria. Extraction of nucleic acids from bacteria may be performed by methods known to those skilled in the art. For example, commercially available DNA extraction kits such as DNeasy Blood & Tissue Kit from QIAGEN, QIAamp DNA Stool Mini Kit, and InstaGene Matrix from Japan BioRad can be used. In addition, DNA is extracted from cells by the Murmur method (Journal of Molecular Biology Vol. 3 p208-218 (1961)), the benzyl chloride method (Nucleic Acid Research Vol. 21 p5279-5280 (1993)), and ribonuclease. DNA can also be extracted by removing RNA. At the time of nucleic acid extraction, it is preferable to add a substance that removes a PCR inhibitor, such as bovine serum albumin. DNA extraction methods are also described in Sambrook et al., “Molecular Cloning A Laboratory Manual, Second Edition”, Cold Spring Harbor Laboratory Press (1989) and the like.

  When the DNA of Bifidobacterium longum subsp. Infantis is amplified by PCR using the primer for nucleic acid amplification of the present technology, for example, 350 to 450 bp, particularly 360 to 420 bp, more particularly 370 to 400 bp. An amplification product of 384 bp is usually obtained. Therefore, the length of the amplified product may be determined in order to more surely confirm whether the obtained amplified product is derived from Bifidobacterium longum subspecies Infantis. The length of the amplification product may be determined appropriately by those skilled in the art, for example, agarose gel electrophoresis.

<3. Nucleic acid amplification kit>
The present technology also provides a kit for nucleic acid amplification. The nucleic acid amplification kit includes a primer for nucleic acid amplification of the present technology. The nucleic acid amplification kit may also include other elements necessary for nucleic acid amplification. Examples of such other elements include, but are not limited to, DNA polymerase, dNTP, and / or buffer. The nucleic acid amplification kit of the present technology is used as a positive control as a chromosomal DNA of Bifidobacterium longum sub-species Infantis or a chromosomal DNA fragment that can be amplified with the nucleic acid amplification primer of the present technology, and / or negative. As a control, a DNA fragment containing a base sequence having a mismatch with respect to 1, 2, 3, 4 or 5 bases among the base sequences of the nucleic acid amplification primer of the present technology may be included.

  The kit for nucleic acid amplification according to the present technology may be for detecting Bifidobacterium longum subspecies infantis. That is, the kit for nucleic acid amplification of the present technology can be used for investigating whether Bifidobacterium longum subspecies infantis or nucleic acid derived from this bacterium is present in a sample. For the investigation, a nucleic acid amplification reaction of a sample using the nucleic acid amplification kit of the present technology can be performed. When an amplification product is obtained by the nucleic acid amplification reaction, it is confirmed that Bifidobacterium longum subspecies infantis or a nucleic acid derived from this bacterium is present in the sample, and no amplification product is obtained. In some cases, it can be confirmed that Bifidobacterium longum subspecies infantis or nucleic acid derived from this bacterium is not present in the sample. In the present technology, the nucleic acid derived from Bifidobacterium longum sub-species Infantis can be, for example, DNA or RNA contained in the bacterium, particularly chromosomal DNA or a fragment thereof.

<4. Bifidobacterium longum subspecies infantis detection and quantification method>
As described below, the present technology also provides a detection method and a quantification method for Bifidobacterium longum subspecies infantis using the oligonucleotide pairs of the present technology.

  That is, in the present technology, a treatment for nucleic acid amplification is performed on a sample using an oligonucleotide pair that is a combination of an oligonucleotide having the base sequence of SEQ ID NO: 1 and an oligonucleotide having the base sequence of SEQ ID NO: 2. And determining the presence or absence of nucleic acid derived from Bifidobacterium longum subspecies infantis or Bifidobacterium longum subspecies infantis in the sample based on the presence or absence of amplification products. Also provided is a method for detecting Fidobacterium longum sub-species Infantis. In the detection method of the present technology, the oligonucleotide pair of the present technology can be used as a primer for amplifying a nucleic acid. As the treatment for nucleic acid amplification, 2. The methods described in the section on primers for nucleic acid amplification can be used. In the detection method of the present technology, the sample may include only one type of bacteria, or may include a plurality of types of bacteria. In the former case, by obtaining an amplification product, the bacterium of the sample is identified as Bifidobacterium longum sub-species Infantis. In the latter case, it can be determined that the sample contains this bacterium by obtaining an amplification product.

  In addition, the present technology uses a pair of oligonucleotides, which is a combination of an oligonucleotide consisting of the base sequence of SEQ ID NO: 1 and an oligonucleotide consisting of the base sequence of SEQ ID NO: 2, to perform a process for nucleic acid amplification on a sample. And quantifying the amount of Bifidobacterium longum subspecies infantis or nucleic acid derived from Bifidobacterium longum subspecies infantis in the sample based on the Ct value of the treated sample There is also provided a method for quantifying Bifidobacterium longum subspecies infantis. In the quantification method of the present technology, the oligonucleotide pair of the present technology can be used as a primer for amplifying a nucleic acid. As the treatment for nucleic acid amplification, 2. The methods described in the section on primers for nucleic acid amplification can be used. The quantification method of the present technology makes it possible to quantify the abundance of Bifidobacterium longum subspecies infantis in a sample.

  Samples to which the detection method or quantification method can be applied include feces, food, and soil, and samples obtained therefrom, such as DNA-containing samples obtained by treating them with a DNA extraction kit. Yes, but not limited to. The sample may or may not contain only Bifidobacterium longum sub-species Infantis, or may contain this and other bacteria. The other bacterium may be, for example, a Bifidobacterium longum subspecies other than Bifidobacterium longum sub-species Infantis, or a Bifidobacterium genus other than Bifidobacterium longum. The bacteria contained in the sample may consist of a single type of separated bacteria, or may contain a plurality of types of bacteria.

  In the detection method, the presence or absence of an amplification product can be determined by a technique known to those skilled in the art. For example, the amplification product can be detected by electrophoresis of the solution after the amplification reaction using QIAxcel (QIAGEN). Alternatively, the amplification product can be detected by staining with ethidium bromide or SYBR Green I after electrophoresis by agarose electrophoresis or capillary electrophoresis. In addition, by performing electrophoresis using an agarose gel containing a fluorescent staining solution in advance, the amplification product can be detected without performing a staining operation after the completion of electrophoresis. The presence or absence of an amplification product is investigated by these techniques, and the presence or absence of Bifidobacterium longum subspecies infantis or a nucleic acid derived from this bacterium in the sample can be determined based on the results. If necessary, after the amplification product is cycle-sequenced, the presence or amount of the amplification product can be confirmed by measuring the base sequence and length using a DNA sequencer.

  In the quantification method, quantification of the amount of Bifidobacterium longum subspecies infantis or nucleic acid derived from this bacterium based on the Ct value can be appropriately performed by those skilled in the art. The Ct value (Threshold Cycle) is the number of cycles when the amplification product reaches a certain amount. The quantification is performed, for example, by performing real-time PCR on a plurality of reference samples having known nucleic acid contents that are serially diluted, and creating a calibration curve indicating the relationship between the Ct value and the nucleic acid content from the results. Can be performed by performing real-time PCR to obtain a Ct value, and applying the Ct value of the test sample to the calibration curve. The amplification reaction can be detected over time by real-time PCR.

  In the quantification method, the amplification product can be detected by fluorescence. Examples of the detection method include, but are not limited to, a method using an intercalator and a method using a fluorescently labeled probe. Examples of fluorescent substances include SYBR Green I, SYBR Gold, SYTO-9, SYTP-13, SYTO-82 (Life Technologies), EvaGreen (Biotium), LCGreen (Idaho), LightCyclerRest However, it is not limited to these.

  Hereinafter, the present technology will be described in more detail based on examples. In addition, the Example described below shows an example of a typical example of the present technology, and the scope of the present technology is not interpreted narrowly.

[Primer design and synthesis]
5 types of Bifidobacterium longum subspecies longum, Bifidobacterium longum subspecies Infantis whose genome sequences have been published in NCBI (National Center for Biotechnology Information, National Center for Biotechnology Information) 23 types of housekeeping genes (dnaB), 10 types, 3 types of Bifidobacterium longum, subspecies, Switzerland, 3 types of Bifidobacterium longum, group C subspecies, and 2 types of Bifidobacterium breve , DnaG, dnaJ, pknB, purF, rpoC, xfp, clpC), the DNA sequence and amino acid sequence comparison software MEGA (http: // w (www.megasoftware.net/home). As a result, a region capable of distinguishing 10 types of Bifidobacterium longum subspecies infantitis from 13 other types was found in the clpC gene. The sequence of the region is shown in FIG. 1, and the region is the primer 1 design region and primer 2 design region in FIG. As primers used to amplify a region sandwiched between these two regions, the following oligonucleotides consisting of the base sequence of SEQ ID NO: 1 and oligonucleotides consisting of the base sequence of SEQ ID NO: 2 were synthesized (hereinafter referred to as “Primer 1”, respectively). And “Primer 2”). As these two oligonucleotides, those synthesized at Thermo Fisher SCIENTIFIC were used. Primer 1 is a forward primer and primer 2 is a reverse primer.

[Preparation of chromosomal DNA of test bacteria]
The Bifidobacterium species listed in Table 1 below were cultured anaerobically at 37 ° C. in MRS liquid medium (with 0.5% L-cysteine hydrochloride monohydrate added). 1 mL of the cultured bacterial solution was centrifuged at 7,500 rpm for 5 minutes, and the supernatant was removed. From the thus obtained bacterial cell precipitate, DNA was extracted using DNeasy Blood & Tissue Kit (QIAGEN) according to the attached protocol.

[Detection of test bacteria by PCR]
A PCR reaction solution having the composition shown in Table 2 below was prepared, and PCR was performed using Primer 1 and Primer 2 in Veriti Thermal Cycler (Thermo Fisher SCIENTIFIC). The PCR reaction conditions were 95 ° C for 3 minutes, 35 cycles of denaturation at 95 ° C for 30 seconds, annealing at 60 ° C for 30 seconds, and extension reaction at 72 ° C for 30 seconds, and finally heating at 72 ° C for 10 minutes. It was.

  After the PCR, the reaction solution after PCR was electrophoresed using a QIAxcel System (QIAGEN). The amplification product was detected using a data analysis software BioCalculator with a DNA marker (QX Alignment Marker 15 bp / 5 kb (QIAGEN)) as an index. The results are shown in Table 3 below. In Table 3, “+” indicates that the amplification product was detected with a fluorescence intensity of 0.1 RFU or more, and “−” indicates that the amplification product was not detected with a fluorescence intensity of 0.1 RFU or more. “RFU” represents a unit of fluorescence intensity corrected by the BioCalculator.

  As shown in Table 3, amplification products were obtained only in 5 types of Bifidobacterium longum subspecies Infantis. The length of the amplified product was 384 bp. Therefore, it was found that Bifidobacterium longum subspecies infantis was specifically detected by using Primer 1 and Primer 2.

  In addition, PCR was performed by the same method for the other five types of Bifidobacterium longum subspecies Infantis possessed by the applicant. As a result, amplification products were detected in all cases using these five types of bacteria. Also from this result, it was confirmed that Bifidobacterium longum subspecies infantis was specifically detected by using Primer 1 and Primer 2.

[Verification of primers described in non-patent literature]
Non-Patent Documents 2 to 8 describe a forward primer and a reverse primer for detecting Bifidobacterium longum subspecies infantis. It was verified whether a sequence in which both the forward primer and the reverse primer matched was present in the genome sequence of the above four subspecies of Bifidobacterium longum.

  Primers used in the above verification were Inf U5 and Inf L6 (Table 2 on page 15 of Non-Patent Document 2), BiINF-1 and BiINF-2 (Table 2 on page 4507 of Non-Patent Document 3), Bil F3 and Inf R5. (Non-Patent Document 4 page 19 Table 2), IFTINF-1 and IFTINF-2 (Non-Patent Document 5 page 9 Table 2), Binf-tF and Binf-tR (Non-Patent Document 6 page M523, Table 2), BINF-23S-F and BINF-23S-R (non-patent document 7, page 99, table 2), and B791f and lm3r (non-patent document 8, page 538, table 2).

  In the verification, the sequence comparison software MEGA was used. The genome sequence used for the above verification was Bifidobacterium longum 21 except for two types of Bifidobacterium breve among the 23 types of Bifidobacterium used in the primer design of Example 1. It was of a kind.

  The results of verification are shown in Table 4 below. In Table 4, “+” indicates that a sequence matching both the forward primer and the reverse primer is present in the genome sequence of the relevant subspecies, and “−” does not exist.

  As shown in Table 4, it was found that the primers described in Non-Patent Documents 2, 4, 5, 7, and 8 are sequences that match the genome sequences of subspecies other than the Infantis subspecies. . Therefore, it is considered that the primers described in Non-Patent Documents 2, 4, 5, 7, and 8 cannot detect only the infantis in the classification of the four subspecies. Then, the following verification was further performed about the primer of a nonpatent literature 3 and 6.

[Verification by quantitative PCR reaction]
It was verified by quantitative PCR reaction whether the primers described in Non-Patent Documents 3 and 6 and the primers 1 and 2 used in Example 1 did not detect Bifidobacterium breve.

The primers described in Non-Patent Document 3 are the oligonucleotide consisting of the base sequence of SEQ ID NO: 3 and the oligonucleotide consisting of the base sequence of SEQ ID NO: 4 described below (hereinafter referred to as “Primer 3” and “Primer 4”, respectively) ).

The primers described in Non-Patent Document 6 are the oligonucleotide consisting of the base sequence of SEQ ID NO: 5 and the oligonucleotide consisting of the base sequence of SEQ ID NO: 6 described below (hereinafter referred to as “Primer 5” and “Primer 6”, respectively) ).

  As the primers 3 to 6, those synthesized by Thermo Fisher SCIENTIFIC were used. As primers 1 and 2, those used in Example 1 were used.

A quantitative PCR reaction solution having the composition shown in Table 5 below was prepared, heated at 95 ° C. for 20 seconds using Primer 1 and Primer 2 in an Applied Biosystems 7500 real-time PCR system (Thermo Fisher SCIENTIFIC), and then denatured at 95 ° C. 3 Real-time PCR was performed under the condition of 40 cycles under the conditions of second, annealing / extension at 60 ° C. for 30 seconds. The conditions of real-time PCR using primer 3 and primer 4 were 40 cycles under the conditions of 95 ° C. for 20 seconds, denaturation at 95 ° C. for 3 seconds, annealing at 55 ° C. for 20 seconds, and extension reaction at 72 ° C. for 30 seconds. The conditions of real-time PCR using primer 5 and primer 6 were 40 cycles under the conditions of 95 ° C. for 20 seconds, denaturation at 95 ° C. for 3 seconds, and annealing / extension at 63 ° C. for 30 seconds. As a template DNA for a calibration curve, Bifidobacterium longum subsp. Infantis JCM1222 DNA (5.8 × 10 ¹ , 5.8 × 10 ² , 5.8 × 10 ³ , 5.8 × 10 ⁴ , and 5.8 × 10 stages of ⁵ copies / μL) were used. Real-time PCR was performed for each of these five dilution series samples, Ct values at specific threshold values were obtained, and a calibration curve was created based on the Ct values. The Ct value obtained by performing real-time PCR using each of primers 1 and 2, primer 3 and 4, and primers 5 and 6 was applied to the calibration curve, and the bifido in the chromosomal DNA solution prepared from the test bacteria The amount of nucleic acid derived from Bacterium longum subspecies Infantis was determined.

The results of the quantitative PCR reaction are shown in Table 6 below. In Table 6, “+” indicates that the target region DNA copy number above the detection limit was detected, and “−” indicates that the target region DNA copy number above the detection limit was not detected. The detection limit is 5.8 × 10 ¹ copy / DNA solution 1 μl.

  As shown in Table 6, in the quantitative PCR using primers 1 and 2, only Bifidobacterium longum subspecies Infantis JCM1222 was detected and cross-reacted with any kind of Bifidobacterium breve. Did not show. Therefore, Bifidobacterium longum subspecies infantis is specifically detected by primers 1 and 2 without cross-reaction with Bifidobacterium breve. On the other hand, quantitative PCR using primers 3 and 4 and primers 5 and 6 detected Bifidobacterium longum subspecies Infantis JCM1222, but showed cross-reactivity with Bifidobacterium breve. Fidobacterium longum subspecies infantis could not be specifically detected.

  Quantitative PCR was carried out by the same method for the other 22 types of bacteria belonging to Bifidobacterium breve held by the applicant. As a result, when using primers 1 and 2, none of the Bifidobacterium breve was detected, but when primers 3 and 4 were used, 7 out of 22 types were detected. When 6 was used, 18 of 22 species were detected. From this result, it was confirmed that by using primers 1 and 2, Bifidobacterium longum subsp. Infantitis was specifically detected without cross-reaction with Fidobacterium breve. .

  This technology enables specific detection of Bifidobacterium longum subspecies infantis. Therefore, the present technology can be used for more reliable evaluation of the effectiveness of this bacterium, and for example, can be used for evaluation of the state of arrival in the intestine and the state of survival in the intestine. In addition, the present technology can also be used for the measurement of the number of bacteria and the management of the fermentation state when industrially producing Bifidobacterium longum sub-species Infantis or a food or drink containing the bacteria.

Claims (11)

  An oligonucleotide consisting of the base sequence of SEQ ID NO: 1.   An oligonucleotide consisting of the base sequence of SEQ ID NO: 2.   An oligonucleotide pair which is a combination of an oligonucleotide consisting of the base sequence of SEQ ID NO: 1 and an oligonucleotide consisting of the base sequence of SEQ ID NO: 2.   A nucleic acid amplification primer comprising an oligonucleotide pair which is a combination of an oligonucleotide having the base sequence of SEQ ID NO: 1 and an oligonucleotide having the base sequence of SEQ ID NO: 2.   The primer for nucleic acid amplification according to claim 4 for detecting Bifidobacterium longum subspecies infantis.   6. The primer for nucleic acid amplification according to claim 5, which does not show a cross-reaction with Bifidobacterium longum subspecies other than Bifidobacterium longum subspecies infantis.   The primer for nucleic acid amplification according to claim 5 or 6, which does not show a cross-reaction with Bifidobacterium breve.   A nucleic acid amplification kit comprising the nucleic acid amplification primer according to any one of claims 4 to 7.   The kit for nucleic acid amplification according to claim 8, for detecting Bifidobacterium longum subspecies infantis.   Using the oligonucleotide pair that is a combination of the oligonucleotide consisting of the base sequence of SEQ ID NO: 1 and the oligonucleotide consisting of the base sequence of SEQ ID NO: 2, the sample is subjected to a process for nucleic acid amplification, and the presence or absence of an amplification product Bifidobacterium longum subspecies infantis or Bifidobacterium longum subspecies infantis nucleic acid derived from the Bifidobacterium longum subspecies infantis Subspecies Infantis detection method. Using the oligonucleotide pair that is a combination of the oligonucleotide consisting of the base sequence of SEQ ID NO: 1 and the oligonucleotide consisting of the base sequence of SEQ ID NO: 2, the sample is subjected to a process for nucleic acid amplification, Bifidobacterium longum sub-species infantis or Bifidobacterium longum sub-species infantis-derived nucleic acid is quantified based on the Ct value of the treated sample. A method for quantifying Fidobacterium longum subspecies infantis.

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