WO2020188909A1 - Primer pair and detection method - Google Patents

Abstract

[Problem] The purpose of the present invention is to provide a novel technology capable of detecting Lactobacillus curvatus CP2998. [Solution] A primer pair for detecting the lactic acid bacterial strain Lactobacillus curvatus CP2998, the primer pair being constituted of a forward primer consisting of the base sequence given by SEQ ID NO: 1 in the Sequence Listing and a reverse primer consisting of the base sequence given by SEQ ID NO: 2 in the Sequence Listing.

Description

Primer pair and detection method

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

In recent years, various foods containing lactic acid bacteria have been developed, and some of these foods exert a function of adjusting physical condition by the action of lactic acid bacteria contained in the food (hereinafter, also referred to as "functional food"). ). 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.

Usually, for identification / detection of a bacterium (strain or strain), morphological observation and physiological / biochemical test by microscopic examination of the test bacterium and identification by phenotypic trait are performed according to the method described in Non-Patent Document 1. Will be done. However, the identification method based on the expression trait is very complicated, requires skill in judgment, and requires a great deal of time and labor. In addition, lactic acid bacteria in foods may be heated and killed in the process of cooking or sterilization, but it is difficult to apply heat-killed bacteria for identification by phenotypic traits.

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

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

Japanese Unexamined Patent Publication No. 03-236799

Bergey's Manual of Determinative Bacteriophage, Williams & Wilkins, Vol. 2, (1986) International Journal of Systematic Bacteriophage, Microbiology Society, Vol. 49, (1999), pp. 997-1007, P997-1007, International Journal of Systematic Bacteriophage, Microbiology Society, Vol. 49, P997-1007

The 16S rRNA sequence analysis method is often used for systematic classification of microorganisms, but the base 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, although the PFGE method is excellent in the ability to discriminate strains, there is a problem that it requires a certain amount of DNA for analysis and is difficult to apply in 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 a Lactobacillus carbatus CP2998 strain.

The gist of the present invention is as follows.

[1] A primer pair for detecting a lactic acid bacterium strain which is Lactobacillus carbatus CP2998 strain (accession number: NITE BP-02033), and a forward primer consisting of the nucleotide sequence represented by SEQ ID NO: 1 in the sequence listing. A primer pair composed of 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 of Lactobacillus carbatus CP2998 strain (accession number: NITE BP-02033), which is a sequence of a forward primer consisting of the nucleotide sequence represented by SEQ ID NO: 1 in the sequence listing and a sequence of the sequence listing. A primer pair composed of a reverse primer consisting of the base sequence represented by No. 2 is used to amplify a specific region of the DNA of the sample, and the amplification product obtained by amplifying the specific region of the DNA is Nla IV. A method for detecting a lactic acid strain, wherein the lactic acid strain is detected based on the length of the base sequence of the amplification product treated with Nla IV.

[3] The lactic acid bacterium strain is compared with the length of the base sequence of the amplification product treated with 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 bacterium strain according to [2].

According to the present invention, it is possible to provide a novel technique capable of detecting the Lactobacillus carbatus CP2998 strain.

It is a figure which shows the analysis result of the electrophoresis of Reference Example 1. FIG. It is an electrophoretic photograph of Reference Example 2. It is a figure which shows the analysis result of the electrophoresis of Example 1. FIG. It is an electrophoretic photograph of Example 2. It is a figure which shows the analysis result of the electrophoresis of Example 2.

First, the background to the completion of the present invention will be explained.

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

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 is a method of amplifying a plurality of different specific regions in DNA by PCR using a plurality of arbitrarily synthesized primers, and identifying and detecting from the length (band pattern) of the base sequence of the amplification product. Is.

However, the CP2998 strain, which became a heat-killed bacterium, could not be detected by the RAPD method. When this result was examined, it was found that the reason why the CP2998 strain could not be detected by the RAPD method was that the DNA of the heat-killed strain CP2998 was decomposed (cut) by heating.

In the process of manufacturing food (for example, cooking process, molding process, sterilization process), heat is usually applied to the food, so that the CP2998 strain becomes a heat-killed bacterium. Therefore, the present inventors considered that in order to detect the CP2998 strain contained in food, it is necessary to be able to detect it regardless of whether or not the DNA is degraded.

Therefore, the present inventors have focused on the PCR-RFLP method (PCR-Retention Fragment Length Polymorphism method). Then, among the DNA of the CP2998 strain, a specific region (hereinafter, also referred to as "polymorphic region") having a base sequence different from that of other lactic acid bacteria strains other than the CP2998 strain (hereinafter, also simply referred to as "other lactic acid bacterium strain") It was examined whether the CP2998 strain contained in food could be detected by using it. Here, the PCR-RFLP method is a method of treating a specific region of DNA amplified by PCR with a restriction enzyme and identifying / detecting it from the length of the base sequence of the DNA fragment.

As a result, among the plurality of polymorphic regions, the CP2998 strain among the lactic acid bacteria strains is a polymorphic region that is easily preserved (that is, difficult to be cleaved) even if DNA is decomposed in the food manufacturing process or the like. We found that there are polymorphic regions that cannot be cleaved with restriction enzymes. Further studies have been made focusing on this polymorphic region, and the present inventors have completed the present invention.

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

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

The primer pair of the present embodiment is a primer pair for detecting the CP2998 strain, and according to the primer pair of the present embodiment, the CP2998 strain contained in the 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 specimen as the CP2998 strain and / or identifying the specimen as not the CP2998 strain. The CP2998 strain is a lactic acid bacterium and is one of the Lactobacillus carbatus strains. The CP2998 strain was transferred to the National Institute of Technology and Evaluation Patent Microorganisms Depositary Center (Room 122, 2-5-8 Kazusakamatari, Kisarazu City, Chiba Prefecture, Japan) on April 15, 2015 under the accession number: NITE P-02033. It has been deposited domestically and has been internationally deposited at the above-mentioned Patent Microorganisms Depositary Center as the deposit number: NITE BP-02033 on April 23, 2019.

The primer pair of the present embodiment consists of a forward primer consisting of a base sequence represented by SEQ ID NO: 1 in the sequence listing (hereinafter, also simply referred to as “SEQ ID NO: 1”) and SEQ ID NO: 2 in the sequence listing (hereinafter, simply referred to as “sequence number 1”). It is a pair of primers composed of a reverse primer consisting of a base sequence represented by (No. 2).

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

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

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

In the present embodiment, substantially homology means homology to the extent that it can function as a primer capable of amplifying a specific region of DNA described later (specific region of FTL gene described later). That is, the primer pair of the present embodiment does not necessarily have to be composed of the forward primer (itself) of the base sequence represented by SEQ ID NO: 1 and the reverse primer (itself) of the base sequence represented by SEQ ID NO: 2. However, each primer may be one in which one or several (five or less) bases are substituted, added or deleted. Since the 3'terminal side of the forward primer may affect the recognition ability of the restriction enzyme described later, the GGTGC corresponding to the recognition sequence of the enzyme, GGNC (N indicates an arbitrary base), is used. It is preferably not substituted, added or deleted.

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

The primer pair of this embodiment is used for nucleic acid amplification using the DNA of a sample as a template. By performing nucleic acid amplification using the DNA of the sample as a template using the primer pair of the present embodiment, a specific region sandwiched between the base sequences corresponding to the sequences of each primer is amplified. Specifically, the specific region amplified by the primer pair of the present embodiment is also referred to as a partial region of the base sequence of the Formate tetrahydrofolate ligase (hereinafter, "specific region of the FTL gene"). ). The nucleotide sequence of the tetrahydrofolate formate ligase gene can be confirmed in the National Center for Biotechnology Information database.

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 CP2998 strain is one base different from the specific region of the FTL gene of other lactic acid bacteria strains, and cannot be cleaved with an 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 cleaved by the FTL restriction enzyme even if it is 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 an FTL restriction enzyme, it is possible to identify whether or not the sample is a CP2998 strain. (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 decomposed by heat or impact. Therefore, according to the primer pair of the present embodiment, the CP2998 strain can be detected even if the DNA is degraded due to the food manufacturing process or the like.

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

The detection method of this embodiment includes (1) amplification step, (2) enzyme treatment step, and (3) detection step.

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

Here, the sample refers to an organism that possesses DNA as genetic information, and specifically refers to a lactic acid bacterium. That is, in the detection method of the present embodiment, in addition to the CP2998 strain, another lactic acid bacterium strain may be used as a sample. The sample may be a live bacterium or a dead bacterium.

The DNA of the sample used in the amplification step may be at least a DNA containing a specific region of the FTL gene, and may be DNA decomposed 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 Neasy Blood & Tissue Kit (manufactured by QIAGEN), it becomes easy to extract DNA from a sample.

When the sample used in the amplification step is a live bacterium, DNA may be extracted (isolated) after culturing the sample. The culture is not particularly limited, but can be carried out using, for example, an agar medium such as MRS (deMan, Rogosa and Sharpe). The temperature of the culture is not particularly limited, but can be, for example, 25 to 37 ° C, preferably 30 ° C from the viewpoint of the optimum growth temperature. The period of culturing can be 1 to 2 days, and is preferably 1 day from the viewpoint of growth rate. DNA extraction can be performed using a sample collected by centrifuging the culture.

Here, when the sample is contained in the food, the sample separated from the food can be used for DNA extraction. For the separation of the sample, a conventional method can be used 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 for amplification of a specific region of the FTL gene. In PCR, DNA denaturation, annealing of each primer to the denatured DNA, and extension reaction of a specific region are performed, and these reactions are repeated for a plurality of cycles.

PCR can be performed by a known method, but it can be performed more easily by using, for example, a PCR reagent kit such as ExTaq (manufactured by Takara Bio Inc.).

When using such a kit, PCR can be performed under the recommended conditions of the kit used. When PCR is performed 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, and in the above-mentioned PCR reagent kit (ExTaq), 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 (ExTaq), for example, assuming that the total amount of the solution used for PCR is 50 μL, 5 μL of the reaction buffer solution attached to the kit and 4 μL of dNTP mixer (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 is preheated by a thermal cycler at 94 ° C. for 3 minutes, and then DNA denaturation is annealing at 94 ° C. for 30 seconds. Is carried out at 55 ° C. for 30 seconds, the extension reaction is carried out at 72 ° C. for 15 seconds, and these processes can be repeated for 35 cycles. The DNA of the sample may be dissolved in a buffer solution (for example, TE buffer solution or the like). In this case, for example, the concentration of DNA can be about 100 ng / μL.

(2) In the enzyme treatment step, the amplification product of the specific region (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 means that the restriction enzyme and the amplification product coexist in the presence of a buffer solution, and does not necessarily mean the treatment of cleaving the amplification product with the restriction enzyme. As the buffer solution, one suitable for the restriction enzyme to be used may be selected, and for example, it can be selected from the Universal Buffer series (manufactured by Takara Bio Inc.) and the RE Reaction Buffer series (manufactured by Nippon Gene Co., Ltd.). 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. Specifically, the FTL restriction enzyme is Nla IV (manufactured by New England Biolabs Japan).

Treatment of the amplified product with an FTL restriction enzyme can be performed by a known method. For example, when Nla IV (manufactured by New England Biolabs Japan) is used, the treatment with the FTL restriction enzyme of the amplification product is attached to Nla IV (manufactured by 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 is different from other lactic acid bacteria strains by one base, so that it is not recognized by the above-mentioned FTL restriction enzyme. Therefore, in the enzyme treatment step, the amplification product of the CP2998 strain is not cleaved with 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. Therefore, in the enzyme treatment 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, the 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 the amplification product subjected to the treatment of the above-mentioned treatment step. Specifically, when the sample is another lactic acid bacterium strain, the amplification product is cleaved by the treatment in the above-mentioned treatment step, so that the amplification product treated with the restriction enzyme refers to a fragment of the amplification product. Further, when the sample is the CP2998 strain, the amplification product is not cleaved by the treatment in the above-mentioned treatment step, so that the amplification product treated with the restriction enzyme refers to 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 the amplification product treated with the restriction enzyme (hereinafter, also referred to as "amplification product (after enzyme treatment)") is changed to the amplification product before the restriction enzyme treatment (hereinafter, "amplification product (hereinafter," amplification product ()). The CP2998 strain can be detected by comparing it with the length of the base sequence (before enzyme treatment) ”.

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

In the method described above, the length of the base sequence of the amplification product (after enzyme treatment) and the length of the amplification product (before enzyme treatment) can be compared by a known method. For example, the lengths of the base sequences can be compared by performing electrophoresis using a gel for electrophoresis and then visualizing the DNA in the gel for electrophoresis. To compare the length of the base sequence of the amplified product (after enzyme treatment) with the length of the amplified product (before enzyme treatment), for example, a microchip-type electrophoresis method using an Agilent 2100 bioanalyzer (Agilent) or the like. May be done by. The microchip-type electrophoresis method is a method for automating electrophoresis using a microchip incorporating a fine flow path.

The electrophoresis gel used for electrophoresis is not particularly limited, and known gels can be used, for example, 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, so that it is 4 from the viewpoint of fragment length. It is preferably 0.0%.

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

When electrophoresis is used, the difference in the length of the base sequence can be determined from the difference in the movement distance of DNA. Specifically, it can be determined that a DNA having a long distance traveled by the migration gel has a longer base sequence than a DNA having a short distance traveled by the migration gel. The position of the DNA may be determined by visually confirming the band of the visualized DNA, or by imaging it.

As a specific method using electrophoresis, for example, a method of performing electrophoresis on an agarose gel using a mixed solution of an amplification product and a staining reagent and observing a band of DNA visualized under a UV lamp is used. Can be mentioned. 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 electrophoresis may be any time as long as the target DNA bands are separated to the extent that they can be comparatively observed. For example, it can be 60 to 120 minutes, and 90 minutes from the viewpoint of comparison of DNA bands. Is preferable.

Here, 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) are compared by comparing the movement distances of the DNAs as described above. However, the lengths of the base sequences of the amplified products (after the enzyme treatment) and the amplified products (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-mentioned detection step, the lengths of the base sequences of the amplification product (after enzyme treatment) and the amplification product (before enzyme treatment) are compared to detect the CP2998 strain, but the method is limited to this method. is not. For example, the length of the base sequence of the amplification product (after enzyme treatment) of another lactic acid bacterium strain is obtained in advance, and the length of this base sequence and the length of the base sequence of the amplification product (after enzyme treatment) of the sample are obtained. You may compare. In this method, when the length of the base sequence of the amplification product (after enzyme treatment) of the sample is longer than the length of the base sequence of the amplification product (after enzyme treatment) of another lactic acid bacterium strain obtained in advance, the sample Can be identified as the CP2998 strain. On the other hand, if the length of the base sequence of the amplification product (after enzyme treatment) of the sample is not longer than the length of the base sequence of the amplification product (after enzyme treatment) of another lactic acid bacterium strain obtained in advance, the sample is It can be identified as another lactic acid 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 the food manufacturing process or the like.

The present invention will be described in more detail 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 bacterium (specimen) of the live bacterium was cultured in Lactobacillus MRS medium (manufactured by Difco) at 30 ° C. for 24 hours. DNA was extracted from the cultured cells according to the protocol recommended by the manufacturer using Neasy Blood & Tissue Kit (manufactured by QIAGEN). The culture solution was used as it was for DNA extraction.

In addition, a test bacterium (specimen) for heat-killed bacteria was prepared. DNA was extracted from the prepared cells according to the protocol recommended by the manufacturer using NeasyBlood & TissueKit (manufactured by QIAGEN).

The test strains used for DNA extraction were all Lactobacillus carbatus strains, but different strains were used. Specifically, Lactobacillus carbatas CP2998 strain, Lactobacillus carbatas 1096 ^T strain, Lactobacillus carbatas A14 strain and Lactobacillus carbatas A20 strain were 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) as a template. PCR was performed according to the manufacturer's recommended protocol.

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). 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.

The analysis results are shown in Fig. 1.

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

(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 the DNA of the heat-killed strain CP2998 was degraded (cut) by heating.

Therefore, in order to confirm the state of the DNA contained in the heat-killed bacteria, each DNA of the infectious cells extracted by the same method as in Reference Example 1 was electrophoresed. The electrophoresis was carried out at 100 V for 40 minutes using a 1 mass% agarose gel.

An electrophoretic photograph is shown in FIG.

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

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

The DNA of the test bacteria (live and dead) was extracted by the same method as in Reference Example 1. A sample for PCR was prepared using each of the extracted DNAs. The composition of the sample is shown in Table 1.

[Table 1]

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

PCR was performed using each of the above-mentioned samples. The PCR conditions are shown in Table 2.

[Table 2]

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

[Table 3]

[Table 4]

The amplified product (after enzyme treatment) and the amplified product (before enzyme treatment) were subjected to DNA analysis by microchip-type electrophoresis using an Agilent DNA1000 kit (manufactured by Agilent). 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 analysis results are shown in Fig. 3. In FIG. 3, "strain name-cut" refers to 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 did not change due to the treatment with the restriction enzyme regardless of whether it was a live bacterium or a heat-killed bacterium, and both were 153 bp. On the other hand, in the lactic acid strains of Lactobacillus carbatus other than the CP2998 strain, the length of the amplification product is shortened by the treatment with the restriction enzyme regardless of whether it is a live bacterium or a heat-killed bacterium, and the amplification product (enzyme treatment). The length of (after) was 135 bp.

From this result, it was understood that the CP2998 strain can be detected according to the primer pair of the present embodiment. Further, according to the detection method of the present embodiment, it was understood that the CP2998 strain can be detected even if the DNA is decomposed by heat.

(Example 2)
The CP2998 strain (heat-killed bacteria) is mixed with cellulose (binder), maltose (excipient), calcium stearate (lubricating agent), and silicon dioxide (anti-caking agent), and the obtained mixture is tableted (compressed). Molded) to obtain tablets. The obtained tablets were placed in a 15 mL tube and 10 mL of sterile water was added. The tablets in the solution is dissolved in an ultrasonic washer, and aliquoted into 1.5mL tube so that the number of bacteria is 2 × 10 ⁹ cells. DNA was extracted from the obtained CP2998 strain by the same method as in Reference Example 1.

In addition, DNA was extracted from the CP2998 strain (CP2998 strain before tableting) and the Lactobacillus carbatus 1096 ^T strain (heat-killed bacteria) used as raw materials for tablets by the same method as in Reference Example 1.

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

The electrophoretic photograph is shown in FIG. 4, and the analysis result by the bioanalyzer is shown in FIG.

As shown in FIGS. 4 and 5, in the CP2998 strain, there was no change in the length of the amplified product by the treatment with the restriction enzyme regardless of whether or not it was tableted, and both were 153 bp. From this result, it was understood that according to the primer pair of the present embodiment, the CP2998 strain can be detected even if the CP2998 strain is impacted.

Claims (3)

A primer pair for detecting a lactic acid bacterium strain, which is a Lactobacillus carbatus CP2998 strain.
A primer pair composed 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 method for detecting a lactic acid bacterium strain, which is a Lactobacillus carbatus CP2998 strain.
A specific region of the DNA of the sample is used by using a primer pair composed 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. Amplifies and
The amplification product obtained by amplifying a specific region of the DNA was treated with Nla IV.
The lactic acid bacterium strain is detected based on the length of the base sequence of the amplification product treated with Nla IV.
Method for detecting lactic acid bacteria strains. The lactic acid bacterium strain is detected by comparing the length of the base sequence of the amplification product treated with 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 bacterium strain according to claim 2.

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