JP2019216707A - Primer pair and detection method - Google Patents

Abstract

To provide novel technologies that can detect Lactobacillus curvatus CP2998 strain.SOLUTION: Provided is a primer pair for detecting a lactic acid bacteria strain that is Lactobacillus curvatus CP2998 strain (Accession number: NITE p-02033), the primer pair being composed of a forward primer consisting of a specific base sequence and a reverse primer consisting of another specific base sequence. Provided is a method for detecting lactic acid bacteria strain comprising amplifying a specific region of the sample DNA using the primer pair; treating with Nla IV the amplified product obtained by amplifying the specific region of the DNA; and detecting lactic acid bacteria strain, based on the base sequence length of the amplification product treated with the Nla IV.SELECTED DRAWING: Figure 3

Description

  The present invention relates to a primer pair for detecting Lactobacillus carvatas CP2998 strain and a method for detecting Lactobacillus carvatas CP2998 strain using the primer pair.

  In recent years, various foods containing lactic acid bacteria have been developed, and among those foods, foods that exert a function of adjusting physical condition by the action of lactic acid bacteria contained in the food (hereinafter, also referred to as “functional foods”) ). In order to provide such a functional food, it is necessary to establish a method for identifying and detecting a strain of lactic acid bacteria contained in the functional food.

  In general, for identification and detection of bacteria (strains and species), morphological observation and physiological / biochemical tests of test bacteria, ie, identification by phenotypic traits, are performed according to the method described in Non-Patent Document 1. Done. However, an identification method based on a phenotypic trait is very complicated, requires a skill of judgment, and requires a great deal of time and labor. In addition, lactic acid bacteria in foods may be killed by heating during cooking or sterilization, but it is difficult to apply heat-killed bacteria to identification by phenotype.

  Therefore, various methods for analyzing and analyzing base sequence information to identify and detect bacteria (strains and species) have been developed. Examples of such techniques include (a) 16S rRNA sequence analysis, (b) RAPD (Randomly Enhanced Polymorphic DNA), (c) PFGE (Pulsed-Field Gel Electrophoresis), and (d) ) PCR-RFLP method (PCR-Restriction Fragment Length Polymorphism method) and the like.

  (A) The 16S rRNA sequence analysis method is described in, for example, Patent Document 1, and is a method of identifying and detecting using a base sequence of 16S rRNA constituting a small subunit of ribosome. (B) The RAPD method is described in, for example, Non-Patent Document 2. A plurality of different specific regions in DNA are amplified by PCR using a plurality of primers arbitrarily synthesized, and the base sequence of the amplification product is amplified. This is a method of identifying and detecting from the length (band pattern). (C) The PFGE method is a method in which genomic DNA is treated with a restriction enzyme and identified and detected from the length (band pattern) of the base sequence of the DNA fragment. (D) The PCR-RFLP method is a method in which a specific region of DNA amplified by PCR is treated with a restriction enzyme and identified and detected from the length (band pattern) of the base sequence of the DNA fragment.

JP-A-03-236799

Bergey's Manual of Deterministic Bacteriology, Williams & Wilkins, Vol. 2, (1986) International Journal of Systematic Bacteriology, Microbiology Society, Vol. 49, (1999), pp. 997-1007.

  Although the 16S rRNA sequence analysis method is frequently used for phylogenetic classification of microorganisms, there is a problem that the nucleotide sequence of 16S rRNA is highly conserved within the same strain, and it is difficult to distinguish between different strains within the same strain. is there. Further, the PFGE method is excellent in discriminating ability of strains, but has a problem that it requires a certain amount of DNA for analysis, and is difficult to apply to food analysis containing only a small amount of lactic acid bacteria.

  An object of the present invention is to provide a novel technique capable of detecting Lactobacillus carvatas CP2998 strain.

  The gist of the present invention is as follows.

  [1] A primer pair for detecting a lactic acid bacterium strain that is Lactobacillus carvatas CP2998 strain (Accession number: NITE p-02033), which comprises a forward primer having a base sequence represented by SEQ ID NO: 1 in the sequence listing. A primer pair comprising a reverse primer consisting of the base sequence represented by SEQ ID NO: 2 in the sequence listing.

  [2] A method for detecting a lactic acid bacterium strain that is Lactobacillus carvatas CP2998 strain (Accession number: NITE p-02033), comprising a forward primer comprising a base sequence represented by SEQ ID NO: 1 in the sequence listing and a sequence in the sequence listing. Using a primer pair consisting of a reverse primer consisting of the base sequence represented by No. 2, a specific region of the sample DNA is amplified, and the amplified product obtained by amplifying the specific region of the DNA is converted into Nla IV. And detecting the lactic acid strain based on the length of the base sequence of the amplification product treated with Nla IV.

  [3] comparing the length of the base sequence of the amplification product treated with the Nla IV with the length of the base sequence of the amplification product obtained by amplifying the specific region of the DNA; The method for detecting a lactic acid bacteria strain according to [2], wherein the strain is detected.

  According to the present invention, a novel technique capable of detecting Lactobacillus carvatas CP2998 strain can be provided.

FIG. 7 is a view showing an analysis result of electrophoresis of Reference Example 1. 9 is an electrophoretic photograph of Reference Example 2. FIG. 3 is a view showing an analysis result of electrophoresis of Example 1. 9 is an electrophoretic photograph of Example 2. FIG. 9 is a view showing an analysis result of electrophoresis of Example 2.

  First, the process of completing the present invention will be described.

  Lactobacillus carvatas CP2998 strain (hereinafter, also simply referred to as “CP2998 strain”), which is a lactic acid bacterium, is known to suppress muscle degradation as described in JP-A-2016-216408. It is being considered to include the strain in foods. Therefore, it was necessary to establish a method for detecting the CP2998 strain contained in foods.

  Therefore, the present inventors first examined whether or not the CP2998 strain could be detected by the RAPD method (randomly amplified polymorphic DNA method), as shown in Examples described later. Here, the RAPD method uses a plurality of arbitrarily synthesized primers to amplify a plurality of different specific regions in DNA by PCR, and identifies and detects the amplified products from the base sequence length (band pattern). It is.

  However, the heat-killed CP2998 strain could not be detected by the RAPD method. When this result was examined, it was found that the CP2998 strain could not be detected by the RAPD method due to the fact that the DNA was degraded (cut) by heating in the heat-killed strain CP2998.

  In the process of producing food (eg, cooking, molding, and sterilizing processes), heat is usually applied to the food, so that the CP2998 strain becomes heat-killed. Therefore, the present inventors thought that in order to detect the CP2998 strain contained in food, it was necessary to be able to detect whether or not DNA was degraded.

  Therefore, the present inventors focused on the PCR-RFLP method (PCR-Restriction Fragment Length Polymorphism method). Then, in the DNA of the CP2998 strain, a specific region (hereinafter, also referred to as “polymorphic region”) having a different base sequence from other lactic acid bacteria strains other than CP2998 strain (hereinafter, also simply referred to as “other lactic acid bacteria strains”). It was examined whether or not CP2998 strain contained in food could be detected. Here, the PCR-RFLP method is a method in which a specific region of DNA amplified by PCR is treated with a restriction enzyme, and the DNA is identified and detected from the length of the base sequence of the DNA fragment.

  As a result, the polymorphic region is a polymorphic region that is easily preserved (that is, hardly cleaved) even if the DNA is degraded in the process of manufacturing food, among the polymorphic regions, and the CP2998 strain among the lactic acid bacteria strains We found that there are polymorphic regions that cannot be cleaved by restriction enzymes. The present inventors have further studied by focusing on this polymorphic region, and completed the present invention.

  Hereinafter, an embodiment of the present invention will be described.

  First, the primer pair of the present embodiment will be described.

  The primer pair of the present embodiment is a primer pair for detecting the CP2998 strain. According to the primer pair of the present embodiment, the CP2998 strain contained in food can be detected.

  In the present embodiment, detection refers to identifying whether or not the sample is the CP2998 strain. In other words, detection refers to identifying the sample as a strain CP2998 and / or identifying the sample as not a strain CP2998. The CP2998 strain is a lactic acid bacterium, and is one of Lactobacillus carbatas strains. The CP2998 strain has been deposited with the Patent Microorganisms Depositary on April 15, 2015 under the accession number: NITE P-02033.

  The primer pair of the present embodiment includes a forward primer consisting of a base sequence represented by SEQ ID NO: 1 (hereinafter, also simply referred to as “SEQ ID NO: 1”) of the sequence listing and a primer having SEQ ID NO: 2 (hereinafter, simply referred to as “SEQ ID NO: 1”). No. 2) is also included.

The nucleotide sequence of SEQ ID NO: 1 is as follows.
5'-GCGATGCTTTTAAAAGGTGC-3 '

The nucleotide sequence of SEQ ID NO: 2 is as follows.
5'- CGCAATATCACCCATTTTTAGG-3 '

  The forward primer consisting of the nucleotide sequence represented by SEQ ID NO: 1 includes not only the forward primer having the nucleotide sequence represented by SEQ ID NO: 1 but also the forward primer having a nucleotide sequence substantially homologous to the nucleotide sequence represented by SEQ ID NO: 1. Including. Similarly, the reverse primer consisting of the base sequence represented by SEQ ID NO: 2 is not only the reverse primer itself having the base sequence represented by SEQ ID NO: 2, but also the base primer having a base sequence substantially homologous to the base sequence represented by SEQ ID NO: 2. Includes reverse primer.

  In the present embodiment, “substantially homologous” means homology to such an extent that it can function as a primer capable of amplifying a specific region of DNA described later (a specific region of FTL gene described later). In other words, the primer pair of the present embodiment does not necessarily need to be constituted by the forward primer of the base sequence represented by SEQ ID NO: 1 (itself) and the reverse primer of the base sequence represented by SEQ ID NO: 2 (itself). Instead, each primer may have one or several (5 or less) bases substituted, added or deleted. Since the 3 'end of the forward primer may affect the recognition ability of a restriction enzyme described later, GGTGC corresponding to GGNNC (N indicates an arbitrary base), which is a recognition sequence of the enzyme, Preferably no substitutions, additions or deletions are made.

  Each primer constituting the primer pair of the present embodiment can be chemically synthesized by, for example, a known nucleic acid synthesis method, or can be synthesized by, for example, a DNA synthesizer.

  The primer pair of the present embodiment is used for nucleic acid amplification using the DNA of the sample as a template. By performing nucleic acid amplification using the DNA of the specimen as a template using the primer pair of the present embodiment, a specific region sandwiched between base sequences corresponding to the sequences of the respective primers is amplified. The specific region amplified by the primer pair of the present embodiment is specifically a partial region of the base sequence of the format tetrahydrofolate ligase (formate tetrahydrofolate ligase) gene (hereinafter, also referred to as “specific region of FTL gene”). ).

  The primer pair of the present embodiment can amplify not only the specific region of the FTL gene of the CP2998 strain but also the specific region of the FTL gene of another lactic acid bacterium strain having the FTL gene. However, the specific region of the FTL gene of the strain CP2998 is different from the specific region of the FTL gene of another lactic acid bacterium by one base, and cannot be cleaved by the FTL restriction enzyme described later. On the other hand, the specific region of the FTL gene of another lactic acid bacterium strain is recognized and cut by the FTL restriction enzyme even in Lactobacillus carbatas, which is the same strain as the CP2998 strain. Therefore, by using the primer pair of the present embodiment to amplify a specific region of the FTL gene of the sample and treating the amplified product with the FTL restriction enzyme, it is possible to identify whether or not the sample is strain CP2998. (That is, the CP2998 strain can be detected).

  Further, the specific region of the FTL gene amplified by the primer pair of the present embodiment is a region that is easily preserved even if the DNA is degraded by heat, impact, or the like. Therefore, according to the primer pair of the present embodiment, the CP2998 strain can be detected even if the DNA has been degraded during the food production process or the like.

  Next, a method for detecting the CP2998 strain using the primer pair of the present embodiment will be described.

  The detection method of the present embodiment includes (1) an amplification step, (2) an enzyme treatment step, and (3) a detection step.

  (1) In the amplification step, a specific region of the sample DNA is amplified using the primer pair of the present embodiment. The specific region of the DNA to be amplified in the amplification step is the above-described specific region of the FTL gene.

  Here, the specimen refers to an organism having DNA as genetic information, and specifically refers to a lactic acid bacterium. That is, in the detection method of the present embodiment, other than the CP2998 strain, another lactic acid bacteria strain may be used as the specimen. The specimen may be a live cell or a dead cell.

  The sample DNA used in the amplification step only needs to contain at least the specific region of the FTL gene, and may be DNA degraded by heat or impact (that is, a DNA fragment). As the DNA of the sample used in the amplification step, for example, DNA extracted (isolated) from the sample can be used.

  DNA extraction can be performed by a known method, and is not particularly limited. For example, by using a reagent kit such as the Easy Blood & Tissue Kit (manufactured by QIAGEN), DNA can be easily extracted from the specimen.

  When the specimen used in the amplification step is a living bacterium, extraction (isolation) of DNA may be performed after culturing the specimen. The culture is not particularly limited, but can be performed using an agar medium such as MRS (de Man, Rogosa and Sharpe). The culture temperature is not particularly limited, but may be, for example, 25 to 37 ° C, and is preferably 30 ° C from the viewpoint of the optimal growth temperature. The culturing period can be 1-2 days, and is preferably 1 day from the viewpoint of the growth rate. Extraction of DNA can be performed using a specimen collected by centrifuging the culture.

  Here, when a sample is contained in food, a sample separated from food can be used for DNA extraction. An ordinary method can be used for the separation of the sample depending on the composition of the food.

  In the amplification step, a nucleic acid amplification reaction, that is, PCR (Polymerase Chain Reaction) can be used to amplify a specific region of the FTL gene. In PCR, denaturation of DNA, annealing of each primer to the denatured DNA, and extension of a specific region are performed, and these reactions are repeated for a plurality of cycles.

  The PCR can be performed by a known method. For example, the PCR can be more easily performed by using a PCR reagent kit such as Ex Taq (manufactured by Takara Bio Inc.).

  When such a kit is used, PCR can be performed under the recommended conditions for the kit used. When performing PCR under the recommended conditions, it is preferable to consider the optimum conditions for the annealing temperature. Further, the extension time (extension reaction time) can be determined in consideration of the length of the base sequence of the amplification product. In the PCR reagent kit (Ex Taq) described above, the expected amplification product length is 1 kb. On the other hand, 60 seconds is recommended.

  When PCR is performed using the above-mentioned PCR reagent kit (Ex Taq), for example, when the total amount of the solution used for PCR is 50 μL, 4 μL of a reaction buffer solution provided with the kit, 4 μL of dNTP mixture (2.5 mM each), A reaction solution containing 1 μM of each primer, 500 ng of template DNA (sample DNA), and 2.5 U of Ex Taq was preheated by a thermal cycler at 94 ° C. for 3 minutes, and then the DNA was denatured at 94 ° C. for 30 seconds. Is carried out at 55 ° C. for 30 seconds, and the extension reaction is carried out at 72 ° C. for 15 seconds, and these treatments can be repeated for 35 cycles. The DNA of the sample may be dissolved in a buffer (for example, a TE buffer or the like). In this case, for example, the concentration of the DNA can be about 100 ng / μL.

  (2) In the enzyme treatment step, the amplification product of the specific region (the specific region of the FTL gene) amplified in the amplification step is treated with a restriction enzyme.

  In the enzyme treatment step, treating the amplification product with a restriction enzyme refers to coexistence of the restriction enzyme and the amplification product in the presence of a buffer, and does not necessarily refer to a treatment of cutting the amplification product with the restriction enzyme. In addition, what is necessary is just to select what is suitable for the restriction enzyme used as a buffer solution, for example, can be selected from Universal Buffer series (made by Takara Bio Inc.) and RE Reaction Buffer series (made by Nippon Gene). For example, CutSmart Buffer (manufactured by New England Biolabs Japan) can be used for Nla IV, which is a restriction enzyme described later.

  As the restriction enzyme, a restriction enzyme that recognizes a specific region of the FTL gene and does not recognize a specific region of the mutated FTL gene (hereinafter, also referred to as “FTL restriction enzyme”) is used. The FTL restriction enzyme is, specifically, Nla IV (manufactured by New England Biolabs Japan).

  The treatment of the amplification product with the FTL restriction enzyme can be performed by a known method. For example, when Nla IV (New England Biolabs Japan) is used, the treatment of the amplification product with the FTL restriction enzyme is performed using CutSmart attached to Nla IV (New England Biolabs Japan) when the total amount of the reaction solution is 50 μL. A reaction solution containing 5 μL of Buffer, 15 μL of amplification product, and 0.5 U of NlaIV may be treated with a thermal cycler at 37 ° C. for 60 minutes, and inactivated at 65 ° C. for 20 minutes.

  In the CP2998 strain, the specific region of the FTL gene differs from other lactic acid bacteria strains by one base, and thus is not recognized by the above-described FTL restriction enzyme. Therefore, in the processing step, the amplification product of the CP2998 strain is not cleaved by the FTL restriction enzyme. On the other hand, in other lactic acid bacteria strains, the FTL restriction enzyme recognizes a specific region of the FTL gene. For this reason, in the processing step, the amplification products of other lactic acid bacteria are cleaved with restriction enzymes to become fragments of the amplification products.

  (3) In the detection step, CP2998 strain is detected based on the length of the base sequence of the amplification product treated with the restriction enzyme (FTL restriction enzyme).

  Here, the amplification product treated with the FTL restriction enzyme refers to an amplification product that has been subjected to the treatment in the above-described treatment step. Specifically, when the specimen is another lactic acid bacteria strain, the amplification product is cleaved by the treatment in the above-described treatment step, and the amplification product treated with the restriction enzyme indicates a fragment of the amplification product. When the specimen is the CP2998 strain, the amplification product is not cleaved by the treatment in the above-described treatment step, and the amplification product treated with the restriction enzyme indicates the amplification product itself.

  In the detection step, the CP2998 strain may be detected using the length of the base sequence of the amplification product treated with the restriction enzyme, and is not particularly limited. For example, the length of the base sequence of an amplification product treated with a restriction enzyme (hereinafter, also referred to as “amplification product (after enzyme treatment)”) is changed to the amplification product before treatment with a restriction enzyme (hereinafter, “amplification product ( CP2998 strain can be detected by comparing the length of the base sequence (also referred to as "before enzyme treatment)").

  In this method, when the length of the base sequence of the amplification product (after the enzyme treatment) is the same as the length of the base sequence of the amplification product (before the enzyme treatment), the specimen can be identified as the CP2998 strain. On the other hand, when the length of the base sequence of the amplification product (after the enzyme treatment) is shorter than the length of the amplification product (before the enzyme treatment), the specimen can be identified as a lactic acid bacteria strain other than the CP2998 strain.

  In the method described above, the length of the base sequence of the amplification product (after the enzyme treatment) can be compared with the length of the amplification product (before the enzyme treatment) by a known method. For example, electrophoresis is performed using a gel for electrophoresis, and then the DNA in the gel for electrophoresis is visualized, whereby the lengths of the base sequences can be compared. The length of the base sequence of the amplification product (after the enzyme treatment) is compared with the length of the amplification product (before the enzyme treatment) by, for example, a microchip electrophoresis method using an Agilent 2100 bioanalyzer (Agilent) or the like. May be performed. Note that the microchip electrophoresis is a method in which electrophoresis is automated using a microchip in which a fine channel is incorporated.

  The gel for electrophoresis used for electrophoresis is not particularly limited, and a known gel can be used. For example, an agarose gel can be used. The concentration of agarose can be, for example, 3.0 to 5.0% by mass with respect to 100% by mass of the gel, and the length of the specific region of the FTL gene is about 150 bp. 0.0% is preferable.

  The method for visualizing DNA in the gel for electrophoresis is not particularly limited, and for example, a staining reagent for staining DNA can be used. For example, when GelRed (registered trademark) (manufactured by Biotium) is used as a staining reagent, it emits fluorescence when irradiated with ultraviolet rays.

  In the case of using electrophoresis, the difference in the length of the base sequence can be determined from the difference in the moving distance of the DNA. Specifically, it can be determined that the DNA that has traveled a long distance through the gel for electrophoresis has a longer base sequence than the DNA that has traveled a short distance through the gel for electrophoresis. The position of the DNA may be determined by visually confirming the visualized band of the DNA, or may be determined by imaging.

  As a specific method using electrophoresis, for example, there is a method of performing electrophoresis on an agarose gel using a mixture of an amplification product and a staining reagent, and observing a DNA band visualized under a UV lamp. No. The electrophoresis voltage can be, for example, 50 to 100 V, and is preferably 100 V from the viewpoint of simplicity of the test. The time for performing the electrophoresis may be a time in which the target DNA band is separated to an extent that can be compared and observed. For example, the time may be 60 to 120 minutes, and from the viewpoint of comparison of the DNA bands, 90 minutes It is preferable that

  Here, the comparison between the length of the base sequence of the amplification product (after the enzyme treatment) and the length of the base sequence of the amplification product (before the enzyme treatment) is performed by comparing the moving distance of the DNA as described above. Alternatively, the lengths of the base sequences of the amplified product (after the enzyme treatment) and the amplified product (before the enzyme treatment) may be compared. The length of the base sequence can be obtained by converting the moving distance of the DNA to be measured into the length of the base sequence.

  In the above-described detection step, the length of the base sequence of the amplified product (after the enzyme treatment) is compared with the length of the base sequence of the amplified product (before the enzyme treatment), and the CP2998 strain is detected. is not. For example, the length of the base sequence of the amplification product of another lactic acid bacterium strain (after the enzyme treatment) is obtained in advance, and the length of this base sequence and the length of the base sequence of the amplification product of the sample (after the enzyme treatment) are determined. You may compare. In this method, when the length of the base sequence of the amplified product of the sample (after the enzyme treatment) is longer than the length of the base sequence of the amplification product of another lactic acid bacterium strain obtained beforehand (after the enzyme treatment), Is a CP2998 strain. On the other hand, if the length of the base sequence of the amplification product of the sample (after the enzyme treatment) is not longer than the length of the base sequence of the amplification product of the other lactic acid bacteria strain (after the enzyme treatment) obtained in advance, It can be identified as another lactic acid bacteria strain.

  According to the detection method of the present embodiment described above, the CP2998 strain can be detected. Further, according to the detection method of the present embodiment, the CP2998 strain can be detected even if the DNA is degraded in a process of manufacturing a food or the like.

  The present invention will be described more specifically with reference to the following examples, but the present invention is not limited thereto.

  First, it was confirmed whether or not the CP2998 strain could be detected by the RAPD method using dead and live bacteria of a plurality of lactic acid bacteria strains.

  (Reference Example 1)

  The test cells (specimens) of live bacteria were cultured at 30 ° C. for 24 hours in a Lactobacilli MRS medium (manufactured by Difco). DNA was extracted from the cultured cells using a Easy Blood & Tissue Kit (manufactured by QIAGEN) according to the protocol recommended by the manufacturer. In addition, the culture solution was used as it was for DNA extraction.

  In addition, test bacteria (specimens) of heat-killed bacteria were prepared. DNA was extracted from the prepared cells using a Easy Blood & Tissue Kit (manufactured by QIAGEN) according to the protocol recommended by the manufacturer.

The test bacteria used for DNA extraction were all Lactobacillus carbatas, but a plurality of different strains were used. Specifically, Lactobacillus Karubatasu CP2998 strain, Lactobacillus Karubatasu 1096 ^T strain, Lactobacillus Karubatasu A14 strain and Lactobacillus Karubatasu A20 strain was used.

  PCR was performed using Ready-To-Go RAPD Analysis Kit (manufactured by GE Healthcare) using each DNA of the extracted cells (live and dead cells) as a template. PCR was performed according to the protocol recommended by the manufacturer.

  DNA analysis by microchip electrophoresis was performed using each amplification product obtained by PCR. DNA analysis by microchip electrophoresis was performed using an Agilent DNA1000 kit (manufactured by Agilent). In addition, GelRed (registered trademark) (manufactured by Biotium) was used as a reagent for staining DNA, and Agarose S (manufactured by Nippon Gene) was used as a gel for electrophoresis.

  FIG. 1 shows the analysis results.

  As can be understood from FIG. 1, the band pattern was different between the live bacteria and the heat-killed bacteria, so that the CP2998 strain could not be detected.

(Reference Example 2)
From the results of Reference Example 1, it was considered that the reason why the CP2998 strain could not be detected by the RAPD method was that DNA was degraded (cut) by heating in the heat-killed CP2998 strain.

  Then, in order to confirm the state of the DNA contained in the heat-killed bacteria, each DNA of the bacteria extracted in the same manner as in Reference Example 1 was subjected to electrophoresis. The electrophoresis was performed at 100 V for 40 minutes using a 1% by mass agarose gel.

  An electrophoresis photograph is shown in FIG.

  As can be understood from FIG. 2, it was confirmed that the DNA was degraded in the heat-killed bacteria.

(Example 1)
Next, it was confirmed whether or not the CP2998 strain could be detected by the PCR-RFLP method using the primer pair of the present embodiment.

  In the same manner as in Reference Example 1, DNA of test bacteria (live and dead) was extracted. A sample for PCR was prepared using each extracted DNA. Table 1 shows the composition of the sample.

[Table 1]

  As the F-primer shown in Table 1, a forward primer (itself) having a base sequence represented by SEQ ID NO: 1 chemically synthesized by a known nucleic acid synthesis method was used. As the R-primer shown in Table 1, a reverse primer (itself) having a base sequence represented by SEQ ID NO: 2 chemically synthesized by a known nucleic acid synthesis method was used. Note that Template shown in Table 1 indicates the extracted DNA.

  PCR was performed using each sample described above. Table 2 shows the PCR conditions.

[Table 2]

  Each of the obtained amplification products was treated with a restriction enzyme (NlaIV). Table 3 shows the composition of the solution used for the treatment with the restriction enzyme. Table 4 shows the conditions of the treatment with the restriction enzyme.

[Table 3]

[Table 4]

  The amplified product (after the enzyme treatment) and the amplified product (before the enzyme treatment) were subjected to DNA analysis by microchip electrophoresis using an Agilent DNA1000 kit (manufactured by Agilent). In addition, GelRed (registered trademark) (manufactured by Biotium) was used for DNA staining, and Agarose S (manufactured by Nippon Gene) was used for the gel for electrophoresis.

  The results of the analysis are shown in FIG. In FIG. 3, “strain name-cut” indicates an amplification product (after enzyme treatment), and “strain name” indicates an amplification product (before enzyme treatment).

  As can be understood from FIG. 3, in the CP2998 strain, the length of the amplified product was unchanged by treatment with the restriction enzyme, both live and heat-killed, and both were 153 bp. On the other hand, in lactic acid bacteria strains of Lactobacillus carbatas other than the CP2998 strain, the length of the amplified product is shortened by the treatment with the restriction enzyme, regardless of whether it is a living bacterium or a heat-killed bacterium. After) the length was 135 bp.

  From these results, it was understood that the primer pair of the present embodiment could detect the strain CP2998. Further, it was understood that according to the detection method of the present embodiment, the CP2998 strain could be detected even if the DNA was degraded by heat.

(Example 2)
CP2998 strain (heat-killed bacteria) was mixed with cellulose (binder), maltose (excipient), calcium stearate (lubricant), and silicon dioxide (anti-caking agent), and the resulting mixture was tableted (compressed). Molding) to obtain tablets. The obtained tablets were placed in a 15 mL tube, and 10 mL of sterilized water was added. The tablets in this solution were dissolved in an ultrasonic cleaner and dispensed into 1.5 mL tubes so that the number of bacteria was 2 × 10 ⁹ . DNA was extracted from the obtained CP2998 strain in the same manner as in Reference Example 1.

Further, CP2998 strain into tablets of raw material (CP2998 strain tableting) from Lactobacillus Karubatasu 1096 ^T strain (heat killed) in the same manner as in Reference Example 1, was extracted DNA.

  Using each extracted DNA, PCR was performed in the same manner as in Example 1, and a treatment with a restriction enzyme (NlaIV) was performed. The amplification product (after the enzyme treatment) and the amplification product (before the enzyme treatment) were subjected to electrophoresis. The electrophoresis was performed at 100 V for 90 minutes using a 4% by mass agarose gel.

  FIG. 4 shows an electrophoresis photograph, and FIG. 5 shows an analysis result by the bioanalyzer.

As shown in FIGS. 4 and 5, in the CP2998 strain, regardless of whether or not tableting was performed, the length of the amplification product was unchanged by treatment with the restriction enzyme, and both were 153 bp. From this result, it was understood that the primer pair of the present embodiment could detect the CP2998 strain even when the CP2998 strain was shocked.

Claims (3)

A primer pair for detecting a lactic acid bacteria strain which is Lactobacillus carvatas CP2998 strain (Accession number: NITE p-02033),
A primer pair comprising a forward primer consisting of the base sequence represented by SEQ ID NO: 1 in the sequence listing and a reverse primer consisting of the base sequence represented by SEQ ID NO: 2 in the sequence listing. A method for detecting a lactic acid bacterium strain that is Lactobacillus carvatas CP2998 strain (Accession number: NITE p-02033),
Using a primer pair consisting of a forward primer consisting of the base sequence represented by SEQ ID NO: 1 in the sequence listing and a reverse primer consisting of the base sequence represented by SEQ ID NO: 2 in the sequence listing, a specific region of the DNA of the specimen Amplify
An amplification product obtained by amplifying the specific region of the DNA is treated with Nla IV,
Detecting the lactic acid strain based on the length of the base sequence of the amplification product treated with the Nla IV,
Method for detecting lactic acid bacteria strain. The length of the base sequence of the amplification product treated with the Nla IV is compared with the length of the base sequence of the amplification product obtained by amplifying the specific region of the DNA to detect the lactic acid bacteria strain. A method for detecting a lactic acid bacterium strain according to claim 2.