JP6968776B2 - Method for discriminating species and strains of Lactobacillus microorganisms - Google Patents

Description

The present invention relates to a method for discriminating a specific species and a specific strain of a Lactobacillus microorganism.

Conventionally, it is a general method to identify bacterial species of bacteria or fungi by using DNA or RNA sequence analysis. As a method for identifying such a general strain, RiboPrinter, Pulsed Field Gel Electrophoresis (PFGE) method, Restion Fragment Length Polymerase (RFLP) method, and RFLP method, using the DNA of the viable cell of the purely cultured strain, are used. Polymorphic DNA (RAPD) method, Repetitive Element Palindromic PCR (Rep-PCR) method, Multilocas sexual typing (MLST) method, Multilocus sexual typing (MLST) method, Multilocus sexual analysis, etc.
Further, as an application of such bacterial species identification, Patent Document 1 discloses a DNA probe for identifying a bacterial strain of Lactobacillus herveticui species in a dairy product, and an attempt to prepare such a probe. ing. Further, in Patent Document 2, a probe prepared from the 16S rRNA gene or 16S rRNA of a novel microorganism belonging to the genus Thalassospira capable of promoting the decomposition and removal of environmental pollutants is used for bacteria having the ability to promote the decomposition of environmental pollutants. It is disclosed that detection and quantification can be performed easily and quickly, and that the polluted environment can be monitored and evaluated.
As described above, techniques for identifying bacterial species and strains of bacteria and fungi are required and developed in various situations.

Japanese Unexamined Patent Publication No. 03-236799 Japanese Unexamined Patent Publication No. 2006-230255

Development of a Tiered Multilocus Sequence Scheme Scheme for Members of the Lactobacillus acidophilus Complex, Applied and Electronic. 7220-7228 December 2013 Volume 79 Number 23

However, in beverages and foods such as soft drinks containing dead lactic acid bacteria, which are currently sold in large numbers, the bacterial cells are subject to pH and heating conditions during the manufacturing process, and pH and temperature conditions during the storage period. Since the DNA is degraded, there is a problem that it is difficult to obtain sufficient DNA necessary for identifying a bacterial species or strain. Then, it is extremely difficult to identify the bacterial species and strains contained in the manufactured products (that is, foods and beverages) required from the viewpoint of quality assurance.
Therefore, it is an object of the present invention to provide a method capable of detecting a specific species of a Lactobacillus microorganism (including dead bacteria) contained in a beverage or a food, or a specific strain of the specific species.

As a result of diligent research, the present inventors have not paid much attention to detect specific species or strains of bacterial cells (including dead bacteria) contained in beverages such as foods and beverages such as refreshing water. We have found that by using an RNA fragment of about 50-about 450 bp of the bacterial cells in the product, the DNA fragment sequence necessary for identifying the bacterial species or strain can be unexpectedly obtained. Completed.
That is, the present invention relates to the following [1] to [10].
[1] A method for detecting a specific species of a Lactobacillus microorganism (lactic acid bacterium) or a specific strain of a specific species of a Lactobacillus microorganism in a sample, which comprises the following steps, as a detection target microorganism, as follows. Process:
i) Step of extracting RNA from a sample,
ii) A step of synthesizing cDNA using the RNA extracted in i) as a template,
iii) Using the cDNA synthesized in ii) as a template, PCR is performed using a primer set consisting of two or more primer pairs capable of amplifying a gene region of about 50 bp to about 450 bp characteristic of the detection target microorganism. And iv) Detected in the sample if any of the sequences in the region amplified by each primer pair in the primer set are identical to each of the sequences of the detection target microorganism amplified by the same primer pair. The process of determining that the target lactic acid bacterium was present,
The method described above.
[2] The method according to [1] above, wherein the sample is a beverage.
[3] The method according to the above [1], wherein the sample is heat sterilized and stored at 25 ° C. for a period of 12 months or less, or is stored under conditions equivalent thereto.
[4] A method for determining a strain of a lactic acid bacterium Lactobacillus amylovorus in a sample.
i) Step of extracting RNA from a sample,
ii) A step of synthesizing cDNA using the RNA extracted in step i) as a template,
iii) Using the cDNA synthesized in step ii) as a template
Primer set A consisting of a primer pair (1) and (3):
Primer pair for amplifying a part of the gyrB gene sequence (1):
Forward primer AAAAAATCGGTACGGTTCCACTTA (SEQ ID NO: 1) and reverse primer TAAAGCGGTCTTAAATCCAGCTTC (SEQ ID NO: 2);
Primer pair for amplifying a part of the pyrG gene sequence (3):
Forward primer AATGCGTCGTGAAGTTGGTGAA (SEQ ID NO: 5) and reverse primer TGGCTTTGGACTTTCAAGGTGC (SEQ ID NO: 6)
Primer set B consisting of primer pairs (1) and (4):
Primer pair for amplifying a part of the gyrB gene sequence (1):
Forward primer AAAAAATCGGTACGGTTCCACTTA (SEQ ID NO: 1) and reverse primer TAAAGCGGTCTTAAATCCAGCTTC (SEQ ID NO: 2);
Primer pair for amplifying a part of the pyrG gene sequence (4):
Forward primer AATGCGTCGTGAAGTTGGTGAA (SEQ ID NO: 5) and reverse primer GCGTCAGTAACTGAAATGTAGGCATC (SEQ ID NO: 7)
Primer set C consisting of primer pairs (2) and (3):
Primer pair for amplifying a part of the gyrB gene sequence (2):
Forward primer CCGATGTTTACAAGTTTGAAGGTG (SEQ ID NO: 3) and reverse primer AAGCGGCTAAAGGTTTCAGAGAAT (SEQ ID NO: 4);
Primer pair for amplifying a part of the pyrG gene sequence (3):
Forward primer AATGCGTCGTGAAGTTGGTGAA (SEQ ID NO: 5) and reverse primer TGGCTTTGGACTTTCAAGGTGC (SEQ ID NO: 6)
And-Primer set D consisting of primer pairs (2) and (4):
Primer pair for amplifying a part of the gyrB gene sequence (2):
Forward primer CCGATGTTTACAAGTTTGAAGGTG (SEQ ID NO: 3) and reverse primer AAGCGGCTAAAGGTTTCAGAGAAT (SEQ ID NO: 4);
Primer pair for amplifying a part of the pyrG gene sequence (4):
Forward primer AATGCGTCGTGAAGTTGGTGAA (SEQ ID NO: 5) and reverse primer GCGTCAGTAACTGAAATGTAGGCATC (SEQ ID NO: 7)
A step of performing PCR using at least one primer set selected from the group consisting of, and
iv): The sequence of the region amplified by the primer pair (1) (excluding the primer sequence) and the sequence of SEQ ID NO: 10 are identical and / or the region amplified by the primer pair (2). When the sequence (excluding the primer sequence) and the sequence of SEQ ID NO: 11 are the same, and the sequence of the region amplified by the primer pair (3) (excluding the primer sequence) and the sequence of SEQ ID NO: 12 are the same. And / or, when the sequence of the region amplified by the primer pair (4) (excluding the primer sequence) and the sequence of SEQ ID NO: 13 are the same, in L. amylovorus. The above method comprising a step of determining a strain.

[5] Further, using the cDNA synthesized in the step ii) as a template,
Primer set F consisting of primer pairs (7) and (9):
Primer pair for amplifying a part of the lepA gene sequence (7):
Forward primer GATGCAGCTTTAGAATTTGAACC (SEQ ID NO: 17) and reverse primer AGGCTCTTGCACTTCCTTGTATTC (SEQ ID NO: 18);
Primer pair for amplifying a part of the gyrA gene sequence (9) :.
Forward primer TTAGCTAAGGCTGAAGCAAGAGCA (SEQ ID NO: 20) and reverse primer CCAAK CCGGTCAAACGAACTAAAC (SEQ ID NO: 21)
Primer set G consisting of primer pairs (7) and (10):
Primer pair for amplifying a part of the lepA gene sequence (7):
Forward primer GATGCAGCTTTAGAATTTGAACC (SEQ ID NO: 17) and reverse primer AGGCTCTTGCACTTCCTTGTATTC (SEQ ID NO: 18);
Primer pair for amplifying a part of the gyrA gene sequence (10) :.
Forward primer GAAGCAAGAGCACACATTCTCGA (SEQ ID NO: 22) and reverse primer CCAAKCCGGTCAAACGAACTAAAC (SEQ ID NO: 21)
Primer set H consisting of primer pairs (8) and (9):
Primer pair for amplifying a part of the lepA gene sequence (8):
Forward primer TGCAGCTTTAGAATTTGAACCTG (SEQ ID NO: 19) and reverse primer AGGCTCTTGCACTTCCTTGTATTC (SEQ ID NO: 18);
Primer pair for amplifying a part of the gyrA gene sequence (9) :.
Forward primer TTAGCTAAGGCTGAAGCAAGAGCA (SEQ ID NO: 20) and reverse primer CCAAKCCGGTCAAACGAACTAAAC (SEQ ID NO: 21)
And-Primer set I consisting of primer pairs (8) and (10):
Primer pair for amplifying a part of the lepA gene sequence (8):
Forward primer TGCAGCTTTAGAATTTGAACCTG (SEQ ID NO: 19) and reverse primer AGGCTCTTGCACTTCCTTGTATTC (SEQ ID NO: 18);
Primer pair for amplifying a part of the gyrA gene sequence (10) :.
Forward primer GAAGCAAGAGCACACATTCTCGA (SEQ ID NO: 22) and reverse primer CCAAKCCGGTCAAACGAACTAAAC (SEQ ID NO: 21)
PCR was performed using at least one primer set selected from the group consisting of
The sequence of the region amplified by the primer pair (7) (excluding the primer sequence) and the sequence of SEQ ID NO: 46 are the same, and / or the sequence of the region amplified by the primer pair (8) (primer). (Excluding the sequence) and the sequence of SEQ ID NO: 47 are the same, and the sequence of the region amplified by the primer pair (9) (excluding the primer sequence) and the sequence of SEQ ID NO: 48 are the same. And / or, when the sequence of the region amplified by the primer pair (10) (excluding the primer sequence) and the sequence of SEQ ID NO: 49 are the same, the strain in L. amylovorus is used. The method according to [4] above, further comprising a step of determining.
[6] A method for detecting the lactic acid bacterium Lactobacillus amylovorus CP1563 strain in a sample.
i) Step of extracting RNA from a sample,
ii) A step of synthesizing cDNA using the RNA extracted in step i) as a template,
iii) Using the cDNA synthesized in step ii) as a template
Primer set E consisting of primer pairs (5) and (6):
Primer pair for amplifying a part of the gyrB gene sequence (5):
Forward primer GTATCGACGTTGAAGTAGCTTTG (SEQ ID NO: 8) and reverse primer TAAAGCGGTCTTAAATCCAGCTTC (SEQ ID NO: 2);
Primer pair for amplifying a part of the pyrG gene sequence (6):
Forward primer AATGCGTCGTGAAGTTGGTGAA (SEQ ID NO: 5) and reverse primer TTGAATACCGATGCTGCGAAG (SEQ ID NO: 9)
And the process of performing PCR using
iv) The sequence of the region amplified by the primer pair (5) (excluding the primer sequence) is the same as the sequence of SEQ ID NO: 14, and the sequence of the region amplified by the primer pair (6) (primer sequence). When is the same as the sequence of SEQ ID NO: 15, L. The process of determining that the amylovourus CP1563 strain was present,
The method described above.
[7] The method according to [6] above, wherein in step iii), PCR using the primer pair (5) and PCR using the primer pair (6) are performed in separate containers.
[8] In step iv), the sequence of the region amplified by the primer pair (6) (excluding the primer sequence) was bound downstream of the sequence of the region amplified by the primer pair (5) (excluding the primer sequence). The method according to [6] above, wherein the identity between the fusion sequence and the sequence of SEQ ID NO: 16 is determined.

[9] L.I. amylovorus CP1563 strain detection kit:
Primer pair for amplifying a part of the gyrB gene sequence (5):
Forward primer GTATCGACGTTGAAGTAGCTTTG (SEQ ID NO: 8) and reverse primer TAAAGCGGTCTTAAATCCAGCTTC (SEQ ID NO: 2),
and,
Primer pair for amplifying a part of the pyrG gene sequence (6):
Forward primer AATGCGTCGTGAAGTTGGTGAA (SEQ ID NO: 5) and reverse primer TTGAATACCGATGCTGCGAAG (SEQ ID NO: 9).
[10] A primer set consisting of the following primer pairs (5) and (6):
Primer pair for amplifying a part of the gyrB gene sequence (5):
Forward primer GTATCGACGTTGAAGTAGCTTTG (SEQ ID NO: 8) and reverse primer TAAAGCGGTCTTAAATCCAGCTTC (SEQ ID NO: 2),
and,
Primer pair for amplifying a part of the pyrG gene sequence (6):
Forward primer AATGCGTCGTGAAGTTGGTGAA (SEQ ID NO: 5) and reverse primer TTGAATACCGATGCTGCGAAG (SEQ ID NO: 9).

According to the method of the present invention, a specific species of a Lactobacillus microorganism (including dead bacteria) contained in a beverage or a specific strain of the specific species can be detected.

It is a figure which briefly schematicized the region amplified by each primer pair (1)-(6) used in this invention. Based on the regions amplified by the primer pairs (1) to (4), Accession Nos. From the sequence, the pyrG gene sequence was connected after the gyrB gene sequence for each strain, processed into one gene sequence for each strain to create comparative sequence data, and created using MEGA6 sequence analysis software. , Shows a phylogenetic tree. The phylogenetic tree created by using MEGA6 sequence analysis software with respect to the sequences of the regions amplified by the primer pairs (5) and (6) (gene sequences of SEQ ID NO: 16 and SEQ ID NOs: 23 to 33) is shown. It is a figure which shows the result of having performed the electrophoresis on the agarose gel with respect to the obtained 4 kinds of PCR products in Example 1. It is a figure which shows the result of having performed the electrophoresis on the agarose gel according to the standard method for each 2 kinds of PCR products obtained from 4 kinds of samples (a) to (d) in Example 8. Based on the regions amplified by the primer pairs (1), (3), (7) and (9), Accession Nos. From the sequence, the pyrG gene sequence is connected after the gyrB gene sequence for each strain, and the Accession No. in Table 2 is further followed. The lepA gene sequence and gyrA gene sequence for each strain were linked from the sequence, processed into one gene sequence for each strain to create comparative sequence data, and created using MEGA6 sequence analysis software. Shows a phylogenetic tree. It is a figure which shows the result of having performed the electrophoresis on the obtained PCR product with the agarose gel according to the standard method in Example 10.

The present inventors used RT-PCR (reverse transcription polymerase chain reaction) as an amplification target for a relatively short region using RNA remaining in the sample as a template, even if the sample has undergone a process such as sterilization treatment or long-term storage. By doing so, it has been found that a specific species of the genus Lactobacillus or a specific strain of a specific species of the genus Lactobacillus can be specifically detected.
In particular, the present inventors have RT-PCR targeting a region relatively short with respect to RNA remaining in the sample, even if the sample has undergone a process such as sterilization and / or long-term storage for several months or longer. By performing L. amylovorus or L. It was found that the amylovourus CP1563 strain can be specifically detected. Long-term storage refers to storage under, for example, 1 month or longer, 3 months or longer, preferably 3 months to 6 months, more preferably 3 months to 12 months or similar conditions, and the sample is particularly for beverages and foods. In the case, "long-term storage" also includes storage for a period from production to expiration date, from production to expiration date, or longer than these. Then, if the sample is placed in a storage environment of 25 ° C. or an environment equivalent thereto, there is no problem if it is within 9 months, at least 6 months, for example, and L. amylovorus or L. The amylovourus CP1563 strain can be specifically detected. The storage temperature is usually 4 to 35 ° C. However, even if the sample is stored under a harsh temperature condition of, for example, 55 ° C., if it is about 3 to 5 days, depending on the type of primer set, L. amylovorus or L. The amylovourus CP1563 strain may be specifically detected.
Therefore, according to the present invention, a specific strain or strain of the genus Lactobacillus can be specifically detected using RT-PCR. In particular, according to the present invention, using RT-PCR, L. amylovorus or L. The amylovourus CP1563 strain can be specifically detected.
According to the present invention, L. amylovorus, especially L. Samples capable of specifically detecting the amylovourus CP1563 strain include foods, beverages, feeds and the like containing lactic acid bacteria (lactic acid bacteria (microbes belonging to the genus Lactobacillus)), particularly foods, beverages and feeds containing dead lactic acid bacteria. Is done. The beverage in the present invention includes fermented milk, fermented milk (sterilized), dairy product lactic acid bacteria beverage, dairy product lactic acid bacteria beverage (sterilized), lactic acid bacteria beverage and refreshing drinking water.
As used herein, "specifically detected" refers to other Lactobacillus species or L. et al. Other strains of amylovorus and L. amylovorus or L. In distinctive feature, L. amylovourus CP1563 strain. amylovorus or L. It refers to detecting a specific strain of amylovorus. For example, "specifically detecting the L. amylovourus CP1563 strain" means that L. amylovorus CP1563 strain is specifically detected. Other strains of amylovorus and L. In distinctive feature from the amylovourus CP1563 strain, L. It refers to detecting the amylovourus CP1563 strain. In addition, "specifically detecting L. amylovourus" means that other Lactobacillus species and L. amylovorus are specifically detected. The L. in the sample is distinguished from amylovorus. It means to detect amylovorus.

In the method of the present invention, using one or more sets of primer pairs, RT-PCR is performed using RNA derived from an organism that can be contained in the sample as a template to determine the sequence of the amplified sequence. If one set of primer pairs does not provide sufficient specificity, then two or more sets of primer pairs (each set of primer pairs may be a combination of further combinations of primer pairs that amplify other regions of the same gene or another gene. , Each consisting of a forward primer and a reverse primer). In the present invention, it is preferable to perform PCR as a primer set by combining two or more sets of primer pairs in order to obtain sufficient specificity. For each primer pair that constitutes a primer set, the sequence amplified by this primer pair is the identification of a specific strain of the detection target (for example, a specific species of the genus Lactobacillus) or a specific strain (for example, a specific species of the specific genus Lactobacillus). Strain) and other strains or strains are selected to be different from at least one. For each primer pair, the gene sequence of the target strain or strain is obtained from a known database, the same gene sequence of another strain or strain is obtained, and these are aligned to obtain the target strain or strain. It can be designed by identifying characteristic sequences, preferably sequences that are as specific as possible. A computer program that outputs primer sequence candidates for a given sequence can also be used for primer design. The length of the region amplified by each primer pair is generally preferably from about 50 bp to about 450 bp, more preferably from about 80 bp to about 450 bp, even more preferably from about 100 bp to about 450 bp, even more preferably from about 100 bp to about 350 bp, in particular. About 100 to about 250 bp is preferable. If the length of the region to be amplified is set to be longer than 450 bp, RNA of that length may not remain in the beverage, and it may not be possible to amplify to a desired amount.

By selecting the sequence and number of primer pairs contained in each primer set as described above, a particular species (eg, a particular species of the genus Lactobacillus), or a particular strain of a particular species (eg, a particular species of the genus Lactobacillus) can be identified. A specific strain of the species) can be identified in the sample.
The PCR-amplified sequence is compared to the sequence of each gene region of the detection target strain or strain (eg, L. amylovourus CP1563 strain) amplified by the same primer set. It is the target strain of interest that the sequence of two or more gene regions amplified from the sample by two or more primer pairs matches the sequence of the gene region of the detection target strain or strain amplified by the same primer pair, respectively. Or it can be an index for identifying a strain. At least one of the (two or more) sequences of two or more gene regions amplified using a set containing two or more primer pairs matches the sequence of the gene region of the detection target strain or strain amplified by the same primer pair. If not, it is not recognized that the detection target strain or strain was present in the sample.

Specifically, one of the following primer sets was used for PCR to L. It is amylovourus, and the strain in it can be specifically detected.
<Primer set A>
Primer pair for amplifying a part of the gyrB gene sequence (1):
Forward primer AAAAAATCGGTACGGTTCCACTTA (SEQ ID NO: 1) and reverse primer TAAAGCGGTCTTAAATCCAGCTTC (SEQ ID NO: 2);
Primer pair for amplifying a part of the pyrG gene sequence (3):
Forward primer AATGCGTCGTGAAGTTGGTGAA (SEQ ID NO: 5) and reverse primer TGGCTTTGGACTTTCAAGGTGC (SEQ ID NO: 6)

<Primer set B>
Primer pair for amplifying a part of the gyrB gene sequence (1):
Forward primer AAAAAATCGGTACGGTTCCACTTA (SEQ ID NO: 1) and reverse primer TAAAGCGGTCTTAAATCCAGCTTC (SEQ ID NO: 2);
Primer pair for amplifying a part of the pyrG gene sequence (4):
Forward primer AATGCGTCGTGAAGTTGGTGAA (SEQ ID NO: 5) and reverse primer GCGTCAGTAACTGAAATGTAGGCATC (SEQ ID NO: 7)

<Primer set C>
Primer pair for amplifying a part of the gyrB gene sequence (2):
Forward primer CCGATGTTTACAAGTTTGAAGGTG (SEQ ID NO: 3) and reverse primer AAGCGGCTAAAGGTTTCAGAGAAT (SEQ ID NO: 4);
Primer pair for amplifying a part of the pyrG gene sequence (3):
Forward primer AATGCGTCGTGAAGTTGGTGAA (SEQ ID NO: 5) and reverse primer TGGCTTTGGACTTTCAAGGTGC (SEQ ID NO: 6)

<Primer set D>
Primer pair for amplifying a part of the gyrB gene sequence (2):
Forward primer CCGATGTTTACAAGTTTGAAGGTG (SEQ ID NO: 3) and reverse primer AAGCGGCTAAAGGTTTCAGAGAAT (SEQ ID NO: 4);
Primer pair for amplifying a part of the pyrG gene sequence (4):
Forward primer AATGCGTCGTGAAGTTGGTGAA (SEQ ID NO: 5) and reverse primer GCGTCAGTAACTGAAATGTAGGCATC (SEQ ID NO: 7)

<Primer set E>
Primer pair for amplifying a part of the gyrB gene sequence (5):
Forward primer GTATCGACGTTGAAGTAGCTTTG (SEQ ID NO: 8) and reverse primer TAAAGCGGTCTTAAATCCAGCTTC (SEQ ID NO: 2);
Primer pair for amplifying a part of the pyrG gene sequence (6):
Forward primer AATGCGTCGTGAAGTTGGTGAA (SEQ ID NO: 5) and reverse primer TTGAATACCGATGCTGCGAAG (SEQ ID NO: 9)

<Primer set F>
Primer pair for amplifying a part of the lepA gene sequence (7):
Forward primer GATGCAGCTTTAGAATTTGAACC (SEQ ID NO: 17) and reverse primer AGGCTCTTGCACTTCCTTGTATTC (SEQ ID NO: 18);
Primer pair for amplifying a part of the gyrA gene sequence (9) :.
Forward primer TTAGCTAAGGCTGAAGCAAGAGCA (SEQ ID NO: 20) and reverse primer CCAAKCCGGTCAAACGAACTAAAC (SEQ ID NO: 21)

<Primer set G>
Primer pair for amplifying a part of the lepA gene sequence (7):
Forward primer GATGCAGCTTTAGAATTTGAACC (SEQ ID NO: 17) and reverse primer AGGCTCTTGCACTTCCTTGTATTC (SEQ ID NO: 18);
Primer pair for amplifying a part of the gyrA gene sequence (10) :.
Forward primer GAAGCAAGAGCACACATTCTCGA (SEQ ID NO: 22) and reverse primer CCAAKCCGGTCAAACGAACTAAAC (SEQ ID NO: 21)

<Primer set H>
Primer pair for amplifying a part of the lepA gene sequence (8):
Forward primer TGCAGCTTTAGAATTTGAACCTG (SEQ ID NO: 19) and reverse primer AGGCTCTTGCACTTCCTTGTATTC (SEQ ID NO: 18);
Primer pair for amplifying a part of the gyrA gene sequence (9) :.
Forward primer TTAGCTAAGGCTGAAGCAAGAGCA (SEQ ID NO: 20) and reverse primer CCAAKCCGGTCAAACGAACTAAAC (SEQ ID NO: 21)

<Primer set I>
Primer pair for amplifying a part of the lepA gene sequence (8):
Forward primer TGCAGCTTTAGAATTTGAACCTG (SEQ ID NO: 19) and reverse primer AGGCTCTTGCACTTCCTTGTATTC (SEQ ID NO: 18);
Primer pair for amplifying a part of the gyrA gene sequence (10) :.
Forward primer GAAGCAAGAGCACACATTCTCGA (SEQ ID NO: 22) and reverse primer CCAAKCCGGTCAAACGAACTAAAC (SEQ ID NO: 21)

<(I) Primer sets A to D>
L. amplified by primer pair (1). The sequence of the gyrB gene region of amylloverus CP1563 is shown in SEQ ID NO: 10 and is amplified by primer pair (2). The sequence of the gyrB gene region of amylloverus CP1563 is shown in SEQ ID NO: 11 and is amplified by primer pair (3). The sequence of the pyrG gene region of amylloverus CP1563 is shown in SEQ ID NO: 12, which is amplified by primer pair (4). The sequence of the pyrG gene region of amylloverus CP1563 is shown in SEQ ID NO: 13 (none of which contains the primer sequence).
In addition, BLAST analysis is performed with a combination of the gyrB gene sequence amplified by the primer pair (1) or (2) and the pyrG gene sequence amplified by the primer pair (3) or (4) by the method described later. Regarding the gyrB gene sequence, the top 12 strains with high sequence identity were L. Amylovourus 11 strain and L. It is a sobrius 1 strain, and its sequence identity is 98-100%. Besides that, L. kitasatonis, L. et al. crispatus, L. et al. gallinarum, L. et al. Although acidophilus and the like can be confirmed at the top, the sequence identity is about 84-91%. Regarding the pyrG gene sequence, the top 11 strains with high sequence identity were L. It is amylovorus and its sequence identity is 99-100%. Besides that, L. acidophilus, L .; gallinarum, L. et al. Helveticus and the like were confirmed at the top, but their sequence identity was about 90-91%.
From the above, when BLAST analysis is performed with a combination of the gyrB gene sequence amplified by the primer pair (1) or (2) and the pyrG gene sequence amplified by the primer pair (3) or (4), it is registered in GeneBank. L. The sequences of the PCR products obtained by any of the above primer sets A to D with the sequences of any of the 12 strains of amylovorus have 98 to 100% sequence identity, but L. It has only about 84 to 91% sequence identity with the sequences of microorganisms of the genus Lactobacillus other than amylovourus.

The region amplified by each primer pair is schematically shown in FIG. Note that FIG. 1 is a simple schematic and does not exactly correspond to the actual primer length. As can be seen from FIG. 1, among the primer sets A to D, the sequence obtained by the primer set D consisting of the combination of the primer pair (2) and the primer pair (4) is the longest, and the primer pair (1) is used. The sequence obtained by the primer set A consisting of the combination of the primer pair (3) is the shortest. L. In order to specifically detect the amylovourus CP1563 strain, it is particularly preferable to use primer set A.

For example, RT-PCR was performed on the sample using the primer set A, and the sequence of the region amplified by the primer pair (1) (excluding the primer sequence) was the same as the sequence of SEQ ID NO: 10 and the primer was used. When the sequence of the region amplified by pair (3) (excluding the primer sequence) is the same as the sequence of SEQ ID NO: 12, the lactic acid bacterium in the sample is L. It can be determined to be the amylovourus CP1563 strain. Further, for example, using a fusion sequence in which the sequence of the region amplified by the primer pair (3) (excluding the primer sequence) is bound downstream of the sequence of the region amplified by the primer pair (1) (excluding the primer sequence). Identity may be determined.

In addition, RT-PCR was performed on the sample, RT-PCR was performed using the primer set B, and the sequence of the region amplified by the primer pair (1) (excluding the primer sequence) was the same as the sequence of SEQ ID NO: 10. When the sequence of the region amplified by the primer pair (4) (excluding the primer sequence) is the same as the sequence of SEQ ID NO: 13, the lactic acid bacterium in the sample is L. It can be determined to be the amylovourus CP1563 strain. Further, for example, using a fusion sequence in which the sequence of the region amplified by the primer pair (4) (excluding the primer sequence) is bound downstream of the sequence of the region amplified by the primer pair (1) (excluding the primer sequence). Identity may be determined.

In addition, RT-PCR was performed on the sample, RT-PCR was performed using the primer set C, and the sequence of the region amplified by the primer pair (2) (excluding the primer sequence) was the same as the sequence of SEQ ID NO: 11. When the sequence of the region amplified by the primer pair (3) (excluding the primer sequence) is the same as the sequence of SEQ ID NO: 12, the lactic acid bacterium in the sample is L. It can be determined to be the amylovourus CP1563 strain. Further, for example, using a fusion sequence in which the sequence of the region amplified by the primer pair (3) (excluding the primer sequence) is bound downstream of the sequence of the region amplified by the primer pair (2) (excluding the primer sequence). Identity may be determined.

In addition, RT-PCR was performed on the sample, RT-PCR was performed using the primer set D, and the sequence of the region amplified by the primer pair (2) (excluding the primer sequence) was the same as the sequence of SEQ ID NO: 11. When the sequence of the region amplified by the primer pair (4) (excluding the primer sequence) is the same as the sequence of SEQ ID NO: 13, the lactic acid bacterium in the sample is L. It can be determined to be the amylovourus CP1563 strain. Further, for example, using a fusion sequence in which the sequence of the region amplified by the primer pair (4) (excluding the primer sequence) is bound downstream of the sequence of the region amplified by the primer pair (2) (excluding the primer sequence). Identity may be determined.

In addition, for example, Accession No. 1 in Table 1 below, which is registered in GeneBank. Download the sequence and L. Match the length of the gene sequence of the region that can be amplified by the above primer set in each strain of amylovorus, connect the pyrrG gene sequence after the gyrB gene sequence for each strain, and make one gene sequence for each strain. It is possible to create a phylogenetic tree by processing to create comparative sequence data and then performing phylogenetic tree analysis using sequence analysis software such as MEGA (see FIG. 2). L. For various strains of amylovourus, the gyrB gene sequence and the pyrG gene sequence can be obtained from the following accession numbers. By this phylogenetic tree analysis, L. It is shown that it is possible to identify any of the various strains of amylovorus.

<(Ii) Primer set FI>
By using PCR for any of the primer sets F to I, in addition to the analysis with the PCR product using any of the primer sets A to D, L. It is possible to further improve the accuracy of specifically detecting the strains that are amylovorus and the strains therein.

For example, RT-PCR was performed on the sample using the primer set F, and the sequence of the region amplified by the primer pair (7) (excluding the primer sequence) was the same as the sequence of SEQ ID NO: 46, and the primer was used. When the sequence of the region amplified by pair (9) (excluding the primer sequence) is the same as the sequence of SEQ ID NO: 48, the lactic acid bacterium in the sample is L. It can be determined to be the amylovourus CP1563 strain. Further, for example, using a fusion sequence in which the sequence of the region amplified by the primer pair (9) (excluding the primer sequence) is bound downstream of the sequence of the region amplified by the primer pair (7) (excluding the primer sequence). Identity may be determined.

In addition, RT-PCR was performed on the sample, RT-PCR was performed using the primer set G, and the sequence of the region amplified by the primer pair (7) (excluding the primer sequence) was the same as the sequence of SEQ ID NO: 46. When the sequence of the region amplified by the primer pair (10) (excluding the primer sequence) is the same as the sequence of SEQ ID NO: 49, the lactic acid bacterium in the sample is L. It can be determined to be the amylovourus CP1563 strain. Further, for example, a fusion sequence in which the sequence of the region amplified by the primer pair (10) (excluding the primer sequence) is bound downstream of the sequence of the region amplified by the primer pair (7) (excluding the primer sequence) is used. Identity may be determined.

In addition, RT-PCR was performed on the sample, RT-PCR was performed using the primer set H, and the sequence of the region amplified by the primer pair (8) (excluding the primer sequence) was the same as the sequence of SEQ ID NO: 47. When the sequence of the region amplified by the primer pair (9) (excluding the primer sequence) is the same as the sequence of SEQ ID NO: 48, the lactic acid bacterium in the sample is L. It can be determined to be the amylovourus CP1563 strain. Further, for example, using a fusion sequence in which the sequence of the region amplified by the primer pair (9) (excluding the primer sequence) is bound downstream of the sequence of the region amplified by the primer pair (8) (excluding the primer sequence). Identity may be determined.

In addition, RT-PCR was performed on the sample, RT-PCR was performed using the primer set I, and the sequence of the region amplified by the primer pair (8) (excluding the primer sequence) was the same as the sequence of SEQ ID NO: 47. When the sequence of the region amplified by the primer pair (10) (excluding the primer sequence) is the same as the sequence of SEQ ID NO: 49, the lactic acid bacterium in the sample is L. It can be determined to be the amylovourus CP1563 strain. Further, for example, using a fusion sequence in which the sequence of the region amplified by the primer pair (10) (excluding the primer sequence) is bound downstream of the sequence of the region amplified by the primer pair (8) (excluding the primer sequence). Identity may be determined.

In addition, Accession No. in Table 2 below, which is registered in GeneBank. In the sequence, L. A fusion sequence of lep gene sequence-gyrA gene sequence may be prepared by matching the lengths of the gene sequences of the regions that can be amplified by the above primer sets F to I in each strain of amylovorus. Then, the fusion sequence of this lep gene sequence-gyrA gene sequence is connected after the fusion sequence of the gyrB gene-pyrG gene sequence for each strain prepared based on the above-mentioned Table 1, and for each strain. It is possible to create a phylogenetic tree by processing it into a single gene sequence to create comparative sequence data, and then performing phylogenetic tree analysis using sequence analysis software such as MEGA (see FIG. 6). Analysis using such regions that can be amplified by at least one of primer sets A to D and at least one primer set of primer sets F to I is a sample at a high level as shown in FIG. Included in L. Various strains of amylovorus can be identified.
In addition, L. The gyrA gene sequence and the lep gene sequence in various strains of amylovorus can be obtained from the following accession numbers.

<(Iii) Primer set E>
RT-PCR was performed on the sample using the primer set E, and the contained cells were L. Is amylovorus, and the strain in it is L. It is possible to specifically detect whether or not it is amylovorus CP1563.

More specifically, RT-PCR was performed on the sample, RT-PCR was performed using the primer set E, and the sequence of the region amplified by the primer pair (5) (excluding the primer sequence) is the SEQ ID NO: When the sequence of 14 is the same and the sequence of the region amplified by the primer pair (6) (excluding the primer sequence) is the same as the sequence of SEQ ID NO: 15, the lactic acid bacterium in the sample is L. It can be determined to be the amylovourus CP1563 strain.
It is preferred that PCR with primer pair (5) and PCR with primer pair (6) be performed in separate containers.

As a result of BLAST analysis on the sequence of the amplified region, the gyrB gene sequence obtained by the primer pair (5) and the pyrG gene sequence obtained by the primer pair (6) are sequences with the existing Lactobacillus amylovourus species sequence. The sequence identity with Lactobacillus acidiphilus species, Lactobacillus gallianum, etc., which have 99-100% identity and are almost the same and have high sequence identity thereafter, is about 85-90%, and the PCR product obtained from the sample. It can be confirmed from the sequence of Lactobacillus amyllovus species.

In another aspect of the invention, a sequence of regions amplified by another set of primer pairs in the same primer set is attached downstream of the sequence of regions amplified by one set of primer pairs in each primer set. A fusion gene sequence can be prepared in silico and this sequence can be compared to a similarly prepared fusion gene sequence using the same primer pair for the detection target strain or strain. When creating a fusion gene sequence, it is preferable to remove the primer sequence because the primer sequences are common.
For example, L. amylovorus CP1563 strain detection target strain the primer set downstream SEQ ID gyrB sequence shown E in amplified SEQ ID NO: 14 (caatatacaaatggctataagacaacgttaatgaccttcgctaacaacattcacacctatgaaggtgggatgcac) using 15 as the pyrG sequence (both primer sequences represented by (Eishitititieijitieishishieishititishititishieitijishieijishitishieishijieieieitijieieijieishitieieijishishieieishitishieieishieititishieijititijishieijieieishititcgcagcatcggtattcaaccaaac) The fusion gene sequence prepared by in silico binding (not included) is as follows.
L. amylloverus_CP1563 (SEQ ID NO: 16)
CAATATACAAATGGCTATAAGACAACGTTAATGACCTTCGCTAACAACATTCACACCTATGAAGGTGGGATGCACACTTTAGTACCACTTCTTCATGCAGCTCACGAAATGAAGACTAAGCCAACTCAACATTCAGTTGCAGAACTTCGCAGCATCGGTATTCAACCAAAC

In addition, L. The fusion gene sequence prepared by in silico binding of the pyrrG sequence downstream of the gyrB sequence amplified using the above primer set E using each of the 11 types of amylovourus as a detection target strain is as follows.
L. amylloverus_ATCC33198 (SEQ ID NO: 23)
CAATATACAAATGGCTATAAGACAACGTTAATGACCTTCGCTAACAACATTCACACCTATGAAGGTGGGATGCACACTTTAGTACCACTTCTTCACGCAGCTCACGAAATGAAGACTAAGCCAACTCAACATTCAGTTGCAGAACTTCGCAGCATCGGTATTCAACCAAAC
L. amylovourus_ATCC33620 (SEQ ID NO: 24)
CAATATACAAATGGCTATAAGACAACGTTAATGACCTTCGCTAACAACATTCACACCTATGAAGGCGGGATGCACACTTTAGTACCACTTCTTCACGCAGCTCATGAAATGAAGACTAAGCCAACTCAACATTCAGTTGCAGAACTTCGCAGCATCGGTATTCAACCAAAC
L. amylloverus_ATCC33321 (SEQ ID NO: 25)
CAATATACAAATGGCTATAAGACAACGTTAATGACCTTCGCTAACAACATTCACACCTATGAAGGCGGGATGCACACTTTAGTACCACTTCTTCACGCAGCTCACGAAATGAAGACTAAGCCAACTCAACATTCAGTTGCAGAACTTCGCAGCATCGGTATTCAACCAAAC
L. amylovourus_ATCC33622 (SEQ ID NO: 26)
CAATATACAAATGGCTATAAGACAACGTTAATGACCTTCGCTAACAACATTCACACCTATGAAGGTGGGATGCACACTTTAGTACCACTTCTTCACGCAGCTCACGAAATGAAGACTAAGCCAACTCAACATTCAGTTGCAGAACTTCGCAGCATCGGTATTCAACCAAAC
L. amylovourus_ATCC537671 (SEQ ID NO: 27)
CAATATACAAATGGCTATAAGACAACGTTAATGACCTTCGCTAACAACATTCACACCTATGAAGGCGGGATGCACACTTTAGTACCACTTCTTCATGCAGCTCACGAAATGAAGACTAAGCCAACTCAACATTCAGTTGCAGAACTTCGCAGCATCGGTATTCAACCAAAC
L. amylovourus_LMG18179 (SEQ ID NO: 28)
CAATATACAAATGGCTATAAGACAACGTTAATGACCTTCGCTAACAACATTCACACCTATGAAGGTGGGATGCACACTTTAGTACCACTTCTTCACGCAGCTCACGAAATGAAGACTAAGCCAACTCAACATTCAGTTGCAGAACTTCGCAGCATCGGTATTCAACCAAAC
L. amylovourus_LMG18180 (SEQ ID NO: 29)
CAATATACAAATGGCTATAAGACAACGTTAATGACCTTCGCTAACAACATTCACACCTATGAAGGTGGGATGCACACTTTAGTACCACTTCTTCACGCAGCTCACGAAATGAAGACTAAGCCAACTCAACATTCAGTTGCAGAACTTCGCAGCATCGGTATTCAACCAAAC
L. amylovourus_LMG18186 (SEQ ID NO: 30)
CAATATACAAATGGCTATAAGACAACGTTAATGACCTTCGCTAACAACATTCACACCTATGAAGGTGGGATGCACACTTTAGTACCACTTCTTCACGCAGCTCACGAAATGAAGACTAAGCCAACTCAACATTCAGTTGCAGAACTTCGCAGCATCGGTATTCAACCAAAC
L. amylovorous_LMG18188 (SEQ ID NO: 31)
CAATATACAAATGGCTATAAGACAACGTTAATGACCTTCGCTAACAACATTCACACCTATGAAGGCGGGATGCACACTTTAGTACCACTTCTTCACGCAGCTCACGAAATGAAGACTAAGCCAACTCAACATTCAGTTGCAGAACTTCGCAGCATCGGTATTCAACCAAAC
L. amylovourus_LMG18190 (SEQ ID NO: 32)
CAATATACAAATGGCTATAAGACAACGTTAATGACCTTCGCTAACAACATTCACACCTATGAAGGTGGGATGCACACTTTAGTACCACTTCTTCACGCAGCTCACGAAATGAAGACTAAGCCAACTCAACATTCAGTTGCAGAACTTCGCAGCATCGGTATTCAACCAAAC
L. amylloverus_LMG18192 (SEQ ID NO: 33)
CAATATACAAATGGCTATAAGACAACGTTAATGACCTTCGCTAACAACATTCACACCTATGAAGGCGGGATGCACACTTTAGTACCACTTCTTCATGCAGCTCACGAAATGAAGACTAAGCCAACTCAACATTCAGTTGCAGAACTTCGCAGCATCGGTATTCAACCAAAC

Regarding the obtained gene sequences of SEQ ID NO: 16 and SEQ ID NOs: 23 to 33, a phylogenetic tree was analyzed using, for example, Neighbor Joining Method using sequence analysis software such as MEGA6, and the created phylogenetic tree is shown in FIG. As can be seen from the phylogenetic tree shown in FIG. 3, the CP1563 strain has a different sequence from all the strains registered in GeneBank, and it can be clearly confirmed that the CP1563 strain is the CP1563 strain. It can be confirmed that it is the strain.

According to the primer set consisting of the primer pair (5) and (6), the cells contained in the sample are L. A kit consisting of the primer set is also included in the present invention because it can be clearly detected whether or not it is an amylovourus CP1563 strain. The region amplified by the primer pair (5) and (6) is very small, 100 to 150 bp, and the target is the RNA portion that is likely to remain in the sample in large quantities, so RT-PCR is successful with high probability. Can be made to. In addition, L. In the comparison between the amylovourus strains, since the region specific to the CP1563 strain is targeted, it is clearly L. It can be determined whether or not the amylovourus CP1563 strain is contained in the sample.

L. can be specifically detected by the present invention. The amylovourus CP1563 strain was released on May 25, 2010 by the Patented Biological Deposit Center (IPOD) (former address: 1-1-1, Higashi, Kisarazu City, Ibaraki Prefecture, 305-8566, Tsukuba Center Central No. 6) (IPOD is 2013 as NITE-IPOD). Moved to the following on April 1: 〒292-0818 Kisarazu City, Chiba Prefecture 2-5-8 Kazusakamatari Room 120) has been internationally deposited and has been given the accession number FERM BP-11255.

In the method of the present invention, RT-PCR can be performed according to a method well known to those skilled in the art. That is, RNA is extracted from a sample according to a conventional method, cDNA is synthesized using RNA as a template using an oligo dT primer or the like according to a conventional method, and PCR (polymerase chain reaction) is performed according to a conventional method using any of the above primer sets. )It can be performed. As the PCR conditions, standard conditions derived from the Tm value of the primer, the length of the region to be amplified, the sequence, etc. can be used. While PCR with two sets of primer pairs included in the primer set (each primer pair consists of forward and reverse primers) can also be carried out in the same vessel, it is necessary to sequence the amplified products individually It is preferable to perform PCR in separate containers for each primer pair. Over amplification products obtained in electrophoresis, to confirm that the fragment of the expected length are amplified, the DNA fragment was extracted according to a conventional method from the gel, sequencing by methods well known in the art Thing.
Those skilled in the art are also familiar with various methods, reagents, kits and devices required for these operations, such as RNA extraction, cDNA synthesis, PCR, extraction of DNA fragments from gels, sequencing, and the like. , Also readily available commercially.

Sequence L. The resulting amplification products amylovorus or L. Although comparisons with sequences derived from the amylovourus CP1563 strain (eg, gyrB and pyrG sequences) can be made visually, various algorithms and programs for sequence comparison are commercially available, and tools such as BLASTN are also available at NCBI, EMBL. And it is provided by the DDBJ Center and is widely available to the public. When using such an algorithm or program, it would be advantageous to compare the above-mentioned fusion gene sequences because the sequence identity can be determined in one analysis.
In addition, L. amylovorus or L. The amylovourus CP1563 strain is found in other lactic acid bacteria species or other L. bacilli strains. In order to make it easier to understand that it is different from the amylovorus strain, a phylogenetic tree can be prepared using the prepared fusion gene sequence. Algorithms and specific programs for creating a phylogenetic tree are well known to those skilled in the art and are commercially available or publicly available. For example, free software such as MEGA and Clustal W for alignment and phylogenetic tree creation provided by NCBI, EMBL and DDBJ Centers are available.

When it is considered that the lactic acid bacteria in the sample are essentially killed by the sterilization treatment or the like, particularly the sterilization treatment and / or long-term storage (for example, at 4 ° C to 35 ° C for 1 month or more or 3 months or more, preferably 3 months or more). Lactic acid bacteria in the sample are killed by 3 months to 6 months, particularly preferably 3 months to 12 months, or storage under the same conditions), and the nucleic acid derived from the lactic acid bacteria is damaged (decomposed, etc.) to a considerable extent. Even in the case where it is considered, according to the present invention, L. amylovorus or L. The amylovourus CP1563 strain can be specifically detected. In the present invention, when the sample is a beverage or food, generally, the period from production to the expiration date, the period from production to the expiration date, or even when stored for a period exceeding these, L. amylovorus or L. The amylovourus CP1563 strain can be specifically detected.

According to the present invention, L. Other lactic acid bacteria that can be distinguished from the amylovourus CP1563 strain and L. Amylovourus strains include, but are not limited to, the following lactic acid bacteria, for example:
L. acidofilus (ATCC314, ATCC4356, ATCC9224, ATCC53544, ATCC4357);
L. amylovourus (ATCC33620, ATCC33321, LMG18188, ATCC33622, ATCC53671, LMG18192, LMG18179, LMG18190, ATCC33198, LMG18180, LMG18186);
L. crispatus (ATCC53545, ATCC55221, JCM5810, LMG11415, LMG17753, ATCC33820, LMG11440, LMG18187, LMG12003, LMG12005, LMG18189);
L. Gallinarum (LMG14751, LMG18181, ATCC53673, LMG14752, LMG14753, LMG14754, LMG14755, ATCC33199);
L. gasseri (ATCC 4963, ATCC 19992, LMG 11413, ATCC 29601, ATCC 9857, ATCC 4962, ATCC 33323, LMG 10771, LMG 18176);
L. jonsonii (LMG18175, LMG18184, LMG18193, NCC533, ATCC332, ATCC33200, LMG18204, LMG18195).

Using the primer pairs known below, L. The results of attempts to detect the amylovourus CP1563 strain but failed are briefly shown as References 1 and 2.
(Reference example 1)
(I) L. Viable cells of the amylovourus CP1563 strain, (ii) L. et al. Amylovourus CP1563 strain was sterilized by heating and dried, and (iii) L. et al. DNA was extracted from three types of samples of bacterial cells (dead bacteria) collected after mixing the amylovorus CP1563 strain in a beverage product and sterilizing by heating by a known method. Using the obtained DNA as a template, six housekeeping genes (fusA, gyrA, gyrA, gyrA A part (about 500 bp to 1000 bp) of lepA, pyrG, recA) was amplified by PCR using the primer pair described in Non-Patent Document 1. As a result, the target amplification product was confirmed from the above samples (i) and (ii), but no sequence-analyzable amplification product was confirmed from the sample (iii), and the strain was analyzed. I couldn't.

(Reference example 2)
(Ii) L. Amylovourus CP1563 strain was sterilized by heating and dried, and (iii) L. et al. The RNA used as a template was used in Reference Example 1 as a template, which was extracted by a known method from two types of cells (dead bacteria) in which the amylovourus CP1563 strain was mixed in a beverage product, sterilized by heating, and then collected. Using the same primer pair as above, a part (about 500 bp to 1000 bp) of 6 housekeeping genes (fusA, gyrA, gyrB, lepA, pyrG, recA) was amplified by RT-PCR. As a result, the desired amplification product could be obtained from the sample (ii) above, but the desired amplification product could not be obtained from the sample (iii), or a non-specific amplification product could be obtained. Was produced, and an amplification product with sufficient purity for base sequence analysis could not be obtained.

The primers used in Reference Examples 1 and 2 were as follows.

Therefore, L. The method and primer described in Non-Patent Document 1 (Applied and Environmental Microbiology, p. 7220-7228 December 2013 Volume 79 Number 23) without testing the specific detection ability for the amylovourus CP1563 strain. can not even be obtained amplification product by first place PCR in (killed) beverage, the object of the present invention was found to be unavailable.
Specific embodiments of the present invention will be described for the purpose of making the present invention easier to understand.

Hereinafter, the present invention will be specifically described with reference to examples, but the present invention is not limited thereto, and various changes and modifications are made without departing from the scope of the present invention. May be good.
In Examples and Comparative Examples, sequence samples were obtained by the following experimental procedure.

<Experimental procedure>
1. 1. Collecting bacteria and cleaning the sample The PET bottle containing the sample beverage as a sample was turned upside down several times before opening and mixed well, and it was confirmed that no precipitate remained at the bottom of the PET bottle. Next, the PET bottle was opened, 10 mL of the sample beverage solution was collected in a 15 mL sterile disposable plastic tube, and the sample was centrifuged at 6000 rpm (4830 × g) for 10 minutes. After discarding the supernatant of the centrifuged sample beverage, 10 mL of sterile water was placed in a tube and resuspended. The resuspension was centrifuged at 6000 rpm (4830 × g) for 10 minutes and the supernatant was discarded. Since there are necessary cells on the lower side of the precipitate and impurities such as milk protein on the upper side, add 5 mL of sterile water and add only the impurities such as milk protein on the upper side so as not to break the pellets of the bacteria. The supernatant was lightly suspended and the supernatant was discarded, and this washing operation was repeated twice. The bacterial precipitate was resuspended in 1 mL of sterile water and collected in a sterile 1.5 mL tube. Then, the resuspension of the bacterial precipitate was centrifuged at 14,000 rpm (19,000 × g) for 2 minutes, 1 mL of sterile water was added, and the upper part of the pellet was washed to prepare a sample for RNA extraction. ..

2. 2. RNA extraction The sample for RNA extraction obtained in 1 above was suspended in 350 μl of Lysis Buffer (LBP) and dissolved well. Then, the DNA was removed using the DNA removal column of the kit. RNA extraction was performed according to the manual of the Nucleospin RNA plus kit to obtain an RNA solution.

3. 3. Amplification of target gene region by RT-PCR From the RNA solution prepared in 2 above, PrimeScript ^TM One Step RT-PCR Kit Ver. 2 was used to amplify the gene region of interest.
A 10 pmol / μl solution of each primer contained in each primer set used in each example was prepared. For each primer, the required sequence was synthesized by requesting synthesis (50 pmol / l) with a PCReady primer manufactured by Eurofin Genomics, and diluted 5-fold with DNase and RNase-free sterile water.

PrimeScript ^TM One Step RT-PCR Kit Ver. Using No. 2, a solution was prepared according to the following reaction composition, and the following composition was placed in a 200 μl volume tube for PCR and mixed well. Although the PCR reaction solution composition was specified to be 50 μl in the protocol of the kit, the total amount was 25 μl and the amount of Template RNA was 4 μl in order to increase the template concentration in the reaction solution. Except for the primer and Template RNA (RNA solution prepared in 2 above), the ones included in the kit were used.

PrimeScript 1 step Enzyme Mix 1 μl
2 x 1 step Buffer 12.5 μl
Forward Primer (10 μM) 1 μl
Reverse Primer (10 μM) 1 μl
Template RNA (RNA solution adjusted in 2) 4 μl
RNase Free dH ₂ O Balance
Total amount 25 μl
When no amplification product was confirmed in the above composition, the amount of Temple RNA was increased to 8 μl, and RT-PCR was performed again.

It was set in the thermal cycler C1000 Touch Thermal Cycler and operated as follows.
STEP1: 50 ° C, 30 minutes,
STEP2: 95 ° C, 2 minutes,
STEP3: 95 ° C, 30 seconds,
STEP4: 55 ° C, 30 seconds,
STEP5: An RT-PCR reaction was carried out under the conditions of 72 ° C. and 30 seconds with STEP3-5 as a total of 40 cycles. The obtained sample was electrophoresed on an agarose gel according to a conventional method, and the target band was confirmed.

4-1. When only the target band is clearly amplified (1) 5 μl of the reaction solution was collected in a new 200 μl volume PCR tube ^{, 2 μl of ExoSAP-IT TM} PCR Product Cleanap Reagent was added, and the mixture was well mixed with a micropipettor.
(2) Incubation was carried out in a thermal cycler at 50 ° C. for 15 minutes → 85 ° C. for 15 minutes to purify the PCR product.

4-2. When Primer dimer and other amplification products of around 100 bp are confirmed in addition to the target band, purification was performed using Nucleospin Gel and PCR cleanup after electrophoresis.
(1) Electrophoresis was carried out on an agarose gel according to a conventional method, and after confirming the target band, a portion of the target band was cut out from the agarose gel and collected in a sterilized 1.5 ml tube.
(2) Add 200 μl of NTI solution and incubate at 50 ° C. for about 5-10 minutes. The gel was completely dissolved by mixing well with Vortex every 2-3 minutes during the incubation.
(3) Necleospin Gel and PCR Clean-up Volume was set in the attached 2 ml collection tube, and the entire amount of the sample solution dissolved in (2) was placed on a column and centrifuged at 11,000 × g for 30 seconds. The filtrate was discarded and set in the collection tube again.
(4) 700 ml of NT3 solution was applied to the column and centrifuged at 11,000 × g for 30 seconds. Discard the filtrate and set it in the collection tube again. This process was repeated once more.
(5) Centrifugation was carried out at 11,000 × g for 1 minute to remove the NT # solution remaining on the column.
(6) The column was set in a new sterilized 1.5 ml tube, 15 μl of NE solution was added, and the mixture was allowed to stand at room temperature for 1 minute.
(7) Centrifugation was carried out at 11,000 × g for 1 minute, and the column was discarded to obtain a purified PCR product for sequencing.

5. Sequence sample preparation A sequence reaction was performed using both the Bigdye Terminator v1.1 Cycle Sequenceing Kit, the purified PCR product, and the primer pair forward and reverse primers of the primer set for each strain.
(1) For the forward and reverse primer 10 pmol / μl solution of the primer pair used for PCR in 3 above, 8 μl was collected in a sterilized 1.5 ml tube, diluted with 92 μl of sterilized water, and 0.8 pmol / μl. The solution was μl.
(2) Using the solution attached to the Bigdye Terminator v1.1 Cycle Sequenceing Kit and the PCR product, primer solution, and sterile water obtained in 4-1 or 4-2, prepare a solution for sequence reaction having the following composition. Prepared in a 200 μl volume PCR tube. Two samples, one for forward analysis and one for reverse analysis, were prepared for each PCR product.

Forward analysis sample purified PCR product 2 μl
Negative Control Water (sterile water) 8 μl
5 x Sequencing Buffer 2 μl
Sequencing RR-100 4 μl
Forward primer (0.8 pmol / μl) 4 μl
20 μl
Reverse analysis sample Purified PCR product 2 μl
Negative Control Water (sterile water) 8 μl
5 x Sequencing Buffer 2 μl
Sequencing RR-100 4 μl
Reverse primer (0.8 pmol / μl) 4 μl
20 μl
(3) Set it in the thermal cycler and
96 ° C, 10 seconds,
50 ° C, 5 seconds,
A total of 25 cycles of reaction were carried out under the conditions of 60 ° C. and 4 minutes.

6. Purification of sequence samples (1) A new sterilized 1.5 mL tube for the number of samples and a Bigdye Xterminator Purification Kit were prepared.
(2) 90 μl of SAM solution and 20 μl of Xterminator solution were collected in a tube, and 20 μl of the above sequence reaction product was added.
(3) The mixture was set in a tube shaker, the stirring speed was set to the maximum value, and the mixture was stirred for 30 minutes.
(4) Centrifugation was carried out at 750 × g for 2 minutes, and 20 μl of the supernatant was used as a sample, HiDi Formamide was mixed with 20 μl, and the mixture was subjected to a DNA sequencer to analyze the sequence.

7. Sequence analysis and sequence identity and bacterial species discrimination (1) Consensus sequences were obtained for the obtained sequence forward and reverse sequences using MEGA (free software) sequence analysis software.
(2) The obtained sequence is sequence-identical with the existing DNA data on the NCBI homepage BLASTN (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastn&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome). Confirmed.
(3) Nucleotide Collection (nr / nt) was selected for the database, and sequence identity was confirmed without setting restrictions on the search target.

8. Strain discrimination by sequence analysis, sequence identity and phylogenetic tree analysis (1) Accession No. 1 shown in Table 1 above registered in GeneBank. The sequence was downloaded, the analyzed gene was compared with the sequence of each gene, and the extra parts before and after were deleted in order to match the length with the sequence of the gene analyzed this time.
(2) Using the gene sequences of the same length, the pyrG gene sequence was linked after the gyrB gene sequence of each strain, and processed into one gene sequence for each strain.
(3) Regarding the gene sequence created by processing, a phylogenetic tree was analyzed using sequence analysis software such as MEGA to create a phylogenetic tree. As a result of phylogenetic tree analysis, it was found that the CP1563 strain had a different sequence from all the strains registered in GeneBank. The phylogenetic trees of FIGS. 2 and 3 are as described above.
(4) In addition, Accession No. 2 shown in Table 2 above. Arrange the above 8. Process in the same manner as in (1), and perform the above 8. In the step (2), the fusion sequence of gyrB gene sequence-pyrG gene sequence is followed by the fusion sequence of lepA gene sequence-gyrA gene sequence, and the fusion sequence is processed into one gene sequence for each strain. It is also possible to create a phylogenetic tree by performing phylogenetic tree analysis using sequence analysis software such as MEGA with respect to the gene sequence created in the above procedure (see FIG. 6).

[Example 1]
As a sample beverage as a sample, a PET bottled body Calpis (registered trademark) stored at 25 ° C. for 6 months was used. For this sample, PCR was performed on each of the primer pairs (1) to (4) in separate containers, and the obtained four types of PCR products were electrophoresed on an agarose gel according to a conventional method. The results are shown in FIG. In FIG. 4, lane 1 shows the PCR product of the primer pair (2), lane 2 shows the PCR product of the primer pair (1), lane 3 shows the PCR product of the primer pair (3), and lane 4 shows the primer pair. The PCR product according to (4) is shown.

[Example 2]
The operation was carried out in the same manner as in Example 1 except that as the sample beverage as a sample, Calpis (registered trademark) in a PET bottle stored at 55 ° C. for 4 days was used. This example corresponds to an accelerated test and corresponds to the condition that the sample beverage is stored at 25 ° C. for 6 months. It was confirmed by agarose gel that the PCR product was obtained.

[Example 3]
The operation was carried out in the same manner as in Example 1 except that the PET bottled Calpis (registered trademark) stored at 25 ° C. for 9 months was used as the sample beverage as a sample. It was confirmed by agarose gel that the PCR product was obtained.

[Example 4]
The operation was carried out in the same manner as in Example 1 except that the PET bottled Calpis (registered trademark) stored at 25 ° C. for 12 months was used as the sample beverage as a sample. It was confirmed by agarose gel that the PCR product was obtained.

[Comparative Example 1]
The procedure was the same as in Example 1 except that the PET bottled Calpis (registered trademark) stored at 55 ° C. for 9 months was used as the sample beverage as a sample, but no PCR product was obtained and analysis was not possible. ..

[Comparative Example 2]
The procedure was the same as in Example 1 except that the PET bottled body Calpis (registered trademark) stored at 55 ° C. for 12 months was used as the sample beverage as a sample, but no PCR product was obtained and analysis was not possible. ..

From the above Examples and Comparative Examples, it is shown that when the primer pairs (1) to (4) are used, it is difficult to obtain the required PCR product for a beverage stored at a high temperature for a long period of time. Was done.

[Example 5]
Sequence samples were prepared using each PCR product obtained in Examples 1 to 4, and sequence analysis and BLAST analysis of the base sequence were performed.
<About the gyrB gene sequence>
The sequence analysis results of the PCR products obtained by using the primer pairs (1) and (2) of the samples of Examples 1 to 4 were all the same. Further, the sequence of the region amplified by the primer pair (1) (excluding the primer sequence) and the sequence of SEQ ID NO: 10 are the same, and the sequence of the region amplified by the primer pair (2) (primer sequence). It was confirmed that (excluding) and the sequence of SEQ ID NO: 11 are the same. Then, as a result of BLAST analysis based on the obtained sequence analysis, the top 12 bacterial species having high sequence identity were L. Amylovourus 11 strain and L. It was a sobrius 1 strain, and its sequence identity was 98-100%. Besides that, L. kitasatonis, L. et al. kalixensis, L. et al. Although acidophilus and the like were confirmed at the top, the sequence identity was about 84-91%. From the above, the analysis sequence of the PCR product obtained by the primer pair (1) and (2) is L.I. It was predicted to be the gyrB gene of amyloborus.
<About the pyrG gene sequence>
The sequence analysis results of the PCR products obtained using the primer pairs (3) and (4) of the samples of Examples 1 to 4 were all the same. Further, the sequence of the region amplified by the primer pair (3) (excluding the primer sequence) and the sequence of SEQ ID NO: 12 are the same, and the sequence of the region amplified by the primer pair (4) (primer sequence). It was confirmed that (excluding) and the sequence of SEQ ID NO: 13 are the same. Then, as a result of BLAST analysis based on the obtained sequence analysis, the top 11 bacterial species having high sequence identity were L. It was amylovorus and its sequence identity was 99-100%. Besides that, L. acidophilus, L .; gallinarum, L. et al. Helveticus and the like could be confirmed at the top, but the sequence identity was about 90-91%. From the above, the analysis sequence of the PCR product obtained by the primer pair (3) and (4) is L.I. It was predicted to be the pyrG gene of amyloborus.
The bacterial species having the relevant sequence included in the samples of Examples 1 to 4 are L. It became clear that the possibility of amylovorus was extremely high.

[Example 6]
<Creation of phylogenetic tree>
L. Nucleotide sequence of PCR product by primer pair (1) and base sequence of PCR product by primer pair (3), base sequence of PCR product by primer pair (1) and base sequence of PCR product by primer pair (4) for amylovorus CP1563 strain Sequence, base sequence of PCR product by primer pair (2) and base sequence of PCR product by primer pair (3), base sequence of PCR product by primer pair (2) and base of PCR product by primer pair (4) The sequences were joined together and connected in silico to create a fusion gene sequence. Similarly, Accession No. of Table 1 registered in GeneBank. Download the sequence and L. The length of the gene sequence of the region that can be amplified by the above primer set in each strain of amylovorus was matched, and the pyrG gene sequence was connected in silico after the gyrB gene sequence for each strain to create a fusion gene sequence. .. A phylogenetic tree was created by processing each strain into one gene sequence to create comparative sequence data, and performing phylogenetic tree analysis using MEGA6 sequence analysis software. The results are shown in FIG. As can be seen from FIG. 2, the DNA sequences obtained from the samples of Examples 1 to 4 analyzed in this test are L.I. It is 100% consistent with the DNA-derived sequence of the amylovourus CP1563 strain, and other L. It was found that the sequence was different from the 12 strains including the amylovourus strain, and the strains contained in the sample were L. It was revealed that it was an amylovrus CP1563 strain.

[Example 7]
The following four types of sample beverages are prepared as samples, and for each sample, a primer pair (5) for amplifying a part of the gyrB gene sequence and a primer pair (5) for amplifying a part of the pyrG gene sequence ( For each of 6), PCR was performed in a separate container.
Sample (a) Body Calpis in a PET bottle (registered trademark) Immediately after production Sample (a) Body Calpis in a PET bottle stored at 55 ° C for 4 days (equivalent to storing at 25 ° C for 6 months) (Registered trademark)
Sample (c) Body Calpis (registered trademark) in a PET bottle stored at 55 ° C for 5 days (equivalent to storing at 25 ° C for 9 months)
Sample (d) Body Calpis in a PET bottle stored at 25 ° C for 9 months (registered trademark)
Each of the two PCR products obtained from the above four samples (a) to (d) was electrophoresed on an agarose gel according to a conventional method. The results are shown in FIG. In FIG. 5, lane 1 shows the PCR product of the primer pair (5) of the sample (a), lane 2 shows the PCR product of the primer pair (5) of the sample (a), and lane 3 shows the primer of the sample (c). The PCR product of pair (5) is shown, lane 4 shows the PCR product of sample (d) primer pair (5), lane 5 shows the PCR product of sample (a) primer pair (6), and lane 6 Shows the PCR product of the sample (a) primer pair (6), lane 7 shows the PCR product of the sample (c) primer pair (6), and lane 8 shows the sample (d) primer pair (6). The PCR product is shown. The amplified chain length was 123 bp with the primer pair (5) for amplifying the gyrB gene sequence and 139 bp with the primer pair (6) for amplifying the pyrG gene sequence.
From this result, it was found that the DNA sequences obtained by the primer pairs (5) and (6) are relatively short, so that a PCR product can be obtained even in a sample after storage at 55 ° C.

[Example 8]
Sequence samples were prepared using each PCR product obtained in Example 7, and sequence analysis and BLAST analysis of the base sequence were performed.
<About the gyrB gene sequence (lanes 1 to 4 in FIG. 5)>
The sequence analysis results of the PCR products obtained using each of the primer pairs (5) of the samples (a) to (d) were the same. Then, as a result of BLAST analysis based on the obtained sequence analysis, the top 12 bacterial species having high sequence identity were L. Amylovourus 11 strain and L. It was a sobrius 1 strain, and the sequence whose sequence identity was confirmed was the entire analyzed sequence 123bp, and the sequence identity was 98-100%. Besides that, L. kitasatonis, L. et al. kalixensis, L. et al. Although acidophilus and the like could be confirmed at the top, the sequence identity was about 89-93%. From the above, the analysis sequence of the PCR product obtained by the primer pair (5) is L. amyloborus or L. It was predicted to be the gyrB gene of sobrius.
<About the pyrG gene sequence (lanes 5 to 8 in FIG. 5)>
The sequence analysis results of the PCR products obtained using each of the primer pairs (6) of the samples (a) to (d) were the same. Then, as a result of BLAST analysis based on the obtained sequence analysis, the top 11 bacterial species having high sequence identity were L. It was amylovourus, and of the 139 bp analyzed, only about 91 bp could be confirmed to have sequence identity. Its sequence identity was 98-100%. In addition, the bacterial species after the 12th position are L. helveticus and L. gallianum, L. et al. It was another bacterial species such as acidophilus, and the sequence identity was about 96% at the highest. From the above, the analysis sequence is L. It was considered to be the pyrG gene of amylovorus.
From the above results, the bacterial species having the relevant sequence contained in the samples (a) to (d) are L. It became clear that the possibility of amylovorus was extremely high.

[Example 9]
<Creation of phylogenetic tree>
L. For the amylovourus CP1563 strain, the pyrG gene sequence amplified using the primer pair (6) is connected in silico downstream of the gyrB gene sequence amplified using the primer pair (5) used in Example 7, and the fusion gene sequence ( SEQ ID NO: 16) was created. Similarly, L.A. registered in GenBank. Similarly, for each of the 12 strains including the amylovourus strain, the pyrG gene sequence amplified using the primer pair (6) is connected and fused in silico downstream of the gyrB gene sequence amplified using the primer pair (5). Gene sequences (SEQ ID NOs: 23-33) were prepared. Using the prepared fusion gene sequence group, a phylogenetic tree was prepared by the Neiberhood-joining method using MEGA6. The results are shown in FIG. As can be seen from FIG. 3, the DNA sequences obtained from all the samples (a) to (d) analyzed in this test are L.I. It is 100% consistent with the DNA-derived sequence of the amylovourus CP1563 strain, and other L. It was found that the sequence was different from the 12 strains including the amylovourus strain, and the strains contained in the samples (a) to (d) were L. It was revealed that it was an amylovrus CP1563 strain.

[Example 10]
As a sample beverage as a sample, a PET bottled body Calpis (registered trademark) stored at 25 ° C. for 6 months was used. For this sample, PCR was performed on each of the primer pairs (7) to (10) in separate containers, and the obtained four types of PCR products were electrophoresed on an agarose gel according to a conventional method. In addition, as a positive control, L. Similarly, for the sample obtained by culturing the amylovourus CP1563 strain by a conventional method, PCR was performed on each of the primer pairs (7) to (10) in separate containers, and the obtained PCR product was electrophoresed on an agarose gel according to a conventional method. Migration was performed. The results are shown in FIG. In addition, each lane in FIG. 7 corresponds as shown in Table 3 below.

[Example 11]
<Creation of phylogenetic tree>
L. For the amylovourus CP1563 strain, the base sequence of the PCR product by the primer pair (1) and the base sequence of the PCR product by the primer pair (3), and the base sequence of the PCR product by the primer pair (7) and the PCR product by the primer pair (9). by connecting the nucleotide sequence, created a fusion gene sequences connected by in silico. Similarly, Accession Nos. In Tables 1 and 2 registered in GeneBank. Download the sequence and L. In each strain of amylovorus, the length of the gene sequence of the region that can be amplified by the above primer set is matched, and the gyrB gene sequence, pyrG gene sequence, lepA gene sequence, and gyrA gene sequence for each strain are connected in silico. To create a fusion gene sequence. A phylogenetic tree was created by processing each strain into one gene sequence to create comparative sequence data, and performing phylogenetic tree analysis using MEGA6 sequence analysis software. The results are shown in FIG. As can be seen from FIG. 6, from the DNA sequence obtained from the sample, the cells contained in the sample were L. It has been clarified that it can be identified as the amylovrus CP1563 strain, and that the other 10 strains can also be identified.

Claims (8)

A method for detecting the lactic acid bacterium Lactobacillus amylovorus species in a sample as a detection target microorganism, and the following steps:
i) Step of extracting RNA from a sample,
ii) A step of synthesizing cDNA using the RNA extracted in i) as a template,
cDNA as a template synthesized in iii) ii), a step of performing PCR using a primer set consisting of the detection target microorganisms characteristic 5 bp to 4 50 bp of gene region capable of amplifying two or more sets of primer pairs, And iv) Detected in the sample if any of the sequences in the region amplified by each primer pair in the primer set are identical to each of the sequences of the detection target microorganism amplified by the same primer pair. The process of determining that the target microorganism was present,
Only including,
The primer set
Primer set A consisting of a primer pair (1) and (3):
Primer pair for amplifying a part of the gyrB gene sequence (1):
Forward primer AAAAATCGGTACGGTTCCACTTA (SEQ ID NO: 1) and
Reverse primer TAAAGCGGTCTTAAATCCAGCTTC (SEQ ID NO: 2);
Primer pair for amplifying a part of the pyrG gene sequence (3):
Forward Primer AATGCGTCGTGAAGTTGGTGAA (SEQ ID NO: 5) and
Reverse primer TGGCTTTGGACTTTCAAGGTGC (SEQ ID NO: 6)

Primer set B consisting of primer pairs (1) and (4):
Primer pair for amplifying a part of the gyrB gene sequence (1):
Forward primer AAAAATCGGTACGGTTCCACTTA (SEQ ID NO: 1) and
Reverse primer TAAAGCGGTCTTAAATCCAGCTTC (SEQ ID NO: 2);
Primer pair for amplifying a part of the pyrG gene sequence (4):
Forward Primer AATGCGTCGTGAAGTTGGTGAA (SEQ ID NO: 5) and
Reverse primer GCGTCAGTAACTGAAATGTAGGCATC (SEQ ID NO: 7)

Primer set C consisting of primer pairs (2) and (3):
Primer pair for amplifying a part of the gyrB gene sequence (2):
Forward primer CCGATGTTTACAAGTTTGAAGGTG (SEQ ID NO: 3) and
Reverse primer AAGCGGCTAAAGGTTTCAGAGAAT (SEQ ID NO: 4);
Primer pair for amplifying a part of the pyrG gene sequence (3):
Forward Primer AATGCGTCGTGAAGTTGGTGAA (SEQ ID NO: 5) and
Reverse primer TGGCTTTGGACTTTCAAGGTGC (SEQ ID NO: 6)
and

Primer set D consisting of primer pairs (2) and (4):
Primer pair for amplifying a part of the gyrB gene sequence (2):
Forward primer CCGATGTTTACAAGTTTGAAGGTG (SEQ ID NO: 3) and
Reverse primer AAGCGGCTAAAGGTTTCAGAGAAT (SEQ ID NO: 4);
Primer pair for amplifying a part of the pyrG gene sequence (4):
Forward Primer AATGCGTCGTGAAGTTGGTGAA (SEQ ID NO: 5) and
Reverse primer GCGTCAGTAACTGAAATGTAGGCATC (SEQ ID NO: 7)
At least one primer set selected from the group consisting of,
The method. The method of claim 1, wherein the sample is a beverage. Sample is heat sterilization, a 12 months period conserved beverages at 25 ° C., The method of claim 1, wherein. Further, as the primer set,
Primer set F consisting of primer pairs (7) and (9):
Primer pair for amplifying a part of the lepA gene sequence (7):
Forward primer GATGCAGCTTTAGAATTTGAACC (SEQ ID NO: 17) and reverse primer AGGCTCTTGCACTTCCTTGTATTC (SEQ ID NO: 18);
Primer pair for amplifying a part of the gyrA gene sequence (9) :.
Forward primer TTAGCTAAGGCTGAAGCAAGAGCA (SEQ ID NO: 20) and reverse primer CCAAKCCGGTCAAACGAACTAAAC (SEQ ID NO: 21)
Primer set G consisting of primer pairs (7) and (10):
Primer pair for amplifying a part of the lepA gene sequence (7):
Forward primer GATGCAGCTTTAGAATTTGAACC (SEQ ID NO: 17) and reverse primer AGGCTCTTGCACTTCCTTGTATTC (SEQ ID NO: 18);
Primer pair for amplifying a part of the gyrA gene sequence (10) :.
Forward primer GAAGCAAGAGCACACATTCTCGA (SEQ ID NO: 22) and reverse primer CCAAKCCGGTCAAACGAACTAAAC (SEQ ID NO: 21)
Primer set H consisting of primer pairs (8) and (9):
Primer pair for amplifying a part of the lepA gene sequence (8):
Forward primer TGCAGCTTTAGAATTTGAACCTG (SEQ ID NO: 19) and reverse primer AGGCTCTTGCACTTCCTTGTATTC (SEQ ID NO: 18);
Primer pair for amplifying a part of the gyrA gene sequence (9) :.
Forward primer TTAGCTAAGGCTGAAGCAAGAGCA (SEQ ID NO: 20) and reverse primer CCAAKCCGGTCAAACGAACTAAAC (SEQ ID NO: 21)
And-Primer set I consisting of primer pairs (8) and (10):
Primer pair for amplifying a part of the lepA gene sequence (8):
Forward primer TGCAGCTTTAGAATTTGAACCTG (SEQ ID NO: 19) and reverse primer AGGCTCTTGCACTTCCTTGTATTC (SEQ ID NO: 18);
Primer pair for amplifying a part of the gyrA gene sequence (10) :.
Forward primer GAAGCAAGAGCACACATTCTCGA (SEQ ID NO: 22) and reverse primer CCAAKCCGGTCAAACGAACTAAAC (SEQ ID NO: 21)
At least one primer set selected from the group consisting of, for use in addition used method of claim 1. A method for detecting the lactic acid bacterium Lactobacillus amylovorus CP1563 strain in a sample.
i) Step of extracting RNA from a sample,
ii) A step of synthesizing cDNA using the RNA extracted in step i) as a template,
iii) Using the cDNA synthesized in step ii) as a template
Primer set E consisting of primer pairs (5) and (6):
Primer pair for amplifying a part of the gyrB gene sequence (5):
Forward primer GTATCGACGTTGAAGTAGCTTTG (SEQ ID NO: 8) and reverse primer TAAAGCGGTCTTAAATCCAGCTTC (SEQ ID NO: 2);
Primer pair for amplifying a part of the pyrG gene sequence (6):
Forward primer AATGCGTCGTGAAGTTGGTGAA (SEQ ID NO: 5) and reverse primer TTGAATACCGATGCTGCGAAG (SEQ ID NO: 9)
And the process of performing PCR using
iv) The sequence of the region amplified by the primer pair (5) (excluding the primer sequence) is the same as the sequence of SEQ ID NO: 14, and the sequence of the region amplified by the primer pair (6) (primer sequence). When is the same as the sequence of SEQ ID NO: 15, L. The process of determining that the amylovourus CP1563 strain was present,
The method described above. The method according to claim 5 , wherein in step iii), PCR using the primer pair (5) and PCR using the primer pair (6) are performed in separate containers. L.A. contains a primer set consisting of the following primer pairs (5) and (6). amylovorus CP1563 strain detection kit:
Primer pair for amplifying a part of the gyrB gene sequence (5):
Forward primer GTATCGACGTTGAAGTAGCTTTG (SEQ ID NO: 8) and reverse primer TAAAGCGGTCTTAAATCCAGCTTC (SEQ ID NO: 2),
and,
Primer pair for amplifying a part of the pyrG gene sequence (6):
Forward primer AATGCGTCGTGAAGTTGGTGAA (SEQ ID NO: 5) and reverse primer TTGAATACCGATGCTGCGAAG (SEQ ID NO: 9). Primer set consisting of the following primer pairs (5) and (6):
Primer pair for amplifying a part of the gyrB gene sequence (5):
Forward primer GTATCGACGTTGAAGTAGCTTTG (SEQ ID NO: 8) and reverse primer TAAAGCGGTCTTAAATCCAGCTTC (SEQ ID NO: 2),
and,
Primer pair for amplifying a part of the pyrG gene sequence (6):
Forward primer AATGCGTCGTGAAGTTGGTGAA (SEQ ID NO: 5) and reverse primer TTGAATACCGATGCTGCGAAG (SEQ ID NO: 9).

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