Primer set and nucleic acid amplification method

Abstract

The present invention provides a primer set specifically for the isothermal nucleic acid amplification of nucleic acids from obligate anaerobic bacteria or lactic acid bacteria. The present invention is: a primer set for detecting obligate anaerobic bacteria, said primer set comprising a forward primer including the base sequence represented by SEQ ID NO: 1 and a reverse primer including the base sequence represented by SEQ ID NO: 2; a primer set for detecting lactic acid bacteria, said primer set comprising a forward primer including the base sequence represented by SEQ ID NO: 3 and a reverse primer including the base sequence represented by SEQ ID NO: 4; a primer set for detecting lactococcus bacteria, said primer set comprising a forward primer including the base sequence represented by SEQ ID NO: 5 and a reverse primer including the base sequence represented by SEQ ID NO: 6; and a nucleic acid amplification method in which a RPA reaction is carried out using a test sample and one of the abovementioned primer sets to amplify a nucleic acid from the rRNA gene of obligate anaerobic bacteria or lactic acid bacteria contained in the test sample.

Description

Primer set and nucleic acid amplification method

[0001] The present invention relates to a primer set for specifically amplifying nucleic acids derived from obligately anaerobic bacteria or lactic acid bacteria, and a method for amplifying nucleic acids derived from obligately anaerobic bacteria or lactic acid bacteria contained in a test sample using the primer set.This application claims priority based on Japanese Patent Application No. 2023-219660, filed in Japan on December 26, 2023, the contents of which are incorporated herein by reference.

[0002] It is necessary to maintain the quality of processed foods and beverages from the time they are produced until they are consumed by the general consumer. In particular, since microorganisms not only cause quality deterioration but also food poisoning, preventing microbial contamination is extremely important from the viewpoint of food safety. One method for testing microorganisms in foods and beverages is, for example, to isolate and culture microorganisms from the food or beverage sample in an appropriate culture medium, and then identify them based on their bacteriological properties. In recent years, a method for performing genetic analysis on the DNA of isolated and cultured bacterial cells has been used as a more rapid method for identifying microorganisms.

[0003] In identifying microorganisms using genetic analysis, the base sequence of rRNA genes is generally used. The base sequence of rRNA genes extracted from a test sample is determined using a sequencer, and the obtained base sequence information is referenced to international base sequence databases such as DDBJ / EMBL / GenBank to identify the microbial species in the test sample. In this case, to efficiently detect microorganisms present in trace amounts in the test sample, a specific region of the rRNA gene is amplified in advance using PCR (polymerase chain reaction) or the like, and the resulting amplified product is then subjected to the sequencer. In recent years, among third-generation sequencers, nanopore sequencers have been developed that identify base sequences by changes in current generated when DNA passes through nanopores (nanoscale holes) (see, for example, Non-Patent Document 1). In particular, the extremely compact nanopore sequencer "MinION" (manufactured by Oxford Nanopore Technologies) is a portable sequencer that enables genetic analysis in a variety of locations.

[0004] By designing primers specific to each organism using PCR, it is possible to determine the presence or absence of a target organism based solely on the presence or absence of a nucleic acid amplification product, and PCR is used in microbial testing for various bacteria, viruses, etc. The species-specific primers used in microbial testing include primers that specifically amplify only the genetic nucleic acid of a specific bacterial species, and primers that specifically amplify the genetic nucleic acid of a specific group of microorganisms. For example, many PCR primers have been developed for specifically detecting lactic acid bacteria (Non-Patent Document 2).

[0005] However, PCR requires a relatively expensive specialized device called a thermal cycler and takes a long reaction time.On the other hand, various isothermal nucleic acid amplification methods, including recombinase polymerase amplification (RPA) (Non-Patent Document 3), are known as nucleic acid amplification methods that do not require a thermal cycler.

[0006] Anonymous, Nature Biotechnology,2018, vol.36, p287.Kim et al., BMC Microbiology, 2020, vol.20, Article:96Li et al., Analyst, 2019, vol.144, p.31-67.

[0007] PCR primers generally only need to have a base sequence of about 20 bases in length. Therefore, it is relatively easy to design not only primers that specifically amplify genes from a specific bacterial species, but also primers that specifically amplify genes from all bacterial species within a specific biological species group consisting of two or more bacterial species (hereinafter referred to as "universal primers"), and many such primers have already been reported, including Non-Patent Document 2.

[0008] On the other hand, RPA primers require a base sequence of 30 bases or more. Generally, the longer the base length, the more specific the base sequence, making it easy to design bacterial species-specific primers for RPA. However, the RPA method is unsuitable for designing universal primers, which require a certain degree of non-specificity. Furthermore, bacterial species identification using base sequences such as rRNA genes generally requires a length of 500 bases or more. However, installing a primer set in which both the forward and reverse primers have universal base sequences at positions 500 bases or more apart and examining whether the set actually amplifies nucleic acids correctly using the RPA method requires extensive experimental verification, which is a challenge. For this reason, developing a universal primer set for RPA is extremely difficult, and in fact, no universal primer set for RPA has been reported to date.

[0009] The present invention aims to provide a primer set for specifically isothermal nucleic acid amplification of nucleic acid derived from obligate anaerobic bacteria or lactic acid bacteria, and a method for amplifying nucleic acid derived from obligate anaerobic bacteria or lactic acid bacteria contained in a test sample using either of the primer sets.

[0010] Based on the base sequence information of the rRNA genes of many microorganisms registered in a database, the present inventors designed candidate sets of forward and reverse primers that can specifically amplify the rRNA genes of a target group of microorganisms by RPA, and experimentally confirmed whether each candidate set was actually an RPA universal primer that amplifies only the target region of the target group of microorganisms.As a result, they discovered a universal primer set for specifically amplifying nucleic acids derived from obligate anaerobic bacteria or lactic acid bacteria by RPA, thereby completing the present invention.

[0011] That is, the present invention is as follows: [1] A primer set for detecting obligately anaerobic bacteria, comprising: a forward primer containing the base sequence represented by SEQ ID NO: 1; and a reverse primer containing the base sequence represented by SEQ ID NO: 2. [2] A primer set for detecting lactic acid bacteria, comprising: a forward primer containing the base sequence represented by SEQ ID NO: 3; and a reverse primer containing the base sequence represented by SEQ ID NO: 4. [3] A primer set for detecting lactic acid bacterium, comprising: a forward primer containing the base sequence represented by SEQ ID NO: 5; and a reverse primer containing the base sequence represented by SEQ ID NO: 6. [4] A nucleic acid amplification method using a test sample and the primer set of [1] to perform a recombinase polymerase amplification reaction to amplify nucleic acid derived from the rRNA gene of obligately anaerobic bacteria contained in the test sample. [5] A nucleic acid amplification method using a test sample and the primer set of [2] to perform a recombinase polymerase amplification reaction to amplify nucleic acid derived from the rRNA gene of lactic acid bacteria contained in the test sample. [6] A nucleic acid amplification method comprising performing a recombinase polymerase amplification reaction using a test sample and the primer set described in [3] above to amplify nucleic acid derived from the rRNA gene of lactic acid bacteria contained in the test sample. [7] A base sequence identification method comprising performing a recombinase polymerase amplification reaction using a test sample and the primer set described in [1] above to amplify nucleic acid derived from the rRNA gene of an obligate anaerobic bacterium contained in the test sample, and identifying the base sequence of the amplification product obtained by the recombinase polymerase amplification reaction. [8] A base sequence identification method comprising performing a recombinase polymerase amplification reaction using a test sample and the primer set described in [2] above to amplify nucleic acid derived from the rRNA gene of lactic acid bacteria contained in the test sample, and identifying the base sequence of the amplification product obtained by the recombinase polymerase amplification reaction. [9] A method for identifying a base sequence, comprising: performing a recombinase polymerase amplification reaction using a test sample and the primer set of [3] to amplify nucleic acid derived from an rRNA gene of a lactic acid bacterium contained in the test sample; and identifying the base sequence of the amplification product obtained by the recombinase polymerase amplification reaction.

[10] The method for identifying a base sequence according to any one of [7] to [9], wherein the base sequence of the amplification product is identified using a sequencer.

[11] The method for identifying a base sequence according to

[10] , wherein the sequencer is a nanopore sequencer.

[12] A method for identifying the species of obligately anaerobic bacteria contained in a test sample, comprising: performing a recombinase polymerase amplification reaction using the test sample and the primer set according to [1] to amplify nucleic acid derived from an rRNA gene of the obligately anaerobic bacteria contained in the test sample; identifying the base sequence of the amplification product obtained by the recombinase polymerase amplification reaction; and identifying the species of the bacteria contained in the test sample based on the base sequence of the amplification product obtained.

[13] The method for identifying the species of obligately anaerobic bacteria according to

[12] , wherein the base sequence of the amplification product is identified using a nanopore sequencer.

[14] A method for identifying the species of lactic acid bacteria contained in a test sample, comprising: performing a recombinase polymerase amplification reaction using the test sample and the primer set of [2] to amplify nucleic acid derived from the rRNA gene of lactic acid bacteria contained in the test sample; identifying the base sequence of the amplification product obtained by the recombinase polymerase amplification reaction; and identifying the species of bacteria contained in the test sample based on the base sequence of the amplification product.

[15] The method for identifying the species of lactic acid bacteria of

[14] , wherein the base sequence of the amplification product is identified using a nanopore sequencer.

[16] A method for identifying a species of lactic acid bacteria contained in a test sample, comprising: performing a recombinase polymerase amplification reaction using the test sample and the primer set of [3] to amplify nucleic acid derived from an rRNA gene of lactic acid bacteria contained in the test sample; identifying the base sequence of the amplification product obtained by the recombinase polymerase amplification reaction; and identifying the species of bacteria contained in the test sample based on the base sequence of the amplification product.

[17] The method for identifying a species of lactic acid bacteria according to

[16] , wherein the base sequence of the amplification product is identified using a nanopore sequencer.

[18] A method for evaluating microbial contamination of processed foods and beverages, comprising: using a processed food or beverage or a semi-finished product collected during the manufacturing process thereof as a test sample; extracting nucleic acid from the test sample; and using the resulting nucleic acid as a template, carrying out a recombinase polymerase amplification reaction using one or more primer sets selected from the group consisting of a primer set for detecting obligately anaerobic bacteria, which comprises a forward primer comprising the base sequence represented by SEQ ID NO: 1 and a reverse primer comprising the base sequence represented by SEQ ID NO: 2; a primer set for detecting lactic acid bacteria, which comprises a forward primer comprising the base sequence represented by SEQ ID NO: 3 and a reverse primer comprising the base sequence represented by SEQ ID NO: 4; and a primer set for detecting lactic acid bacteria, which comprises a forward primer comprising the base sequence represented by SEQ ID NO: 5 and a reverse primer comprising the base sequence represented by SEQ ID NO: 6; and evaluating that semi-finished products and finished products in the same manufacturing lot as the test sample are contaminated with obligately anaerobic bacteria when an amplification product is obtained using the primer set for detecting obligately anaerobic bacteria. A method for evaluating microbial contamination, wherein, when an amplification product is obtained using the primer set for detecting lactic acid bacteria, semi-finished products and finished products from the same production lot as the test sample are evaluated to be contaminated with lactic acid bacteria, and when an amplification product is obtained using the primer set for detecting lactic acid bacteria, semi-finished products and finished products from the same production lot as the test sample are evaluated to be contaminated with lactic acid bacteria.

[19] A method for evaluating microbial contamination of processed foods and beverages, comprising: using a processed food or beverage or a semi-finished product collected during its production process as a test sample; extracting nucleic acid from the test sample; and using the resulting nucleic acid as a template, performing a recombinase polymerase amplification reaction using one or more primer sets selected from the group consisting of a primer set for detecting obligate anaerobic bacteria, which comprises a forward primer comprising the base sequence represented by SEQ ID NO: 1 and a reverse primer comprising the base sequence represented by SEQ ID NO: 2; a primer set for detecting lactic acid bacteria, which comprises a forward primer comprising the base sequence represented by SEQ ID NO: 3 and a reverse primer comprising the base sequence represented by SEQ ID NO: 4; and a primer set for detecting lactic acid bacteria, which comprises a forward primer comprising the base sequence represented by SEQ ID NO: 5 and a reverse primer comprising the base sequence represented by SEQ ID NO: 6; analyzing the base sequence of the amplification product of the recombinase polymerase amplification reaction to identify the microbial species of the amplification product; and if the microbial species of the amplification product is the causative bacterium of contamination of the processed food or beverage, evaluating semi-finished products and finished products in the same production lot as the test sample as being contaminated with the causative bacterium.

[20] The method for evaluating microbial contamination according to

[18] or

[19] above, wherein the processed food or drink is a packaged beer-taste beverage.

[0012] The primer set for detecting obligate anaerobic bacteria, the primer set for detecting lactic acid bacteria, and the primer set for detecting lactic acid cocci according to the present invention are primer sets capable of amplifying only rRNA genes derived from obligate anaerobic bacteria, lactic acid bacteria, and lactic acid cocci, respectively, by RPA. Therefore, by performing RPA using these primer sets, obligate anaerobic bacteria, lactic acid bacteria, and lactic acid cocci, respectively, can be detected based on the presence or absence of amplification products. Furthermore, the primer set for detecting obligate anaerobic bacteria, the primer set for detecting lactic acid bacteria, and the primer set for detecting lactic acid cocci according to the present invention are also useful for identifying the species of obligate anaerobic bacteria, lactic acid bacteria, and lactic acid cocci, respectively, in test samples.

[0013] The primer set for detecting obligate anaerobic bacteria according to the present invention comprises a forward primer (hereinafter sometimes referred to as a "PM forward primer") containing the base sequence represented by SEQ ID NO: 1 and a reverse primer (hereinafter sometimes referred to as a "PM reverse primer") containing the base sequence represented by SEQ ID NO: 2. The base sequences represented by SEQ ID NO: 1 and SEQ ID NO: 2 are each homologous or complementary to base sequences commonly present in the rRNA genes of bacteria belonging to obligate anaerobic bacteria, and the distance between the two in the 16S rRNA gene is approximately 500 bases. Therefore, when RPA is performed using this primer set for detecting obligate anaerobic bacteria and the genomic DNA of obligate anaerobic bacteria as a template, a 559 bp DNA fragment is obtained as an amplification product. In addition, when genomic DNA from a species other than obligate anaerobic bacteria is used as a template, no amplification product is obtained even when RPA is performed using this primer set for detecting obligate anaerobic bacteria. In other words, the primer set for detecting obligate anaerobic bacteria according to the present invention is a universal primer set for RPA for detecting obligate anaerobic bacteria.

[0014]

[0015] The obligate anaerobic bacteria detected using the primer set for detecting obligate anaerobic bacteria according to the present invention are not particularly limited, and examples of obligate anaerobic bacteria include bacteria of the genera Pectinatus and Megasphaera, which are major causes of microbial contamination in foods and beverages.

[0016] The primer set for detecting lactic acid bacteria according to the present invention comprises a forward primer (hereinafter sometimes referred to as the "La forward primer") containing the nucleotide sequence represented by SEQ ID NO: 3 and a reverse primer (hereinafter sometimes referred to as the "La reverse primer") containing the nucleotide sequence represented by SEQ ID NO: 4. The nucleotide sequences represented by SEQ ID NO: 3 and SEQ ID NO: 4 are homologous or complementary to nucleotide sequences commonly present in the rRNA genes of Lactobacillus and some Lactococcus species, which belong to the lactic acid bacteria family, and the distance between the two sequences in the 16S rRNA genes is approximately 500 bases. Therefore, when RPA is performed using this primer set for detecting lactic acid bacteria and genomic DNA of lactic acid bacteria as a template, a 503-bp DNA fragment is obtained as an amplified product. In addition, when genomic DNA from a species other than lactic acid bacteria is used as a template, no amplified product is obtained even when RPA is performed using this primer set for detecting lactic acid bacteria. In other words, the primer set for detecting lactic acid bacteria according to the present invention is a universal primer set for RPA for detecting lactic acid bacteria.

[0017]

[0018] The primer set for detecting lactic acid bacteria according to the present invention can detect Lactobacillus and some Lactococcus species. Among the Lactobacillus species that can be detected using the primer set for detecting lactic acid bacteria according to the present invention, Lactobacillus species are not particularly limited. Examples of lactic acid bacteria include bacteria of the genus Lactobacillus, Fructilactobacillus, Lacticaseibacillus, Lactiplantibacillus, Lentilactobacillus, Limosilactobacillus, Liquorilactobacillus, Loigolactobacillus, and Secundilactobacillus, which are the main causes of microbial contamination in foods and beverages. Among the lactic acid bacteria detected using the primer set for detecting lactic acid bacteria of the present invention, the lactic acid coccus is preferably a bacterium of the genus Pediococcus, which is a major cause of microbial contamination in food and beverages, such as Pediococcus claussenii and Pediococcus pentosaceus.

[0019] The primer set for detecting lactic acid bacteria according to the present invention comprises a forward primer (hereinafter sometimes referred to as a "Pe forward primer") containing the nucleotide sequence represented by SEQ ID NO: 5 and a reverse primer (hereinafter sometimes referred to as a "Pe reverse primer") containing the nucleotide sequence represented by SEQ ID NO: 6. The nucleotide sequences represented by SEQ ID NO: 5 and SEQ ID NO: 6 are homologous or complementary to nucleotide sequences commonly present in the rRNA genes of some bacteria belonging to the genus Lactococcus, and the distance between the two in the 23S rRNA gene is approximately 500 bases. Therefore, when RPA is performed using this primer set for detecting lactic acid bacteria and genomic DNA of lactic acid bacteria as a template, a 422-bp DNA fragment is obtained as an amplified product. In addition, when genomic DNA from a species other than lactic acid bacteria is used as a template, no amplified product is obtained even when RPA is performed using this primer set for detecting lactic acid bacteria. In other words, the primer set for detecting lactic acid bacteria according to the present invention is a universal primer set for RPA for detecting lactic acid bacteria.

[0020]

[0021] The lactic acid bacillus detected using the primer set for detecting lactic acid bacillus according to the present invention is preferably a bacterium of the genus Pediococcus, and examples of the genus Pediococcus include Pediococcus damnosus, Pediococcus inopinatus, and Pediococcus parvulus.

[0022] The nucleic acid amplification method using a primer set for detecting obligate anaerobic bacteria according to the present invention is a method of performing an RPA reaction using a test sample and the primer set for detecting obligate anaerobic bacteria. The RPA reaction amplifies nucleic acids derived from the rRNA genes of obligate anaerobic bacteria contained in the test sample. If the test sample contains obligate anaerobic bacteria, a target RPA amplification product of approximately 500 bases in length is obtained. However, if the test sample does not contain obligate anaerobic bacteria, no RPA amplification product is obtained. Thus, obligate anaerobic bacteria in the test sample can be detected by performing RPA using the test sample and the primer set for detecting obligate anaerobic bacteria and detecting the amplification product.

[0023] The nucleic acid amplification method using a primer set for detecting lactic acid bacteria according to the present invention involves performing an RPA reaction using a test sample and the primer set for detecting lactic acid bacteria. The RPA reaction amplifies nucleic acids derived from the rRNA genes of lactic acid bacteria contained in the test sample. If the test sample contains lactic acid bacteria, a target RPA amplification product of approximately 500 bases in length is obtained. However, if the test sample does not contain lactic acid bacteria, no RPA amplification product is obtained. Thus, lactic acid bacteria in the test sample can be detected by performing RPA using the test sample and the primer set for detecting lactic acid bacteria and detecting the amplification product.

[0024] The nucleic acid amplification method using a primer set for detecting lactic acid bacteria according to the present invention involves performing an RPA reaction using a test sample and the primer set for detecting lactic acid bacteria. The RPA reaction amplifies nucleic acids derived from the rRNA genes of lactic acid bacteria contained in the test sample. If the test sample contains lactic acid bacteria, a target RPA amplification product of approximately 500 bases in length is obtained. However, if the test sample does not contain lactic acid bacteria, no RPA amplification product is obtained. Thus, lactic acid bacteria in the test sample can be detected by performing RPA using the test sample and the primer set for detecting lactic acid bacteria and detecting the amplification product.

[0025] The RPA reaction can be carried out by conventional methods, except that the primer set used is the primer set for detecting obligate anaerobic bacteria, the primer set for detecting lactic acid bacteria, or the primer set for detecting lactic acid cocci according to the present invention. For example, the RPA reaction is carried out under isothermal conditions by unwinding double-stranded DNA of a template using a recombinase and a single-stranded DNA-binding protein, annealing the primers, and using this as a starting point to obtain an amplification product using a strand-displacing DNA polymerase. The three proteins used in the reaction can be any of the various proteins commonly used in RPA (see Non-Patent Document 2). In addition, commercially available RPA kits can also be used in the present invention.

[0026] In the present invention and this specification, "isothermal conditions" refers to temperature conditions in which the reaction temperature range (the difference between the highest and lowest reaction temperatures) from the start of the reaction to the end of the reaction is within 10°C (or 10°C or less), preferably 5°C or less. The RPA reaction using the primer set for detecting obligately anaerobic bacteria, the primer set for detecting lactic acid bacteria, or the primer set for detecting lactic acid cocci according to the present invention may be carried out in a temperature-controlled environment using a thermostatic device, or in a non-temperature-controlled environment. For example, the RPA reaction can also be carried out at ambient temperature without using a thermostatic device.

[0027] The reaction temperature of the RPA reaction is not particularly limited as long as it is isothermal, and the reaction may be carried out within a temperature range in which the enzyme used in the RPA exhibits enzymatic activity. When using a commercially available RPA kit, the reaction may be carried out within the temperature range recommended in the instruction manual attached to the kit. The RPA reaction using the primer set for detecting obligate anaerobic bacteria, the primer set for detecting lactic acid bacteria, or the primer set for detecting lactic acid cocci according to the present invention may be carried out within a temperature range of, for example, 15 to 65°C, preferably within a temperature range of 20 to 60°C, more preferably within a temperature range of 30 to 50°C, even more preferably within a temperature range of 34 to 45°C, and even more preferably within a temperature range of 37 to 42°C.

[0028] The nucleic acid amplification products contained in the reaction products after the RPA reaction can be detected by various methods known for detecting nucleic acid amplification products obtained in various nucleic acid amplification reactions such as PCR, etc. Examples of such detection methods include gel electrophoresis, size exclusion chromatography, and fluorescence detection using an intercalator.

[0029] Furthermore, the base sequence of the nucleic acid amplification product obtained by the RPA reaction can also be identified. The base sequence of the nucleic acid amplification product can be identified by nucleic acid analysis using various DNA sequencers. The DNA sequencer is not particularly limited, and may be an electrophoretic sequencer or capillary sequencer using the Sanger method, a next-generation sequencer (NGS) that fragments the template DNA into approximately 300 bases in advance and then identifies the base sequence in parallel, or a third-generation sequencer such as a nanopore sequencer. The identification of the base sequence using a nanopore sequencer can be performed, for example, with reference to the method described in International Publication No. WO 2020 / 255587.

[0030] After identifying the base sequence of the nucleic acid amplification product obtained by an RPA reaction using the primer set for detecting obligate anaerobic bacteria according to the present invention, the species of obligate anaerobic bacteria contained in the test sample used as a template can be identified based on the base sequence of the obtained amplification product. After identifying the base sequence of the nucleic acid amplification product obtained by an RPA reaction using the primer set for detecting lactic acid bacteria according to the present invention, the species of lactic acid bacteria contained in the test sample used as a template can be identified based on the base sequence of the obtained amplification product. After identifying the base sequence of the nucleic acid amplification product obtained by an RPA reaction using the primer set for detecting lactic acid bacteria according to the present invention, the species of lactic acid bacteria contained in the test sample used as a template can be identified based on the base sequence of the obtained amplification product. Bacterial species identification can be performed, for example, by performing BLAST (Basic Local Alignment Search Tool) analysis on the identified base sequence.

[0031] Lactobacillus can be detected by RPA using the primer set for detecting lactic acid bacteria according to the present invention and identifying the base sequence of the resulting nucleic acid amplification product. Lactococcus can be detected by RPA using either the primer set for detecting lactic acid bacteria according to the present invention or the primer set for detecting lactic acid bacteria according to the present invention and identifying the base sequence of the resulting nucleic acid amplification product. Therefore, by performing detection on a test sample using the primer set for detecting lactic acid bacteria according to the present invention and detection using the primer set for detecting lactic acid bacteria according to the present invention, it is possible to detect all lactobacillus and lactic acid bacteria in the test sample.

[0032] In identifying the species of lactic acid bacteria in a test sample, if the lactic acid bacteria in the test sample are cultured in a medium for culturing lactic acid bacteria and the lactic acid bacteria that grow are bacilli, the species of the lactic acid bacteria can be identified by performing detection using only the primer set for detecting lactic acid bacteria according to the present invention. On the other hand, if the lactic acid bacteria that grow are cocci, the species of the lactic acid bacteria can be identified by using both the primer set for detecting lactic acid bacteria according to the present invention and the primer set for detecting lactic acid bacteria according to the present invention. Whether the lactic acid bacteria cultured in a medium for culturing lactic acid bacteria are bacilli or cocci can be determined by observing the shape of the bacteria under a microscope.

[0033] The test sample used in the present invention is not particularly limited as long as it is a sample that may contain nucleic acids derived from microorganisms. Examples of the test sample include foods and beverages and semi-finished products in the course of their production, pharmaceuticals and semi-finished products in the course of their production, and biological samples collected from animals, etc. Nucleic acid samples extracted and purified from these samples may also be used. Nucleic acid extraction and purification can be performed by conventional methods.

[0034] Because RPA is an isothermal reaction, a relatively simple incubator is sufficient, an expensive thermal cycler is not required, and amplification products can be obtained in a very short time of about 20 minutes. Therefore, the nucleic acid amplification method using the primer set for detecting obligate anaerobic bacteria, the primer set for detecting lactic acid bacteria, or the primer set for detecting lactic acid cocci according to the present invention is particularly suitable for use in evaluating microbial contamination in processed foods and beverages, where early detection of microbial contamination is highly desirable.

[0035] In assessing microbial contamination of processed foods and beverages, the processed foods and beverages or semi-finished products collected during their manufacturing process are used as test samples. For example, microorganisms that cause microbial contamination problems in processed foods and beverages are used as contaminants, and if the test sample contains contaminants, the semi-finished products and finished products from the same manufacturing lot as the test sample are evaluated as being contaminated with the contaminants. On the other hand, if the test sample does not contain contaminants, the semi-finished products and finished products from the same manufacturing lot as the test sample are evaluated as not being contaminated with the contaminants. A semi-finished product refers to an incomplete product that is in the middle of manufacturing, and is not particularly limited as long as it is an item in the middle of manufacturing.

[0036] Specifically, nucleic acids are first extracted from the test sample. The resulting nucleic acids are used as a template to perform an RPA reaction using any one of the primer sets according to the present invention, namely, the primer set for detecting obligate anaerobic bacteria, the primer set for detecting lactic acid bacteria, and the primer set for detecting lactic acid cocci. The presence or absence of microbial contamination in the test sample is evaluated based on whether an amplification product is obtained by the RPA reaction.

[0037] When an amplification product is obtained using a primer set for detecting obligate anaerobic bacteria, semi-finished products and finished products from the same production lot as the test sample are evaluated as being contaminated with obligate anaerobic bacteria.When an amplification product is obtained using a primer set for detecting lactic acid bacteria, semi-finished products and finished products from the same production lot as the test sample are evaluated as being contaminated with lactic acid bacteria.When an amplification product is obtained using a primer set for detecting lactic acid bacteria, semi-finished products and finished products from the same production lot as the test sample are evaluated as being contaminated with lactic acid bacteria.

[0038] The species of contaminating bacteria can also be identified by identifying the base sequence of the resulting amplification product. A nanopore sequencer is preferably used to identify the base sequence, and a portable sequencer such as the "MinION" (manufactured by Oxford Nanopore Technologies) is particularly preferred. For example, the base sequence of the amplification product of the RPA reaction is analyzed to identify the microbial species of the amplification product (the microbial species from which the DNA used as the template for RPA is derived). If the identified microbial species is the causative bacterium of the processed food or beverage being tested, then semi-finished products and finished products from the same production lot as the test sample used for analysis are assessed as being contaminated with the causative bacterium.

[0039] Examples of bacteria that may cause contamination in beer-taste beverages include bacteria of the genus Lactobacillus, such as Levilactobacillus brevis, Fructilactobacillus lindneri, Secundilactobacillus paracollinoides, Lacticaseibacillus paracasei, and Lactiplanibacillus plantarum, bacteria of the genus Pediococcus, such as Pediococcus claussenii and Pediococcus damnosus, bacteria of the genus Pectinatus, such as Pectinatus frisingensis and Pectinatus cerevisiiphilus, and bacteria of the genus Megasphaera, such as Megasphaera cerevisiae. Therefore, the primer set for detecting obligately anaerobic bacteria, the primer set for detecting lactic acid bacteria, and the primer set for detecting lactococci according to the present invention are particularly useful for evaluating microbial contamination in packaged beer-taste beverages.

[0040] The term "beer-flavored beverage" refers to a beverage that has a beer-like flavor. Furthermore, "beer-like" refers to a flavor or taste that evokes beer, regardless of the product name or labeling. In other words, a beer-flavored beverage refers to a beverage that has a flavor, taste, and texture equivalent to or similar to that of beer, and that has high thirst-quenching properties and drinkability, regardless of the alcohol content, whether or not malt and hops are used, whether or not fermented, and whether or not it is sparkling.

[0041] The container into which the beer-taste beverage is filled is not particularly limited. Specific examples include glass bottles, cans, barrels, and flexible containers. Flexible containers include containers made by molding flexible resins such as PE (polyethylene), PP (polypropylene), EVOH (ethylene-vinyl alcohol copolymer), and PET (polyethylene terephthalate). Flexible containers may be made of a single-layer resin or a multi-layer resin.

[0042] The present invention will now be described in more detail with reference to examples, but the present invention is not limited to the following examples.

[0043] Example 1 Using the primer set for detecting obligate anaerobic bacteria (a set of PM forward primer and PM reverse primer), the primer set for detecting lactic acid bacteria (a set of La forward primer and La reverse primer), and the primer set for detecting lactic acid bacteria (a set of Pe forward primer and Pe reverse primer) according to the present invention, RPA was performed using the genomic DNA of various bacteria as templates to examine whether amplification products could be obtained.

[0044] (1) Bacterial Culture For lactic acid bacteria, each isolated strain was inoculated onto MRS (de Man, Rogosa, Sharpe) agar medium and cultured at 25-28°C to form colonies. For obligate anaerobic bacteria, each isolated strain was inoculated onto TGC (thioglycollate) agar medium and cultured at 25-28°C to form colonies. For bacteria other than obligate anaerobic bacteria and lactic acid bacteria, each isolated strain was inoculated onto standard agar medium and cultured at 30-37°C to form colonies.

[0045] (2) DNA Extraction 25 μL of "Prepman Ultra reagent" (Thermo Fisher Scientific) was poured into a 200 μL tube, and a small amount of bacterial cells was collected from one colony of each isolated strain using a sterilized tip to prepare a suspension of isolated and cultured microbial cells. The 200 μL tube was placed in a thermal cycler and reacted using a program set to 98°C for 10 minutes. After the reaction was completed, the tube was centrifuged for 3 minutes using a tabletop centrifuge. Next, 20 μL of the resulting supernatant was collected in a new tube, and 80 μL of sterile water was added and mixed. The resulting DNA solution was used as a template DNA solution for each bacterium.

[0046] (3) RPA Reaction The RPA reaction was performed using a commercially available RPA kit, "TwistAmp® Basic DNA Amplification Kit" (manufactured by TwistDx), according to the manufacturer's recommended protocol. Specifically, 2.4 μL of forward primer solution (10 μM), 2.4 μL of reverse primer solution (10 μM), 29.5 μL of the "Primer-free rehydration buffer" buffer provided with the RPA kit, and 8.2 μL of water were mixed to prepare 42.5 μL of primer solution. This primer solution was added to the "TwistAmp Basic reaction pellets tube" provided with the RPA kit and thoroughly suspended by pipetting. 5 μL of template DNA solution was then added and further mixed thoroughly by pipetting. Next, 2.5 μL of MgOAc provided with the RPA kit was added to the reaction solution in the tube and mixed. The tube was then immediately placed in a 39°C heat block and incubated for 4 minutes. After 4 minutes, the tube was vortexed to mix well, then spun down and returned to the heat block, where it was incubated for another 16 minutes at 39° C. The tube was then removed from the heat block and promptly stored on ice.

[0047] (4) Purification of RPA product The tube containing the reaction solution after the RPA reaction was placed in a 65°C heat block and incubated for 10 minutes. The tube was then cooled on ice for 1 minute and centrifuged (10,000 rpm, 3 minutes). The collected supernatant was used as a DNA sample.

[0048] (5) Detection of RPA products. The obtained DNA samples were subjected to 2% agarose gel electrophoresis to detect RPA product bands. The size of the detected DNA bands was estimated using a DNA marker (100 bp DNA ladder) electrophoresed at the same time.

[0049]

[0050] The base length of the RPA reaction product obtained using each primer set is shown in Table 4. Tables 5 to 8 show whether a band of the desired size was detected in the RPA product obtained using the genomic DNA of each bacterium as a template. In the tables, "Yes" means that a band was detected, "No" means that a band was not detected, and "NA" means that measurement was not performed.

[0051]

[0052]

[0053]

[0054]

[0055] As shown in Tables 5 to 8, when the primer set for detecting obligate anaerobic bacteria was used, bands were detected only when DNA derived from obligate anaerobic bacteria was used as a template, confirming that RPA using this primer set can specifically detect obligate anaerobic bacteria, distinguishing them from other bacteria, including lactic acid bacteria. Furthermore, when the primer set for detecting lactic acid bacteria was used, bands were detected only when DNA derived from lactic acid bacteria, including Lactobacillus spp., was used as a template, confirming that RPA using this primer set can specifically detect lactic acid bacteria, distinguishing them from other bacteria, including obligate anaerobic bacteria. When the primer set for detecting lactococci was used, bands were detected only when DNA derived from lactic acid bacteria, including Pediococcus spp., was used as a template, confirming that RPA using this primer set can specifically detect lactic acid bacteria, distinguishing them from other bacteria, including lactobacillus.

[0056] (6) Base sequence analysis The RPA products obtained using the obligate anaerobic bacteria detection primer set with the DNA of the obligate anaerobic bacteria listed in Table 5 as a template, the RPA products obtained using the lactic acid bacteria detection primer set with the DNA of lactobacillus listed in Table 6 as a template, and the RPA products obtained using the lactic acid bacteria detection primer set or the lactic acid bacteria detection primer set with the DNA of lactococcus listed in Table 7 as a template were analyzed using a nanopore sequencer "MinION" (manufactured by Oxford Nanopore Technologies). Analysis using "MinION" was performed using "Rapid Barcoding (SQK-kit series)" (manufactured by Oxford Nanopore Technologies) in accordance with the method described in WO 2020 / 255587.

[0057] The prepared DNA library was loaded into the flow cell of the MinION nanopore sequencer, and the base sequence of the DNA was determined based on the change in current that occurred when a single DNA molecule passed through the nanopore, resulting in base sequence output data (FASTQ). The determined base sequence of each DNA molecule is called a "read."

[0058] The base sequence output data was analyzed using analysis software such as "Geneious." Specifically, the obtained base sequence output data was filtered to select reads of 300 to 550 bp, and then de novo assembly was performed to identify the base sequence of each read. The obtained base sequence data was analyzed using the Basic Local Alignment Search Tool (BLAST) to identify the microbial species of each read.

[0059] As a result, it was possible to perform base sequence analysis on all of the RPA products. Furthermore, for all of the RPA products whose base sequences were analyzed, the bacterial species used as the template matched the microbial species identified by the base sequence analysis, confirming that the desired RPA products were obtained.

Claims

1. A primer set for detecting obligate anaerobic bacteria, comprising a forward primer containing the nucleotide sequence represented by SEQ ID NO: 1 and a reverse primer containing the nucleotide sequence represented by SEQ ID NO:

2.

2. A primer set for detecting lactic acid bacteria, comprising a forward primer containing the nucleotide sequence represented by SEQ ID NO: 3 and a reverse primer containing the nucleotide sequence represented by SEQ ID NO:

4.

3. A primer set for detecting Lactococcus lactis, comprising a forward primer containing the nucleotide sequence represented by SEQ ID NO: 5 and a reverse primer containing the nucleotide sequence represented by SEQ ID NO:

6.

4. A nucleic acid amplification method, comprising performing a recombinase polymerase amplification reaction using a test sample and the primer set according to claim 1 to amplify a nucleic acid derived from the rRNA gene of obligate anaerobic bacteria contained in the test sample.

5. A nucleic acid amplification method, comprising performing a recombinase polymerase amplification reaction using a test sample and the primer set according to claim 2 to amplify a nucleic acid derived from the rRNA gene of lactic acid bacteria contained in the test sample.

6. A nucleic acid amplification method, comprising performing a recombinase polymerase amplification reaction using a test sample and the primer set according to claim 3 to amplify a nucleic acid derived from the rRNA gene of Lactococcus lactis contained in the test sample.

7. A method for identifying a nucleotide sequence, comprising performing a recombinase polymerase amplification reaction using a test sample and the primer set according to claim 1 to amplify a nucleic acid derived from the rRNA gene of obligate anaerobic bacteria contained in the test sample, and identifying the nucleotide sequence of the amplification product obtained by the recombinase polymerase amplification reaction.

8. A method for identifying a nucleotide sequence, comprising performing a recombinase polymerase amplification reaction using a test sample and the primer set according to claim 2 to amplify a nucleic acid derived from the rRNA gene of lactic acid bacteria contained in the test sample, and identifying the nucleotide sequence of the amplification product obtained by the recombinase polymerase amplification reaction.

9. A method for identifying a nucleotide sequence, comprising performing a recombinase polymerase amplification reaction using a test sample and the primer set according to claim 3 to amplify a nucleic acid derived from the rRNA gene of Lactococcus lactis contained in the test sample, and identifying the nucleotide sequence of the amplification product obtained by the recombinase polymerase amplification reaction.

10. The method for identifying the nucleotide sequence according to any one of claims 7 to 9, wherein the nucleotide sequence of the amplification product is identified using a sequencer.

11. The method for identifying the nucleotide sequence according to claim 10, wherein the sequencer is a nanopore sequencer.

12. A method for identifying the bacterial species of obligate anaerobic bacteria contained in a test sample, comprising: performing a recombinase polymerase amplification reaction using the test sample and the primer set according to claim 1 to amplify the nucleic acid derived from the rRNA gene of the obligate anaerobic bacteria contained in the test sample; identifying the nucleotide sequence of the amplification product obtained by the recombinase polymerase amplification reaction; and identifying the bacterial species of the bacteria contained in the test sample based on the nucleotide sequence of the obtained amplification product. The method for identifying the bacterial species of obligate anaerobic bacteria.

13. The method for identifying the bacterial species of obligate anaerobic bacteria according to claim 12, wherein the nucleotide sequence of the amplification product is identified using a nanopore sequencer.

14. A method for identifying the bacterial species of lactic acid bacteria contained in a test sample, comprising: performing a recombinase polymerase amplification reaction using the test sample and the primer set according to claim 2 to amplify the nucleic acid derived from the rRNA gene of the lactic acid bacteria contained in the test sample; identifying the nucleotide sequence of the amplification product obtained by the recombinase polymerase amplification reaction; and identifying the bacterial species of the bacteria contained in the test sample based on the nucleotide sequence of the obtained amplification product. The method for identifying the bacterial species of lactic acid bacteria.

15. The method for identifying the bacterial species of lactic acid bacteria according to claim 14, wherein the nucleotide sequence of the amplification product is identified using a nanopore sequencer.

16. A method for identifying the bacterial species of Lactococcus contained in a test sample, comprising: performing a recombinase polymerase amplification reaction using the test sample and the primer set according to claim 3 to amplify the nucleic acid derived from the rRNA gene of the Lactococcus contained in the test sample; identifying the nucleotide sequence of the amplification product obtained by the recombinase polymerase amplification reaction; and identifying the bacterial species of the bacteria contained in the test sample based on the nucleotide sequence of the obtained amplification product. The method for identifying the bacterial species of Lactococcus.

17. The method for identifying the bacterial species of Lactococcus according to claim 16, wherein the nucleotide sequence of the amplification product is identified using a nanopore sequencer.

18. A method for evaluating microbial contamination of processed foods and beverages, comprising: using a semi-finished product collected during the production process of the processed food or beverage as a test sample; extracting nucleic acid from the test sample; using the obtained nucleic acid as a template, and performing a recombinase polymerase amplification reaction using at least one primer set selected from the group consisting of a primer set for detecting obligate anaerobic bacteria, which consists of a forward primer containing the nucleotide sequence represented by SEQ ID NO: 1 and a reverse primer containing the nucleotide sequence represented by SEQ ID NO: 2; a primer set for detecting lactic acid bacteria, which consists of a forward primer containing the nucleotide sequence represented by SEQ ID NO: 3 and a reverse primer containing the nucleotide sequence represented by SEQ ID NO: 4; and a primer set for detecting Lactococcus lactis, which consists of a forward primer containing the nucleotide sequence represented by SEQ ID NO: 5 and a reverse primer containing the nucleotide sequence represented by SEQ ID NO: 6; when an amplification product is obtained using the primer set for detecting obligate anaerobic bacteria, evaluating that the semi-finished products and finished products of the same production lot as the test sample are contaminated with obligate anaerobic bacteria; when an amplification product is obtained using the primer set for detecting lactic acid bacteria, evaluating that the semi-finished products and finished products of the same production lot as the test sample are contaminated with lactic acid bacteria; and when an amplification product is obtained using the primer set for detecting Lactococcus lactis, evaluating that the semi-finished products and finished products of the same production lot as the test sample are contaminated with Lactococcus lactis. A method for evaluating microbial contamination.

19. A method for evaluating microbial contamination of processed food and drink, comprising: using a semi-finished product collected from the processed food and drink or during its manufacturing process as a test sample; extracting nucleic acid from the test sample; using the obtained nucleic acid as a template, and performing a recombinase polymerase amplification reaction using one or more primer sets selected from the group consisting of a primer set for detecting obligate anaerobic bacteria comprising a forward primer containing the nucleotide sequence represented by SEQ ID NO: 1 and a reverse primer containing the nucleotide sequence represented by SEQ ID NO: 2, a primer set for detecting lactic acid bacteria comprising a forward primer containing the nucleotide sequence represented by SEQ ID NO: 3 and a reverse primer containing the nucleotide sequence represented by SEQ ID NO: 4, and a primer set for detecting Lactococcus lactis comprising a forward primer containing the nucleotide sequence represented by SEQ ID NO: 5 and a reverse primer containing the nucleotide sequence represented by SEQ ID NO: 6; analyzing the nucleotide sequence of the amplification product of the recombinase polymerase amplification reaction to identify the microbial species of the amplification product; and when the microbial species of the amplification product is the causative bacterium of the contamination of the processed food and drink, evaluating that the semi-finished products and finished products of the same production lot as the test sample are contaminated by the causative bacterium. A method for evaluating microbial contamination.

20. The method for evaluating microbial contamination according to claim 18 or 19, wherein the processed food and drink is a canned beer-flavored beverage.

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