Primer set for specific detection of Lactobacillus LQ80 strain, kit using the primer set, and detection method.

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

Technical Field

[0001] The present invention relates to a method for detecting specific lactic acid bacteria strains. Specifically, it relates to a primer set for detecting nucleic acids derived from the lactic acid bacterium Lactiplantibacillus plantarum LQ80 strain, a kit using the primer set, and a detection method.

Background Art

[0002] Lactic acid bacteria have long been known to have various effects. In recent years, as probiotics, they have various health maintenance effects such as intestinal regulation effects, immunity improvement effects, and anti-tumor effects by improving the intestinal flora in the digestive tract. Therefore, they are used as useful microbial resources that are deeply related to human health. Among them, the Lactiplantibacillus plantarum species is widely present in nature, such as in plants, foods, and animal bodies. It also grows well in synthetic media, has a wide range of sugar utilization properties, and high fermentation ability. Therefore, it is used worldwide as a probiotic for humans and animals and as a starter for fermented foods.

[0003] The LQ80 strain belonging to the Lactiplantibacillus plantarum species is a lactic acid bacterium for preparing fermented liquid feed separated from liquid feed raw materials for pigs (Patent Document 1). So far, improvements in intestinal flora in weaned piglets, reduction of chlorotetracycline-resistant Escherichia coli (Non-Patent Document 1), and effects of promoting intestinal villus elongation in pigs have been reported (Non-Patent Document 2). In addition, compared with other strains of the same species, it is excellent in extracellular polysaccharide productivity. Therefore, as a probiotic with excellent gastric acid and bile acid resistance, as a starter that can be expected to have effects on stabilizing fermented dairy products and improving texture, and as a functional food material with an immunostimulatory effect, it has attracted attention. However, since not all strains belonging to the Lactiplantibacillus plantarum species exhibit the same effects and functions, technology is needed to distinguish the LQ80 strain from other Lactiplantibacillus plantarum strains. In particular, for the LQ80 strain, technology is needed to prove that the LQ80 strain is present when it is intended to be used as an additive in feed, etc. Furthermore, while the LQ80 strain is currently expected to be used as a livestock probiotic, the Lactobacillus plantarum species is a type of lactic acid bacterium of the Lactobacillus genus that is GRAS (Generally Recognized as Safe Substances) (in 2020, it was reclassified as Lactiplantibacillus plantarum due to a reclassification of Lactobacillus lactic acid bacteria). Therefore, if safety tests are conducted, the possibility of use as a human probiotic can also be considered, and technology to specifically detect the LQ80 strain will become even more important.

[0004] A known method for detecting the lactic acid bacterium Lactiplantibacillus plantarum involves extracting a DNA sample from the lactic acid bacterium and performing species-specific PCR to confirm the presence or absence of the species (Patent Document 2). However, the methods described in these documents are for detecting the Lactiplantibacillus plantarum species and cannot be used to specifically detect only the LQ80 strain. Furthermore, live bacterial cells themselves could not be directly subjected to the PCR reaction; DNA had to be extracted from the live cells first before being subjected to the PCR reaction. Furthermore, in recent years, the use of heat-killed lactic acid bacteria as food additives has become more common, so there is a need for a method to detect heat-killed LQ80 strain bacteria. [Prior art documents] [Patent Documents]

[0005] [Patent Document 1] Japanese Patent Publication No. 2007-82468 [Patent Document 2] Japanese Patent Publication No. 2005-176809 [Non-patent literature]

[0006] [Non-Patent Document 1] Kobashi Y. et al., Anaerobe, 2008, Vol.14, No.4, pp.201-204 [Non-Patent Document 2] Yoshida Y.et al., Animal Science Journal, 2009, Vol.80, p.709-715 [Overview of the project] [Problems that the invention aims to solve]

[0007] The present invention aims to provide a primer set, a kit using the primer set, and a detection method that can specifically, rapidly, and simply identify the LQ80 strain without extracting DNA from live or heat-dead bacterial cells. [Means for solving the problem]

[0008] As a result of diligent research to solve the above problems, the inventors have discovered three sets of detection primers containing oligonucleotides having a base sequence specific to the LQ80 strain, and have completed the present invention.

[0009] The present invention is summarized in the following terms: 1. A primer set for detecting nucleic acids derived from Lactobacillus plantarum strain LQ80, consisting of a pair of oligonucleotides 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 acids derived from Lactobacillus plantarum strain LQ80, consisting of a pair of oligonucleotides 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 acids derived from Lactiplantibacillus plantarum strain LQ80, consisting of a pair of oligonucleotides containing the base sequence of SEQ ID NO: 1 and an oligonucleotide containing the base sequence of SEQ ID NO: 5. A nucleic acid detection kit derived from Lactiplantibacillus plantarum LQ80 strain, containing one, two, or three of the primer sets described in 4.1 to 4.3. 5. A primer set for detecting nucleic acids derived from heat-killed Lactobacillus plantarum LQ80 strain, consisting of a pair of oligonucleotides 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 nucleic acids derived from heat-killed Lactobacillus plantarum LQ80 strain, consisting of a pair of oligonucleotides containing the base sequence of SEQ ID NO: 3 and an oligonucleotide containing the base sequence of SEQ ID NO: 4. A kit for detecting nucleic acids derived from heat-killed Lactiplantibacillus plantarum LQ80 strain, containing one or two of the primer sets described in 7.5 and 6. 8. A method for detecting nucleic acids derived from Lactiplantibacillus plantarum LQ80 strain, comprising the following steps (1) and (2). (1) A step of amplifying nucleic acid fragments at an annealing temperature of 63-68°C using the primer set described in 1. or 2. (2) Step to detect nucleic acid fragments obtained in step (1) 9. A method for detecting nucleic acids derived from Lactiplantibacillus plantarum LQ80 strain, comprising the following steps (1) and (2). (1) Using the primer set described in 3., amplify the nucleic acid fragment at an annealing temperature of 55 - 66°C (2) Detect the nucleic acid fragment obtained in step (1)

Advantages of the Invention

[0010] According to the present invention, it is possible to specifically, simply, and rapidly detect the LQ80 strain with certainty without extracting DNA from the bacterial cell sample, which is very useful.

Brief Description of the Drawings

[0011] [Figure 1] It is a photograph of the agarose gel electrophoresis of the PCR product in Example 1. [Figure 2] It is a photograph of the agarose gel electrophoresis of the PCR product using the Lactiplantibacillus plantarum reference strain JCM1149 in Examples 1 - 3. [Figure 3] It is a photograph of the agarose gel electrophoresis of the PCR product in Example 2. [Figure 4] It is a photograph of the agarose gel electrophoresis of the PCR product in Example 3. [Figure 5] It is a photograph of the agarose gel electrophoresis of the PCR product in Example 4.

Modes for Carrying Out the Invention

[0012] The primer set of the present invention can amplify a chromosomal DNA region having a base sequence specific to the LQ80 strain by PCR (polymerase chain reaction), specifically recognize the LQ80 strain without recognizing strains closely related to the LQ80 strain, and specifically detect the LQ80 strain. Note that the LQ80 strain in the present invention refers to the lactic acid bacterium Lactiplantibacillus plantarum LQ80 strain, which has been deposited as LQ80 at the Patent Microorganisms Depositary, National Institute of Technology and Evaluation, and its accession number is NITE P-03564 (date of deposit: December 1, 2021).

[0013] The primer set of the present invention was designed based on the following method. First, six strains (WCFS1, JDM1, ST-III, SN35N, HFC8, ZJ316) with distant positions in the genomic phylogenetic tree within the L. plantarum species were selected from the L. plantarum strains whose genomes had been decoded (hereinafter, L. plantarum). Then, the genomic sequences of those six strains and the LQ80 strain were aligned using ProgressiveMauve (http: / / darlinglab.org / mauve / user-guide / introduction.html). As a result, 25 specific indels (insertions and deletions) in the LQ80 strain were detected, and 85 primer sets were designed in a form containing each indel. The primers were designed using Primer3 (https: / / github.com / primer3-org / primer3). Next, the genomic sequences of all 523 strains (as of 2020) registered with L. plantarum in NCBI were obtained, and the specificity of each of the above 85 primer sets was confirmed using MFEprimer (https: / / www.mfeprimer.com / ). As a result, 28 primer sets designed in a form containing 9 indels were expected to amplify only in the LQ80 strain and did not amplify in other L. plantarum strains. From among those 28 primer sets, 12 primer sets containing indels of multiple bases were further selected. Subsequently, PCR was performed using 12 primer sets according to the method described in the examples below. Only the three primer sets of the present invention showed specific amplification of LQ80 strain DNA. This led to the final determination of the three primer sets of the present invention.

[0014] <About the Primer Set of the Invention> The primer set of the present invention is summarized in Table 1 below. The primer set 1 of the present invention consists of a pair of oligonucleotides containing the base sequence of SEQ ID NO: 1 and an oligonucleotide containing the base sequence of SEQ ID NO: 2. The primer set 2 of the present invention consists of a pair of oligonucleotides containing the base sequence of SEQ ID NO: 3 and an oligonucleotide containing the base sequence of SEQ ID NO: 4. The primer set 3 of the present invention consists of a pair of oligonucleotides: one containing the base sequence of SEQ ID NO: 1 and the other containing the base sequence of SEQ ID NO: 5.

[0015] [Table 1]

[0016] Furthermore, the primer sets that can be used in the detection method of the present invention also include primer sets consisting of oligonucleotides having a base sequence substantially homologous to the base sequences of SEQ ID NOs: 1 to 5, depending on the base length, PCR conditions, etc. Here, substantially homologous means having homology to a fragment length sufficient to function as a PCR primer, etc. For example, the primer set of the present invention does not necessarily need to have 100% homology to the base sequences of SEQ ID NOs: 1-5, depending on the intended use and conditions. A few bases may differ near the 5' end of the primer in the target region. Even when using such primers, it is possible to appropriately amplify the target DNA fragment by considering factors such as the annealing temperature. For more details, when high specificity is required for the detection of the LQ80 strain, it is advisable to use completely homologous sequence sites (nucleotide sequences of SEQ ID NOs. 1-5) and select PCR conditions that only anneal with such sequences. 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. 1-5 and select PCR conditions that still allow annealing.

[0017] The primer set of the present invention can be synthesized by conventional DNA synthesis methods well known to those skilled in the art, for example, using a DNA synthesizer. Alternatively, the primers of the present invention can be obtained by commissioning a DNA synthesis company to perform the synthesis. By using the primer set of the present invention, LQ80 strain can be detected in a sample by performing PCR using the chromosomal DNA of the test bacterium contained in the sample as a template and determining the presence or absence of amplification products. In other words, when a PCR reaction specific to the primer sequence occurs, the region (target region) in the chromosomal DNA of the LQ80 strain that is sandwiched between sequences corresponding to the sequences of each pair of primers is amplified. Therefore, if an amplification product is obtained using the primer set of the present invention, the test bacterium is identified as strain LQ80, or it is determined that the test bacterium contains strain LQ80. Furthermore, if amplification products are obtained, determining the presence or absence of amplification products may include determining the length of the amplification products. For example, when using primer sets 1-3, if the test bacteria include strain LQ80, amplification products of 415 bp, 351 bp, and 756 bp are usually obtained, respectively. In this invention, the detection of the LQ80 strain includes detecting whether or not the LQ80 strain is present in the sample, and, if the test bacterium is a single species, identifying whether or not that test bacterium is the LQ80 strain.

[0018] Here, the sample used for detecting the LQ80 strain can be any sample in which the LQ80 strain may be present, such as pig feed, mixed cultures of lactic acid bacteria, feces of experimental animals such as mice and rats or humans, and bacteria present in the environment such as soil. The test bacterium may be a single isolated species or a mixture containing multiple bacterial species. Live or heat-dead bacterial cell samples can be used as samples as they are. Alternatively, DNA can be extracted from bacterial cell samples and used as a sample. Methods for extracting DNA from these samples are not limited to those used in general, such as degrading the cell wall with enzymes like lysozyme or physically destroying it with beads, and then using commercially available extraction kits (e.g., DNeasy Blood & Tissue kit (QIAGEN), Isogen).

[0019] <About the Primer Kit of the Present Invention> The primer kit of the present invention can be combined with other elements to form a kit for detecting the LQ80 strain. These other elements include, for example, one or more reagents necessary for nucleic acid extraction, PCR, and detection of amplification products. The primer kit may contain only one primer set, or a combination of two or three primer sets. Using multiple primer sets can improve the accuracy of LQ80 strain detection. Furthermore, this LQ80 strain detection kit may include, as a positive control, a DNA fragment having a portion of the chromosomal DNA sequence of the LQ80 strain and being amplified by the primer set of the present invention, and / or, as a negative control, a DNA fragment corresponding to the primer set of the present invention but having a nucleotide sequence with one or several nucleotide mismatches. As described above, the primer kit of the present invention can be used for the detection of the LQ80 strain.

[0020] <Regarding the detection method of the present invention> The method for detecting the LQ80 strain of the present invention includes (1) a step of amplifying nucleic acid fragments at an annealing temperature of 63 to 68°C using primer sets 1 and 2 of the present invention, or a step of amplifying nucleic acid fragments at an annealing temperature of 55 to 66°C using primer set 3 of the present invention, and (2) a step of detecting the nucleic acid fragments obtained in step (1). Specific examples of detection methods of the present invention include PCR, FISH, Southern hybridization, and dot hybridization. Among these, PCR is preferred.

[0021] In the PCR method for detecting the LQ80 strain, it is possible to use commonly used PCR reagents and equipment such as DNA polymerase, in addition to using the primer set of the present invention, and is not particularly limited. While there are no particular limitations on the DNA polymerase used in PCR, preferred examples include ExTaq (manufactured by Takara Bio Inc.), KOD DNA polymerase (manufactured by Toyobo Co., Ltd.), and KOD-plus-polymerase (manufactured by Toyobo Co., Ltd.). The PCR reaction conditions can be set appropriately depending on the PCR equipment used, the optimal temperature of the DNA polymerase, the length and type of DNA to be synthesized, etc. However, for primer sets 1 and 2, a preferred condition is to perform a total of 20 to 40 cycles, with one cycle consisting of "90-98°C for 5-30 seconds (thermal denaturation / dissociation) → 63-68°C for 5-30 seconds (annealing) → 65-80°C for 30-60 seconds (synthesis / extension)". An annealing temperature of 65°C is more preferred. For primer set 3, a preferred condition is to perform a total of 20 to 40 cycles, with one cycle consisting of "90-98°C for 5-30 seconds (thermal denaturation / dissociation) → 55-66°C for 5-30 seconds (annealing) → 65-80°C for 30-60 seconds (synthesis / extension)". An annealing temperature of 58°C is more preferred. In this case, the preferred ratio of template DNA to primer is approximately 0.24 μM of each primer for a total amount of 10-30 ng of genomic DNA. The presence or size of amplification products obtained by PCR can be determined by conventional nucleic acid detection methods. For example, amplification products can be detected by electrophoresis using agarose electrophoresis followed by staining with ethidium bromide or SYBRGreenI. The amount of amplification product can be determined by fluorescence intensity, and the molecular weight can be determined by comparison with a molecular weight marker. The presence or absence of amplification products can also be confirmed by cycle sequencing of the PCR products and measuring the base sequence and length using a DNA sequencer. Furthermore, the amplification reaction can be detected over time using real-time PCR. [Examples]

[0022] The present invention will be described below with reference to examples, but the technical scope of the present invention is not limited thereto. <Example 1: Confirmation of the specificity of primer set 1> (1) Template DNA As template DNA, we used DNA extracted from L. plantarum strain 38 and L. plantarum reference strain JCM1149, both held by the Livestock Research Division of our research institute, as well as DNA extracted from live and heat-dead cells of L. plantarum strain LQ80. For the creation of heat-killed bacterial cells, a heating condition of 105°C for 30 minutes was used. However, it has been confirmed that heating at 90°C or higher for 15 minutes or more reliably kills the bacteria, so the above heating conditions are not the only ones that can be used.

[0023] (2)DNA preparation In this example, a kit is used for convenience, but DNA may be prepared using other methods. Purified DNA is preferable because it provides better PCR reproducibility. (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 using the DNeasy Blood & Tissue Kit (QIAGEN). Details are as follows: (b-1) 0.1 mL of culture medium (turbidity of approximately 1.0 at OD 600 nm) was centrifuged at 8000 rpm for 5 minutes to collect the cells. The supernatant was discarded. (b-2) 180 μL of lysis solution (prepared by adding 20 mg lysozyme and 12 μL Triton X-100 to 1 mL of 2 × TE (20 mM Tris-HCl, 2 mM EDTA)) was added to the above-mentioned collected bacterial cells, suspended, and incubated at 37°C for 30 minutes. (b-3) After incubation, 25 μL of Proteinase K and 200 μL of Buffer AL were added to the above suspension, mixed, and incubated at 56°C for 30 minutes. (b-4) 200 μL of 100% ethanol was added to this. (b-5) The solution from (b-4) above was added to the spin column and centrifuged at 8000 rpm for 1 minute to adsorb the DNA onto the column. The filtration solution and tubing were discarded. (b-6) The column was placed in a new tube, 500 μL of Buffer AW1 was added, and the column was centrifuged at 8000 rpm for 1 minute. The phosphate and tube were discarded. (b-7) The column was placed in a new tube, 500 μL of Buffer AW2 was added, and the column was centrifuged at 14000 rpm for 2 minutes. (b-8) The column was placed in a 1.5 mL microtube, 200 μL of Buffer AE was added, and the mixture was centrifuged at 8000 rpm for 1 minute. (b-9) Measure the absorbance of the eluate containing the obtained DNA at OD260nm and determine the DNA concentration (OD 260 (×50 μg / mL) was diluted to 20 ng / μL and used as the template DNA solution.

[0024] (3) PCR reaction Using the DNA solutions obtained in (2) above as templates, a PCR reaction was performed according to the method of the PCR reaction reagent kit ExTaq (manufactured by Takara Bio Inc.) using primer set 1, which consists of oligonucleotide pairs containing the base sequences of SEQ ID NO: 1 and SEQ ID NO: 2. Assuming a total volume of PCR reaction solution of 50 μL, the kit included 0.25 μL of TaKaRa Taq, 5 μL of 10× PCR Buffer, 4 μL of dNTP mixture, 2.5 μL of template DNA, 0.6 μL of F primer (20 μM), 0.6 μL of R primer (20 μM), and 37.05 μL of MiliQ water. Using the GeneAmp PCR System 9700 (manufactured by Applied Biosystems Inc.), the mixture was preheated at 94°C for 240 seconds, followed by denaturation at 94°C for 30 seconds, annealing at 65°C for 30 seconds, and extension at 72°C for 45 seconds, repeated for 40 cycles. After that, it was heated at 72°C for 420 seconds.

[0025] (4) Agarose electrophoresis The PCR products obtained by the PCR reaction were subjected to electrophoresis on a 1.0% agarose gel at 100V for 30 minutes. Next, the agarose gel was stained with ethidium bromide (0.5 μg / mL), and the bands indicating amplification of the PCR product were observed under blue LED light. The results are shown in Figures 1 and 2, and in Tables 2 (Set 1) and 3 (Set 1; lanes 4-6). In the tables, "+" indicates that the PCR product was amplified to 415 bp after reaction with the primer, and "-" indicates that it did not react with the primer.

[0026] <Example 2: Confirmation of the specificity of primer set 2> The procedure was carried out in the same manner as in Example 1, except that a primer set 2 consisting of a pair of oligonucleotides containing the nucleotide 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 in Tables 2 (Set 2) and 3 (Set 2; lanes 1-3). In the tables, a "+" indicates that the product reacted with the primer and amplified to a length of 351 bp, while a "-" indicates that it did not react with the primer.

[0027] [Table 2]

[0028] [Table 3]

[0029] As shown in Figures 1-3 and Tables 2 and 3 (Sets 1 and 2; lanes 1-6), primer sets 1 and 2 showed amplification by PCR reaction only with samples using DNA extracted from live and heat-killed LQ80 strain cells, demonstrating their ability to specifically detect the LQ80 strain. Simultaneously, annealing temperature was investigated during the PCR reaction, and non-specific amplification was observed at temperatures lower than 63°C.

[0030] <Example 3: Confirmation of the specificity of primer set 3> The procedure was carried out in the same manner as in Example 1, except that a primer set 3 consisting of oligonucleotide pairs containing the base sequences of SEQ ID NO: 1 and SEQ ID NO: 5 was used as the primer set, the annealing temperature was set to 58°C, and electrophoresis was performed on a 1.8% agarose gel. The results are shown in Figures 2 and 4, and in Table 3 (Set 3; lanes 7-9) and Table 4. In the table, a "+" indicates that the product reacted with the primer and amplified to a length of 756 bp, while a "-" indicates that it did not react with the primer.

[0031] [Table 4]

[0032] As shown in Figures 2 and 4 and Tables 3 (Set 3; lanes 7-9) and 4, primer set 3 showed amplification by PCR reaction only with samples using DNA extracted from live LQ80 strain cells, demonstrating its ability to specifically detect the LQ80 strain. Simultaneously, the annealing temperature in the PCR reaction was investigated, and non-specific amplification was observed at temperatures lower than 55°C. Furthermore, because primer set 3 produces a longer chain length in the PCR reaction compared to primer sets 1 and 2, specific detection at lower annealing temperatures became possible. Furthermore, as can be seen from lane 42 in Figures 1, 3, and 4, and lane 42 in Tables 2 and 4, heat treatment of lactic acid bacteria usually denatures and breaks the DNA, weakening PCR amplification and making detection difficult. With primer set 3, it was not possible to amplify and detect DNA extracted from heat-killed LQ80 strain cells by PCR reaction. However, with primer sets 1 and 2, by shortening the chain length of the PCR reaction, it became clear that it was possible to amplify and detect DNA from LQ80 strain cells extracted from heat-killed cells by PCR reaction.

[0033] <Example 4: PCR using the bacterial cells themselves> Except for using live LQ80 strain cells themselves and heated LQ80 strain cells themselves as samples, and performing electrophoresis on a 1.8% agarose gel, the procedure was the same as in Example 1 for primer sets 1 and 2, and the same as in Example 3 for primer set 3. The results are shown in Figure 5 and Table 5. In the table, "+" indicates that the PCR products of 415 bp, 351 bp, and 756 bp were amplified by reacting with each primer, while "-" indicates that the product did not react with the primer.

[0034] [Table 5]

[0035] As shown in Figure 5 and Table 5, primer sets 1 and 2 show amplification by PCR reaction even when using live LQ80 strain cells and heated LQ80 strain cells themselves. Primer set 3 also shows amplification even when using live cells. This result means that when using the primer sets of the present invention, the LQ80 strain can be detected without extracting DNA from the LQ80 strain, which is extremely useful. [Industrial applicability]

[0036] The LQ80 strain was isolated from food waste used for livestock feed. In addition to its potential use as a livestock probiotic, safety testing could potentially lead to its use as a human probiotic. [Accession Number]

[0037] NITE P-03564

Claims

1. A primer set for detecting nucleic acids derived from Lactobacillus plantarum LQ80 strain, consisting of a pair of oligonucleotides: one with the base sequence of SEQ ID NO: 1 and the other with the base sequence of SEQ ID NO:

2.

2. A primer set for detecting nucleic acids derived from Lactobacillus plantarum LQ80 strain, consisting of a pair of oligonucleotides: one with the base sequence of SEQ ID NO: 3 and the other with the base sequence of SEQ ID NO:

4.

3. A primer set for detecting nucleic acids derived from Lactobacillus plantarum LQ80 strain, consisting of a pair of oligonucleotides: one with the base sequence of SEQ ID NO: 1 and the other with the base sequence of SEQ ID NO:

5.

4. A kit for detecting nucleic acids derived from Lactobacillus plantarum LQ80 strain, comprising one, two, or three of the primer sets described in claims 1 to 3.

5. A primer set for detecting nucleic acids derived from heat-killed Lactobacillus plantarum LQ80 strain, consisting of a pair of oligonucleotides: one with the base sequence of SEQ ID NO: 1 and the other with the base sequence of SEQ ID NO:

2.

6. A primer set for detecting nucleic acids derived from heat-killed Lactobacillus plantarum LQ80 strain, consisting of a pair of oligonucleotides: one with the base sequence of SEQ ID NO: 3 and the other with the base sequence of SEQ ID NO:

4.

7. A kit for detecting nucleic acids derived from heat-killed Lactobacillus plantarum LQ80 strain, comprising one or two of the primer sets described in claims 5 and 6.

8. A method for detecting nucleic acids derived from Lactobacillus plantarum LQ80 strain, comprising the following steps (1) and (2). (1) A step of amplifying nucleic acid fragments at an annealing temperature of 63 to 68°C using the primer set described in claim 1 or 2. (2) Step to detect nucleic acid fragments obtained in step (1)

9. A method for detecting nucleic acids derived from Lactobacillus plantarum LQ80 strain, comprising the following steps (1) and (2). (1) A step of amplifying nucleic acid fragments at an annealing temperature of 55 to 66°C using the primer set described in claim 3. (2) Step to detect nucleic acid fragments obtained in step (1)

Images