JP2023106944A - Primer set which specifically detects lactic acid bacteria lq80 strain, kit including primer set, and detection method using primer set - Google Patents

Abstract

To provide a primer set which can identify specifically the lactic acid bacteria Lactiplantibacillus plantarum LQ80 strain quickly and readily, without extracting DNA from a bacterial sample, a kit including the primer set, and a detection method using the primer set.SOLUTION: A primer set for detecting a nucleic acid derived from the lactic acid bacteria Lactiplantibacillus plantarum LQ80 strain, includes a pair of oligonucleotides containing a specific base sequence.SELECTED DRAWING: Figure 5

Description

The present invention relates to a method for detecting specific lactic acid bacteria strains. Specifically, it relates to a primer set for detecting nucleic acid derived from Lactiplantibacillus plantarum LQ80 strain of lactic acid bacterium, a kit and a detection method using the primer set.

Lactic acid bacteria have been known to have various effects for a long time, and in recent years, various health maintenance effects such as intestinal regulation effect by improving the bacterial flora in the gastrointestinal tract, immunity improvement effect, antitumor effect as a probiotic , it is used as a useful microbial resource deeply related to human health.
Among them, Lactiplantibacillus plantarum species exist widely in the natural world, such as in plants, foods, and animals. and as a probiotic for animals and as a fermented food starter worldwide.

The LQ80 strain, which belongs to the species Lactiplantibacillus plantarum, is a lactic acid bacterium for the preparation of fermented liquid feed isolated from raw materials for swine liquid feed (Patent Document 1), and has been shown to improve intestinal flora in weaned piglets and reduce chlortetracycline-resistant E. coli. (Non-Patent Document 1), an intestinal villus elongation promoting effect in pigs, etc. have been reported (Non-Patent Document 2). In addition, compared to other strains of the same species, it has excellent exopolysaccharide productivity, so it can be expected to stabilize fermented milk products and improve texture as a probiotic with excellent gastric acid and bile acid resistance. It is attracting attention as a starter and as a functional food material with an immunostimulatory effect.
However, not all strains belonging to the Lactiplantibacillus plantarum species exhibit similar effects and functions, so a technique for distinguishing the LQ80 strain from other Lactiplantibacillus plantarum strains is required. In particular, with regard to the LQ80 strain, there is a need for a technique to prove that the LQ80 strain is present when adding it to feed or the like. In addition, the LQ80 strain is currently expected to be used as a livestock probiotic, but the Lactobacillus plantarum species is a type of lactic acid bacterium of the genus Lactobacillus that is considered to be GRAS (Generally Recognized as Safe Substances) It has been classified as Lactiplantibacillus plantarum), so it is possible that it can be used as a human probiotic by conducting safety tests, and the technology to specifically detect the LQ80 strain will become even more important. I think that the.

As a method for detecting the lactic acid bacterium Lactiplantibacillus plantarum, a method is known in which the presence or absence of the lactic acid bacterium is confirmed by performing bacterial species-specific PCR using a DNA sample extracted from the lactic acid bacterium (Patent Document 2).
However, the methods described in these documents detect Lactiplantibacillus plantarum species and cannot be used for strain-specific detection of the LQ80 strain alone. Moreover, the viable cells themselves cannot be subjected to the PCR reaction as they are, and it is necessary to first extract the DNA from the viable cells and then subject the DNA to the PCR reaction.
In recent years, heat-killed cells of lactic acid bacteria are often used as food additives, so a method for detecting heat-killed cells of the LQ80 strain is also desired.

Japanese Unexamined Patent Application Publication No. 2007-82468 JP 2005-176809 A

Kobashi Y. et al. , Anaerobe, 2008, Vol. 14, No. 4, p. 201-204 Yoshida Y. et al. , Animal Science Journal, 2009, Vol. 80, p. 709-715

The present invention provides a primer set that can specifically identify the LQ80 strain quickly and easily without extracting DNA from viable cells and heat-killed cells, a kit using the primer set, and a detection method. is an issue.

As a result of intensive research to solve the above problems, the present inventors have found three sets of detection primer sets containing oligonucleotides having nucleotide sequences specific to the LQ80 strain, and have completed the present invention. .

Specifically, the gist of the present invention is as follows.
1. A primer set for detecting nucleic acid derived from lactic acid bacterium Lactiplantibacillus plantarum LQ80 strain, comprising a pair of an oligonucleotide containing the base sequence of SEQ ID NO: 1 and an oligonucleotide containing the base sequence of SEQ ID NO: 2.
2. A primer set for detecting nucleic acid derived from lactic acid bacterium Lactiplantibacillus plantarum LQ80 strain, comprising a pair of an oligonucleotide containing the base sequence of SEQ ID NO:3 and an oligonucleotide containing the base sequence of SEQ ID NO:4.
3. A primer set for detecting nucleic acid derived from lactic acid bacterium Lactiplantibacillus plantarum LQ80 strain, comprising a pair of an oligonucleotide containing the base sequence of SEQ ID NO: 1 and an oligonucleotide containing the base sequence of SEQ ID NO: 5.
4.1. ~3. A kit for detecting a nucleic acid derived from the lactic acid bacterium Lactiplantibacillus plantarum LQ80 strain, containing one, two, or three of the primer sets described in .
5. A primer set for detecting heat-killed cell-derived nucleic acid of lactic acid bacterium Lactiplantibacillus plantarum LQ80 strain, comprising a pair of an oligonucleotide containing the base sequence of SEQ ID NO: 1 and an oligonucleotide containing the base sequence of SEQ ID NO: 2.
6. A primer set for detecting heat-killed cell-derived nucleic acid of lactic acid bacterium Lactiplantibacillus plantarum LQ80 strain, comprising a pair of an oligonucleotide containing the nucleotide sequence of SEQ ID NO: 3 and an oligonucleotide containing the nucleotide sequence of SEQ ID NO: 4.
7.5. 6. A kit for detecting nucleic acid derived from heat-killed cells of the lactic acid bacterium Lactiplantibacillus plantarum LQ80 strain, comprising one or two of the primer sets described in .
8. A method for detecting a nucleic acid derived from the lactic acid bacterium Lactiplantibacillus plantarum LQ80 strain, comprising the following steps (1) and (2).
(1) 1. or 2. Step (2) of amplifying the nucleic acid fragment at an annealing temperature of 63 to 68° C. using the primer set described in 9. of detecting the nucleic acid fragment obtained in step (1). A method for detecting a nucleic acid derived from the lactic acid bacterium Lactiplantibacillus plantarum LQ80 strain, comprising the following steps (1) and (2).
(1)3. Step (2) of amplifying the nucleic acid fragment at an annealing temperature of 55 to 66° C. using the primer set described in (2) Step of detecting the nucleic acid fragment obtained in Step (1)

INDUSTRIAL APPLICABILITY According to the present invention, it is possible to specifically detect the LQ80 strain simply, quickly, and reliably without extracting DNA from a bacterial sample, which is extremely useful.

1 is a photograph of agarose gel electrophoresis of PCR products in Example 1. FIG. FIG. 2 is a photograph of agarose gel electrophoresis of PCR products using Lactiplantibacillus plantarum type strain JCM1149 strain in Examples 1 to 3. FIG. 2 is a photograph of agarose gel electrophoresis of PCR products in Example 2. FIG. 2 is a photograph of agarose gel electrophoresis of PCR products in Example 3. FIG. 2 is a photograph of agarose gel electrophoresis of PCR products in Example 4. FIG.

The primer set of the present invention can amplify a chromosomal DNA region having a nucleotide sequence specific to the LQ80 strain by PCR (polymerase chain reaction), and can recognize the LQ80 strain without recognizing strains related to the LQ80 strain. It is possible to specifically recognize and specifically detect the LQ80 strain.
In addition, the LQ80 strain in the present invention means the lactic acid bacterium Lactiplantibacillus plantarum LQ80 strain, and as LQ80, it has been deposited at the National Institute of Technology and Evaluation Patent Microorganisms Depositary Center, and the accession number is NITE P-03564 ( Acceptance date: December 1, 2021).

The primer set of the present invention is designed based on the following method.
First, among the genome-encoded Lactiplantibacillus plantarum (hereinafter, L. plantarum) strains, L. Six strains (WCFS1, JDM1, ST-III, SN35N, HFC8, ZJ316) with distant positions in the genome phylogenetic tree within the plantarum species were selected.
Then, the genome sequences of the 6 strains and the LQ80 strain were aligned using Progressive Mauve (http://darlinglab.org/mauve/user-guide/introduction.html).
As a result, 25 indels (insertion deletions) specific to the LQ80 strain were detected, and 85 sets of primers were designed including each indel. Primers were designed using Primer3 (https://github.com/primer3-org/primer3).
Next, from NCBI to L.I. Obtain the genome sequences of all 523 strains (as of 2020) registered in plantarum, and use MFEprimer (https://www.mfeprimer.com/) for each of the 85 primer sets described above to determine specificity. Confirmed. As a result, 28 pairs of primer sets designed to contain 9 indels were expected to amplify only in the LQ80 strain, and other L. plantarum strain did not amplify. From the 28 sets of primer sets, 12 sets of primer sets containing indels of multiple bases were further selected.
Thereafter, PCR was performed using the 12 sets of primer sets by the method described in Examples below, and only the 3 sets of primer sets of the present invention were the only primer sets in which amplification specific only to LQ80 strain DNA was actually confirmed. rice field. As a result, the three primer sets of the present invention were finally determined.

<Regarding the primer set of the present invention>
The primer sets of the present invention are summarized in Table 1 below.
The primer set 1 of the present invention consists of a pair of an oligonucleotide containing the nucleotide sequence of SEQ ID NO: 1 and an oligonucleotide containing the nucleotide sequence of SEQ ID NO:2.
The primer set 2 of the present invention consists of a pair of an oligonucleotide containing the base sequence of SEQ ID NO:3 and an oligonucleotide containing the base sequence of SEQ ID NO:4.
Primer set 3 of the present invention consists of a pair of an oligonucleotide containing the nucleotide sequence of SEQ ID NO: 1 and an oligonucleotide containing the nucleotide sequence of SEQ ID NO:5.

In addition, the primer set that can be used in the detection method of the present invention also includes a primer set consisting of oligonucleotides having a nucleotide sequence substantially homologous to the nucleotide sequences of SEQ ID NOs: 1 to 5, depending on the nucleotide length, PCR conditions, and the like. included. Here, "substantially homologous" means having fragment length and homology to the extent that it can function as a PCR primer or the like.
For example, the primer set of the present invention does not necessarily have 100% homology with the nucleotide sequences of SEQ ID NOs: 1 to 5, depending on the purpose and conditions of use. A few bases may differ in the vicinity. Even if such primers are used, it is possible to appropriately amplify the target DNA fragment by considering the annealing temperature and the like.
Specifically, in the detection of the LQ80 strain, if high specificity is required, use a completely homologous sequence site (nucleotide sequences of SEQ ID NOS: 1 to 5), and anneal only with such a sequence PCR conditions may be selected. On the other hand, if conditions with relatively low specificity are acceptable, it is possible to use sequences that differ by a few nucleotides from the nucleotide sequences of SEQ ID NOs: 1 to 5, and to select PCR conditions that still allow annealing. can.

The primer set of the present invention can be synthesized by a conventional DNA synthesis method well known to those skilled in the art, for example, using a DNA synthesizer. The primers of the present invention can also be obtained by entrusting synthesis to a DNA synthesizer.
Using the primer set of the present invention, PCR is performed using the chromosomal DNA of the test bacterium contained in the sample as a template, and the presence or absence of an amplified product is determined to detect the LQ80 strain in the sample. That is, when a PCR reaction specific to the sequences of the primers occurs, a region (target region) sandwiched between sequences corresponding to the sequences of the paired primers in the chromosomal DNA of the LQ80 strain is amplified.
Therefore, if an amplification product is obtained using the primer set of the present invention, the test bacterium is identified as the LQ80 strain, or it is determined that the test bacterium contains the LQ80 strain.
Determining the presence or absence of an amplification product may also include determining the length of the amplification product, if an amplification product is obtained. For example, when primer sets 1 to 3 are used, amplification products of 415 bp, 351 bp, and 756 bp are usually obtained if the strain LQ80 is included in the test bacteria.
In the present invention, the detection of the LQ80 strain means detecting whether the LQ80 strain is present in the sample, and when the test bacterium is a single species, the test bacterium is the LQ80 strain. including identifying whether or not

Here, the sample used for detecting the LQ80 strain may be any sample in which the LQ80 strain can be present. Bacteria present in environments such as experimental animal and human feces, soil, and the like can be mentioned. The test bacterium may be an isolated single species or a mixture containing multiple bacterial species.
A sample of live cells or heat-killed cells can be used as a sample as it is. DNA can also be extracted from a bacterial cell sample and used as a sample. As a method for extracting DNA from these samples, for example, the cell wall is degraded with an enzyme such as Lysozyme or physically disrupted with beads or the like, which is generally used for the preparation of genomic DNA of lactic acid bacteria, followed by commercially available extraction. A kit (DNeasy Blood & Tissue kit (manufactured by QIAGEN), Isogen, etc.) can be used, and is not particularly limited.

<Regarding the primer kit of the present invention>
The primer kit of the present invention can be combined with other elements to form an LQ80 strain detection kit. Said other elements include, for example, any one or more of the reagents necessary for nucleic acid extraction, PCR, and amplification product detection. The primer set contained in the primer kit may be of only one type, or may be a combination of two or three types. The use of multiple primer sets can increase the precision of detection of the LQ80 strain. In addition, this LQ80 strain detection kit includes, as a positive control, a DNA fragment that has a part of the chromosomal DNA sequence of the LQ80 strain and can be amplified by the primer set of the present invention, and / or as a negative control, the , but may contain a DNA fragment having a base sequence with one or several base mismatches.
The primer kit of the present invention can be used to detect the LQ80 strain, as described above.

<Regarding the detection method of the present invention>
The method for detecting the LQ80 strain of the present invention includes (1) the step of amplifying a nucleic acid fragment at an annealing temperature of 63 to 68 ° C. using the primer sets 1 and 2 of the present invention, or the annealing temperature using the primer set 3 of the present invention It comprises the step of amplifying the nucleic acid fragment at 55-66°C, and the step of (2) detecting the nucleic acid fragment obtained in step (1).
Specific examples of the detection method of the present invention include PCR, FISH, Southern hybridization, dot hybridization, and the like. Among them, the PCR method is preferred.

Among them, in the detection method of the LQ80 strain by PCR method, except for using the primer set of the present invention, it is possible to use commonly used PCR reagents and equipment such as DNA polymerase, and there is no particular limitation. .
The DNA polymerase used in PCR is not particularly limited, but ExTaq (manufactured by Takara Bio), KOD DNA polymerase (manufactured by Toyobo), KOD-plus-polymerase (manufactured by Toyobo) and the like are preferred.
The PCR reaction conditions may be appropriately set according to the PCR equipment used, the optimum temperature of the DNA polymerase, the length and type of DNA to be synthesized, etc. Seconds (thermal denaturation/dissociation) → 5 to 30 seconds at 63 to 68°C (annealing) → 30 to 60 seconds at 65 to 80°C (synthesis/extension) are preferably performed for a total of 20 to 40 cycles. The annealing temperature is more preferably 65°C. For primer set 3, "5 to 30 seconds at 90 to 98°C (thermal denaturation/dissociation) → 5 to 30 seconds at 55 to 66°C (annealing) → 30 to 60 seconds at 65 to 80°C (synthesis/extension )” as one cycle, a total of 20 to 40 cycles is preferable. The annealing temperature is more preferably 58°C. In this case, the quantitative ratio of template DNA and primers is preferably about 0.24 μM of each primer for 10-30 ng of genomic DNA.
The presence or absence or size of an amplification product obtained by PCR can be determined by a conventional nucleic acid detection method. For example, after electrophoresis by agarose electrophoresis, the amplified product can be detected by staining with ethidium bromide or SYBR Green I. Also, the amount of amplification product can be determined by fluorescence intensity, and the molecular weight can be determined by comparison with molecular weight markers. The presence or absence of an amplification product can also be confirmed by measuring the base sequence and length using a DNA sequencer after cycle sequencing the PCR product. Moreover, according to the real-time PCR method, the amplification reaction can be detected over time.

EXAMPLES The present invention will be described below with reference to examples, but the technical scope of the present invention is not limited by these examples.
<Example 1: Confirmation of specificity of primer set 1>
(1) Template DNA
As a template DNA, L. spp. plantarum 38 strain and L. DNA extracted from the JCM1149 type plantarum strain, and DNA extracted from the live and heat-killed cells of the LQ80 strain were used.
In addition, the heating conditions of 105° C. and 30 minutes were adopted when the heat-killed cells were prepared. However, it has already been confirmed that heating at 90° C. or higher for 15 minutes or longer will surely kill the cells, and the heating conditions are not limited to those described above.

(2) DNA preparation In this example, a kit is used because it is convenient, but other DNA preparation methods may be used. It is preferable to use purified DNA because of good reproducibility of PCR.
(a) The test strain was cultured overnight in MRS liquid medium.
(b) DNA was prepared according to the DNA preparation protocol for Gram-positive bacteria of DNeasy Blood & Tissue Kit (manufactured by QIAGEN). Details are as follows.
(b-1) 0.1 mL of the culture solution (turbidity of OD 600 nm approximately 1.0) was centrifuged at 8000 rpm for 5 minutes to collect the cells. This supernatant was discarded.
(b-2) 180 μL of lysis solution (prepared by adding 20 mg of lysozyme and 12 μL of Triton X-100 to 1 mL of 2×TE (20 mM Tris-HCl, 2 mM EDTA)) was added to the above collected cells and suspended. After turbidity, it was incubated at 37°C for 30 minutes.
(b-3) 25 μL of Proteinase K and 200 μL of Buffer AL were added to the suspension after incubation, mixed, and incubated at 56° C. for 30 minutes.
(b-4) To this, 200 μL of 100% ethanol was added.
(b-5) The solution of (b-4) above was added to a spin column and centrifuged at 8000 rpm for 1 minute to adsorb DNA onto the column. The column flow-through and tubing were discarded.
(b-6) The column was set in a new tube, 500 μL of Buffer AW1 was added, and centrifuged at 8000 rpm for 1 minute. The column flow-through and tubing were discarded.
(b-7) The column was set in a new tube, 500 μL of Buffer AW2 was added, and centrifuged at 14000 rpm for 2 minutes.
(b-8) The column was set in a 1.5 mL microtube, 200 μL of Buffer AE was added, and centrifuged at 8000 rpm for 1 minute.
(b-9) The absorbance at OD260 nm of the resulting eluate containing DNA was measured, and the DNA concentration (OD ₂₆₀ ×50 µg/mL) was adjusted to 20 ng/µL, which was used as a template DNA solution.

(3) PCR reaction Using each DNA solution obtained in (2) above as a template, using a primer set 1 consisting of a pair of oligonucleotides containing the base sequences of SEQ ID NO: 1 and SEQ ID NO: 2, PCR reaction reagent kit ExTaq (manufactured by Takara Bio Inc.), a PCR reaction was performed. Assuming that the total volume of the PCR reaction solution is 50 μL, TaKaRa Taq attached to the kit 0.25 μL, 10×PCR Buffer 5 μL, dNTP mixture 4 μL, template DNA 2.5 μL, F primer (20 μM) 0.6 μL, R primer (20 μM) 0 .6 μL and 37.05 μL of MiliQ water were preheated at 94° C. for 240 seconds using GeneAmp PCR System 9700 (manufactured by Applied Biosystems), denatured at 94° C. for 30 seconds, and annealed at 65° C. for 30 seconds. , extension was performed at 72° C. for 40 cycles of 45 seconds. After that, it was heated at 72° C. for 420 seconds.

(4) Agarose electrophoresis The PCR product obtained by the PCR reaction was electrophoresed on a 1.0% agarose gel at 100 V for 30 minutes. Next, after staining the agarose gel with ethidium bromide (0.5 μg/mL), a band indicating amplification of the PCR product was observed under blue LED light. The results are shown in Figures 1 and 2 and Tables 2 (set 1) and 3 (set 1; lanes 4-6). In the table, "+" indicates that a 415 bp PCR product was amplified by reaction with the primers, and "-" indicates that it did not react with the primers.

<Example 2: Confirmation of specificity of primer set 2>
Everything was carried out in the same manner as in Example 1, except that primer set 2 consisting of a pair of oligonucleotides containing the base sequences of SEQ ID NO:3 and SEQ ID NO:4 was used as the primer set.
The results are shown in Figures 2 and 3 and Tables 2 (set 2) and 3 (set 2; lanes 1-3). In the table, "+" indicates that a 351 bp PCR product was amplified by reaction with the primers, and "-" indicates that it did not react with the primers.

As shown in FIGS. 1 to 3 and Tables 2 and 3 (sets 1 and 2; lanes 1 to 6), primer sets 1 and 2 used DNA extracted from live cells and heat-killed cells of the LQ80 strain. Amplification by the PCR reaction was observed only in the sample, and it became clear that the LQ80 strain could be specifically detected. At the same time, the annealing temperature was also examined in the PCR reaction, but non-specific amplification was observed at temperatures lower than 63°C.

<Example 3: Confirmation of specificity of primer set 3>
Using a primer set 3 consisting of a pair of oligonucleotides containing the base sequences of SEQ ID NO: 1 and SEQ ID NO: 5 as a primer set, annealing temperature was set to 58 ° C., and electrophoresis was performed on a 1.8% agarose gel. Everything else was performed in the same manner as in Example 1.
The results are shown in FIGS. In the table, "+" indicates that a 756 bp PCR product was amplified by reaction with the primers, and "-" indicates that it did not react with the primers.

As shown in FIGS. 2, 4 and Table 3 (set 3; lanes 7-9) and Table 4, primer set 3 is only a sample using DNA extracted from living cells of LQ80 strain, and amplification by PCR reaction is not performed. It was found that the LQ80 strain can be specifically detected. At the same time, the annealing temperature was also examined in the PCR reaction, but non-specific amplification was observed at temperatures lower than 55°C. Also, when primer set 3 was used, the chain length of the PCR reaction was longer than when primer sets 1 and 2 were used, so specific detection was possible at a lower annealing temperature.
In addition, as can be seen from lane 42 in FIGS. 1, 3, and 4, and lane 42 in Tables 2 and 4, heat treatment of lactic acid bacteria normally denatures and cuts DNA, resulting in weak amplification by PCR. , making it difficult to detect. With primer set 3, the DNA extracted from the heat-killed strain LQ80 could not be amplified and detected by PCR reaction, but with primer sets 1 and 2, by shortening the chain length of the PCR reaction, heat-killed cells were found. It was clarified that the DNA of the LQ80 strain extracted from the cells can be amplified and detected by PCR reaction.

<Example 4: PCR using bacterial cells themselves>
In the case of primer sets 1 and 2, Example 1 and primer Set 3 was carried out in the same manner as in Example 3.
The results are shown in FIG. 5 and Table 5. In the table, PCR products of 415 bp, 351 bp, and 756 bp that were amplified by reaction with the respective primers are indicated as "+", and those that did not react with the primers are indicated as "-".

As shown in FIG. 5 and Table 5, primer sets 1 and 2 are amplified by PCR even when LQ80 strain live cells and LQ80 strain heated cells themselves are used. Amplification of primer set 3 is observed even when viable cells are used. This result means that the LQ80 strain can be detected without extracting DNA from the LQ80 strain when using the primer set of the present invention, which is very useful.

The source of the isolation of the LQ80 strain is food residue used for livestock feed, but in addition to livestock probiotics, it is also possible to use it as a human probiotic by conducting safety tests.

NITE P-03564

Claims (9)

A primer set for detecting nucleic acid derived from lactic acid bacterium Lactiplantibacillus plantarum LQ80 strain, comprising a pair of an oligonucleotide containing the base sequence of SEQ ID NO: 1 and an oligonucleotide containing the base sequence of SEQ ID NO: 2. A primer set for detecting nucleic acid derived from lactic acid bacterium Lactiplantibacillus plantarum LQ80 strain, comprising a pair of an oligonucleotide containing the base sequence of SEQ ID NO:3 and an oligonucleotide containing the base sequence of SEQ ID NO:4. A primer set for detecting nucleic acid derived from lactic acid bacterium Lactiplantibacillus plantarum LQ80 strain, comprising a pair of an oligonucleotide containing the base sequence of SEQ ID NO: 1 and an oligonucleotide containing the base sequence of SEQ ID NO: 5. A kit for detecting a nucleic acid derived from the lactic acid bacterium Lactiplantibacillus plantarum LQ80 strain, comprising one, two, or three of the primer sets according to any one of claims 1 to 3. A primer set for detecting heat-killed cell-derived nucleic acid of lactic acid bacterium Lactiplantibacillus plantarum LQ80 strain, comprising a pair of an oligonucleotide containing the base sequence of SEQ ID NO: 1 and an oligonucleotide containing the base sequence of SEQ ID NO: 2. A primer set for detecting heat-killed cell-derived nucleic acid of lactic acid bacterium Lactiplantibacillus plantarum LQ80 strain, comprising a pair of an oligonucleotide containing the nucleotide sequence of SEQ ID NO: 3 and an oligonucleotide containing the nucleotide sequence of SEQ ID NO: 4. A kit for detecting nucleic acid derived from heat-killed cells of lactic acid bacterium Lactiplantibacillus plantarum LQ80 strain, comprising one or two of the primer sets according to claims 5 and 6. A method for detecting a nucleic acid derived from the lactic acid bacterium Lactiplantibacillus plantarum LQ80 strain, comprising the following steps (1) and (2).
(1) A step of amplifying a nucleic acid fragment at an annealing temperature of 63 to 68° C. using the primer set of claim 1 or 2; (2) A step of detecting the nucleic acid fragment obtained in step (1); A method for detecting a nucleic acid derived from the lactic acid bacterium Lactiplantibacillus plantarum LQ80 strain, comprising the following steps (1) and (2).
(1) A step of amplifying a nucleic acid fragment at an annealing temperature of 55 to 66° C. using the primer set of claim 3. (2) A step of detecting the nucleic acid fragment obtained in step (1).