Universal primer set and nucleic acid amplification method

Abstract

The present invention provides a universal primer set for the isothermal nucleic acid amplification of nucleic acids from bacteria or fungi. The present invention is: a universal primer set for detecting bacteria, said universal primer set comprising a forward primer including a region of at least 30 consecutive bases in the base sequence represented by SEQ ID NO: 1 and a reverse primer including a region of at least 30 consecutive bases in the base sequence represented by SEQ ID NO: 2; and a universal primer set for detecting fungi, said universal primer set comprising a forward primer including a region of at least 30 consecutive bases in the base sequence represented by SEQ ID NO: 3 and a reverse primer including a region of at least 30 consecutive bases in the base sequence represented by SEQ ID NO: 4.

Description

Universal primer set and nucleic acid amplification method

[0001] The present invention relates to a universal primer set for nucleic acid amplification of bacterial genes, a universal primer set for nucleic acid amplification of fungal genes, and a method for amplifying nucleic acids derived from bacteria or fungi contained in a test sample using these primer sets.This application claims priority based on Japanese Patent Application No. 2023-219659, 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:96.Li 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] An object of the present invention is to provide a universal primer set for isothermal nucleic acid amplification of nucleic acid derived from bacteria or fungi, and a method for amplifying nucleic acid derived from bacteria or fungi contained in a test sample using any 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 a target group of microorganisms.As a result, they discovered a universal primer set for specifically amplifying nucleic acids derived from bacteria or fungi by RPA, and completed the present invention.

[0011] That is, the present invention is as follows. [1] A universal primer set for detecting bacteria, comprising: a forward primer including a region of 30 or more contiguous bases in the base sequence represented by SEQ ID NO: 1; and a reverse primer including a region of 30 or more contiguous bases in the base sequence represented by SEQ ID NO: 2. [2] A universal primer set for detecting fungi, comprising: a forward primer including a region of 30 or more contiguous bases in the base sequence represented by SEQ ID NO: 3; and a reverse primer including a region of 30 or more contiguous bases in the base sequence represented by SEQ ID NO: 4. [3] A nucleic acid amplification method, comprising: using a test sample and the primer set of [1] to perform a recombinase polymerase amplification reaction, thereby amplifying nucleic acid derived from a bacterial 16S rRNA gene contained in the test sample. [4] A nucleic acid amplification method, comprising: using a test sample and the primer set of [2] to perform a recombinase polymerase amplification reaction, thereby amplifying nucleic acid derived from a fungal ITS gene contained in the test sample. [5] A method for identifying a base sequence, comprising: performing a recombinase polymerase amplification reaction using a test sample and the primer set of [1] to amplify nucleic acid derived from a bacterial 16S rRNA gene contained in the test sample; and identifying the base sequence of the amplification product obtained by the recombinase polymerase amplification reaction. [6] A method for identifying a base sequence, comprising performing a recombinase polymerase amplification reaction using a test sample and the primer set of [2] to amplify nucleic acid derived from a fungal ITS gene contained in the test sample; and identifying the base sequence of the amplification product obtained by the recombinase polymerase amplification reaction. [7] The method for identifying a base sequence of [5] or [6], wherein the identification of the base sequence of the amplification product is performed using a sequencer. [8] The method for identifying a base sequence of [7], wherein the sequencer is a nanopore sequencer.[9] A method for identifying a bacterial species contained in a test sample, comprising: performing a recombinase polymerase amplification reaction using the test sample and the primer set of [1] to amplify nucleic acid derived from a 16S rRNA gene of the bacteria contained in the test sample; identifying the base sequence of the amplification product obtained by the recombinase polymerase amplification reaction; and identifying the bacterial species contained in the test sample based on the base sequence of the amplification product.

[10] The method for identifying a bacterial species of [9], wherein the base sequence of the amplification product is identified using a nanopore sequencer.

[11] A method for identifying a fungal species contained in a test sample, comprising: performing a recombinase polymerase amplification reaction using the test sample and the primer set of [2] above to amplify nucleic acid derived from an ITS gene of the fungus contained in the test sample; identifying the nucleotide sequence of the amplification product obtained by the recombinase polymerase amplification reaction; and identifying the fungal species contained in the test sample based on the nucleotide sequence of the amplification product obtained.

[12] The method for identifying a fungal species of

[11] above, wherein the nucleotide sequence of the amplification product is identified using a nanopore sequencer.

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

[0013] 1 shows nucleic acid stained images of gels obtained by agarose gel electrophoresis of RPA products obtained using bacterial genomic DNA as a template in Example 3. FIG. 2 shows nucleic acid stained images of gels obtained by agarose gel electrophoresis of RPA products obtained using fungal genomic DNA as a template in Example 4.

[0014] The universal primer set for bacteria detection according to the present invention comprises a forward primer (hereinafter sometimes referred to as a "Pro forward primer") comprising a region of 30 or more consecutive bases in the base sequence represented by SEQ ID NO: 1, and a reverse primer (hereinafter sometimes referred to as a "Pro reverse primer") comprising a region of 30 or more consecutive bases in the base sequence represented by SEQ ID NO: 2. The base sequences represented by SEQ ID NO: 1 and SEQ ID NO: 2 are homologous or complementary to base sequences commonly present in the rRNA genes of various bacteria, and the distance between the two in the 16S rRNA gene is approximately 500 bases. Therefore, when RPA is performed using this universal primer set for bacteria detection and bacterial genomic DNA as a template, a DNA fragment of approximately 500 bp is obtained as an amplified product. In addition, when genomic DNA from a species other than bacteria is used as a template, no amplified product is obtained even when RPA is performed using this universal primer set for bacteria detection. In other words, the universal primer set for bacteria detection according to the present invention is a universal primer set for RPA for detecting microorganisms broadly classified as bacteria.

[0015]

[0016] The RPA primer may be at least 30 bases in length. For example, the base sequence of the Pro forward primer used in the present invention may be a primer having the same base sequence as the region of bases 1 to 30 of the base sequence of SEQ ID NO: 1, a primer having the same base sequence as the region of bases 1 to 35, a primer having the same base sequence as the region of bases 1 to 40, a primer having the same base sequence as the region of bases 1 to 45 (the entire length of the base sequence of SEQ ID NO: 1), a primer having the same base sequence as the region of bases 16 to 45, or a primer having the same base sequence as the region of bases 10 to 45. Similarly, the nucleotide sequence of the Pro reverse primer used in the present invention may be a primer consisting of the same nucleotide sequence as the region of positions 1 to 30 of the nucleotide sequence of SEQ ID NO: 2, a primer consisting of the same nucleotide sequence as the region of positions 1 to 35, a primer consisting of the same nucleotide sequence as the region of positions 1 to 40, a primer consisting of the same nucleotide sequence as the region of positions 1 to 45, a primer consisting of the same nucleotide sequence as the region of positions 28 to 72 (the entire length of the nucleotide sequence of SEQ ID NO: 2), a primer consisting of the same nucleotide sequence as the region of positions 33 to 72, a primer consisting of the same nucleotide sequence as the region of positions 43 to 72, a primer consisting of the same nucleotide sequence as the region of positions 16 to 45, a primer consisting of the same nucleotide sequence as the region of positions 21 to 50, or a primer consisting of the same nucleotide sequence as the region of positions 11 to 55. The base lengths of the regions recognizing template DNA in the Pro forward primer and Pro reverse primer constituting the universal primer set for bacteria detection may be the same or different.

[0017] Examples of Pro forward primers include 337f_B1_1 primer, 337f_B1_2 primer, and 337f_B1_3 primer. Examples of Pro reverse primers include 805r_B1_1 primer, 805r_B1_2 primer, and 805r_B1_3 primer. These Pro forward primers and Pro reverse primers can be used in any combination.

[0018]

[0019] Bacteria to be detected using the universal primer set for bacteria detection according to the present invention are not particularly limited, and may be, for example, normal bacteria or bacteria that are widely distributed in the natural environment. Specifically, Acetobacter, Bacillus, Enterobacter, Escherichia, Fructilactobacillus, Klebsiella, Lacticaseibacillus, Lactiplantibacillus, Lactobacillus, Lactococcus, Lecrercia, Lentilactobacillus, Leuconostoc, Levilactobacillus, and Limosilactobacillus. , Liquorilactobacillus spp., Loigolactobacillus spp., Megasphaera spp., Micrococcus spp., Novosphingobium spp., Paenibacillus spp., Pantoea spp., Pectinatus spp. atus, Pediococcus, Pseudomonas, Schleiferilactobacillus, Secundilactobacillus, Staphylococcus, Serratia, Zymomonas, and the like.

[0020] The universal primer set for detecting fungi according to the present invention comprises a forward primer (hereinafter sometimes referred to as the "Eu forward primer") comprising a region of 30 or more consecutive bases in the base sequence represented by SEQ ID NO: 3, and a reverse primer (hereinafter sometimes referred to as the "Eu reverse primer") comprising a region of 30 or more consecutive bases in the base sequence represented by SEQ ID NO: 4. The base sequences represented by SEQ ID NO: 3 and SEQ ID NO: 4 are each homologous or complementary to base sequences commonly present in the rRNA genes of various fungi, such as yeast and mold, and the distance between the two in the ITS gene is approximately 500 bases. Therefore, when RPA is performed using this universal primer set for detecting fungi and fungal genomic DNA as a template, a DNA fragment of approximately 500 bp is obtained as an amplified product. In addition, when genomic DNA from a species other than fungi is used as a template, no amplified product is obtained even when RPA is performed using this universal primer set for detecting fungi. In other words, the universal primer set for detecting fungi according to the present invention is a universal primer set for RPA for detecting microorganisms broadly classified as fungi.

[0021]

[0022] The base sequence of the Eu forward primer used in the present invention may be a primer consisting of the same base sequence as the region of positions 1 to 30 of the base sequence represented by SEQ ID NO: 3, a primer consisting of the same base sequence as the region of positions 1 to 35, a primer consisting of the same base sequence as the region of positions 1 to 40, a primer consisting of the same base sequence as the region of positions 1 to 45, a primer consisting of the same base sequence as the region of positions 15 to 59, a primer consisting of the same base sequence as the region of positions 20 to 59, a primer consisting of the same base sequence as the region of positions 25 to 59, a primer consisting of the same base sequence as the region of positions 30 to 59, a primer consisting of the same base sequence as the region of positions 16 to 45, or a primer consisting of the same base sequence as the region of positions 10 to 44. Similarly, the base sequence of the Eu reverse primer used in the present invention may be a primer consisting of the same base sequence as the region from bases 1 to 30 of the base sequence represented by SEQ ID NO: 4, a primer consisting of the same base sequence as the region from bases 1 to 40, a primer consisting of the same base sequence as the region from bases 1 to 46, a primer consisting of the same base sequence as the region from bases 7 to 46, a primer consisting of the same base sequence as the region from bases 17 to 46, or a primer consisting of the same base sequence as the region from bases 7 to 40. The base lengths of the regions recognizing the template DNA of the Eu forward primer and Eu reverse primer constituting the universal primer set for fungal detection may be the same or different.

[0023] Examples of Eu forward primers include 6F_ITS(5.8s)_Fwd1 primer and 6F_ITS(5.8s)_Fwd2 primer. Examples of Eu reverse primers include 53R_ITS_Rev1 primer and 53R_ITS_Rev2 primer. These Eu forward primers and Eu reverse primers can be used in any combination.

[0024]

[0025] The fungi detected using the universal primer set for fungal detection according to the present invention are not particularly limited, and may be, for example, resident fungi or fungi that are widely distributed in the natural environment, and preferably yeasts used in fermented foods, such as beer yeast, sake yeast, wine yeast, and baker's yeast.

[0026] The nucleic acid amplification method using a universal primer set for bacteria detection according to the present invention involves performing an RPA reaction using a test sample and the universal primer set for bacteria detection. The RPA reaction amplifies nucleic acids derived from the 16sRNA gene of bacteria contained in the test sample. If the test sample contains bacteria, a target RPA amplification product of approximately 500 bases in length is obtained regardless of the specific biological species of the bacteria. However, if the test sample does not contain bacteria, no RPA amplification product is obtained. In this way, bacteria in the test sample can be detected by performing RPA using the test sample and the universal primer set for bacteria detection and detecting the amplification product.

[0027] The nucleic acid amplification method using a universal primer set for fungal detection according to the present invention involves performing an RPA reaction using a test sample and the universal primer set for fungal detection. The RPA reaction amplifies nucleic acid derived from the ITS gene of a fungus contained in the test sample. If the test sample contains a fungus, a target RPA amplification product of approximately 500 bases in length is obtained regardless of the specific species of the fungus. However, if the test sample does not contain a fungus, no RPA amplification product is obtained. In this way, fungi in the test sample can be detected by performing RPA using the test sample and the universal primer set for fungal detection and detecting the amplification product.

[0028] The RPA reaction can be carried out by conventional methods, except that the primer set used is the universal primer set for bacterial detection or the universal primer set for fungal detection according to the present invention. For example, the RPA reaction is carried out under isothermal conditions by unwinding the double-stranded DNA template DNA 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, a commercially available RPA kit can also be used in the present invention.

[0029] 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 universal primer set for detecting bacteria or the universal primer set for detecting fungi 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.

[0030] 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 universal primer set for detecting bacteria or the universal primer set for detecting fungi according to the present invention may be carried out within a temperature range of, for example, 15 to 65°C, preferably 20 to 60°C, more preferably 20 to 50°C, even more preferably 25 to 45°C, still more preferably 25 to 42°C, and particularly preferably 30 to 42°C.

[0031] 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.

[0032] 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 electrophoresis 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. In particular, it is more preferable to use a portable sequencer such as "MinION" (manufactured by Oxford Nanopore Technologies). 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.

[0033] After identifying the base sequence of the nucleic acid amplification product obtained by an RPA reaction using the universal primer set for detecting bacteria according to the present invention, the bacterial species contained in the test sample used as a template can be identified based on the base sequence of the obtained amplification product. Similarly, after identifying the base sequence of the nucleic acid amplification product obtained by an RPA reaction using the universal primer set for detecting fungi according to the present invention, the fungal species 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.

[0034] 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.

[0035] 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 universal primer set for detecting bacteria or the universal primer set for detecting fungi 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.

[0036] 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.

[0037] [Example 1] Using the universal primer set for bacteria detection according to the present invention, RPA was performed using the genomic DNA of 42 types of bacteria as a template to investigate whether amplification products could be obtained. The universal primer set for bacteria detection used was a set consisting of primers 337f_B1_1 and 805r_B1_1.

[0038] (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.

[0039] (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.

[0040] (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.

[0041] (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.

[0042] (5) MinION sequencing The base sequence of the RPA product in the DNA sample was analyzed using a nanopore sequencer "MinION" (Oxford Nanopore Technologies). Analysis using "MinION" was performed according to the method described in WO 2020 / 255587.

[0043] 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."

[0044] 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 base sequence output data of the RPA product obtained using the universal primer set for bacterial detection was filtered to remove contaminating sequencing reads, and then further filtered to select reads of 300 to 550 bp. The obtained base sequence data was analyzed using the Basic Local Alignment Search Tool (BLAST) to identify the microbial species of each read.

[0045] Tables 5 and 6 show the average percent homology of each read to the microbial species identified by sequence analysis for the RPA products obtained using the universal primer set for bacterial detection. In the "Microbial species identified by sequence analysis" column in the tables, "N / A" means that sequence analysis was not possible. In addition, in Tables 5 and 6, bacterial species for which two microbial species were identified as a result of sequence analysis were closely related and therefore could not be distinguished by sequence analysis.

[0046]

[0047]

[0048] As shown in Tables 5 and 6, when genomic DNA from 42 types of bacteria was used as a template, base sequence analysis was possible for 37 types of bacteria, and the template bacteria or their closely related species were identified in almost all cases. Furthermore, when RPA products from five types of bacteria for which base sequence analysis was not possible were subjected to 2% agarose gel electrophoresis, DNA bands of the desired size were detected, confirming that amplification products could be obtained using the universal primer set for bacterial detection even when DNA from these five types of bacteria was used as a template. These results demonstrated that the universal primer set for bacterial detection used can amplify target regions in the genomic DNA of various bacteria. The RPA enzyme used in this experiment was isolated and extracted from E. coli, and E. coli-derived DNA was inevitably contaminated. Therefore, following the manufacturer's recommendations for the RPA kit used, the filter process to remove contaminating sequencing reads primarily excluded base sequences from E. coli-derived DNA. The five types of bacteria in Tables 5 and 6 for which base sequence analysis of the RPA products was not possible were all Escherichia coli or its closely related species. Therefore, it was presumed that the base sequence data of the RPA products derived from these bacteria was excluded by the filtering process, resulting in the result "base sequence analysis not possible (N / A)."

[0049] [Example 2] Using the universal primer set for fungal detection according to the present invention, RPA was performed using the genomic DNA of 69 types of fungi as a template to examine whether amplification products could be obtained. The universal primer set for fungal detection used was a set consisting of the 6F_ITS(5.8s)_Fwd1 primer and the 53R_ITS_Rev1 primer.

[0050] Fungi (yeast and mold) were cultured by inoculating each isolated strain onto PD agar medium or YPD agar medium and culturing at 25° C. to form colonies. As in Example 1, DNA was extracted from the resulting colonies, and an RPA reaction was performed using this as a template. The RPA product obtained was subjected to base sequence analysis using "MinION."

[0051] For the RPA products obtained using the universal primer set for fungal detection, the average homology (%) of each read to the microbial species identified by base sequence analysis is shown in Tables 7 to 9. In addition, in Tables 7 to 9, the fungal species for which two microbial species were identified as a result of base sequence analysis were closely related and could not be distinguished by base sequence analysis.

[0052]

[0053]

[0054]

[0055] As shown in Tables 7 to 9, when the genomic DNA of 69 types of fungi was used as a template, base sequence analysis was possible for all fungi, and the fungus used as the template or its closely related species was identified in all cases. These results demonstrate that the universal primer set for fungal detection used can amplify target regions in the genomic DNA of various fungi.

[0056] [Example 3] From the results of Example 1, it was confirmed that the set consisting of the 337f_B1_1 and 805r_B1_1 primers can obtain amplification products by RPA using genomic DNA derived from various bacteria as a template, and that the set is useful as a universal primer for RPA for bacterial detection. Therefore, we investigated whether primers with slightly shifted target regions in bacterial rRNA genes could also function as universal primers for bacterial detection.

[0057] Specifically, an RPA reaction was carried out in the same manner as in Example 1 using a set consisting of the 337f_B1_1 primer and the 805r_B1_1 primer, a set consisting of the 337f_B1_2 primer and the 805r_B1_2 primer, a set consisting of the 337f_B1_2 primer and the 805r_B1_3 primer, a set consisting of the 337f_B1_3 primer and the 805r_B1_2 primer, or a set consisting of the 337f_B1_3 primer and the 805r_B1_3 primer, and using the genomic DNA of Fructilactobacillus lindneri as a template, and the resulting RPA product was purified to obtain a DNA sample.

[0058] The resulting DNA samples were subjected to 2% agarose gel electrophoresis to detect bands of RPA products. The size of the detected DNA bands was estimated using a DNA marker (100 bp DNA ladder) run simultaneously.

[0059] Figure 1 shows a nucleic acid stained image of the gel in which each DNA sample was electrophoresed. Table 10 lists the forward and reverse primers used to prepare the RPA products run in each lane of the gel in Figure 1. As shown in Figure 1, a single band of DNA amplification product was obtained from the RPA reaction with any combination of primers. These results clearly demonstrate that any combination of forward and reverse primers shown in Table 2 is useful as a universal primer for RPA for bacterial detection.

[0060]

[0061] [Example 4] From the results of Example 2, it was confirmed that the set consisting of the 6F_ITS(5.8s)_Fwd1 primer and the 53R_ITS_Rev1 primer can obtain amplification products by RPA using genomic DNA derived from various fungi as a template, and is useful as a universal primer for RPA for fungal detection. Therefore, we investigated whether primers with slightly shifted target regions in fungal rRNA genes would also function as universal primers for fungal detection.

[0062] Specifically, an RPA reaction was carried out in the same manner as in Example 1, except that a set consisting of the 6F_ITS(5.8s)_Fwd1 primer and the 53R_ITS_Rev1 primer, or a set consisting of the 6F_ITS(5.8s)_Fwd2 primer and the 53R_ITS_Rev2 primer, and the genomic DNA of Dekkera anomala was used as a template, and the resulting RPA product was purified to obtain a DNA sample.

[0063] The resulting DNA samples were subjected to 2% agarose gel electrophoresis to detect bands of RPA products. The size of the detected DNA bands was estimated using a DNA marker (100 bp DNA ladder) run simultaneously.

[0064] Figure 2 shows a nucleic acid stained image of the gel in which each DNA sample was electrophoresed. Table 11 shows the forward and reverse primers used to prepare the RPA products run in each lane of the gel in Figure 2. As shown in Figure 2, regardless of the primer combination, a DNA amplification product from the RPA reaction was obtained as a single band. These results clearly demonstrate that any combination of forward and reverse primers shown in Table 4 is useful as a universal primer for RPA for fungal detection.

[0065]

Claims

1. A universal primer set for bacterial detection, comprising a forward primer containing 30 or more consecutive regions in the nucleotide sequence represented by SEQ ID NO: 1 and a reverse primer containing 30 or more consecutive regions in the nucleotide sequence represented by SEQ ID NO:

2.

2. A universal primer set for fungal detection, comprising a forward primer containing 30 or more consecutive regions in the nucleotide sequence represented by SEQ ID NO: 3 and a reverse primer containing 30 or more consecutive regions in the nucleotide sequence represented by SEQ ID NO:

4.

3. 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 16S rRNA gene of bacteria contained in the test sample.

4. 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 ITS gene of fungi contained in the test sample.

5. 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 16S rRNA gene of bacteria contained in the test sample, and identifying the nucleotide sequence of the amplification product obtained by the recombinase polymerase amplification reaction.

6. 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 ITS gene of fungi contained in the test sample, and identifying the nucleotide sequence of the amplification product obtained by the recombinase polymerase amplification reaction.

7. The method for identifying a nucleotide sequence according to claim 5 or 6, wherein the identification of the nucleotide sequence of the amplification product is performed using a sequencer.

8. The method for identifying a nucleotide sequence according to claim 7, wherein the sequencer is a nanopore sequencer.

9. A method for identifying the bacterial species 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 16S rRNA gene of the 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 contained in the test sample based on the nucleotide sequence of the obtained amplification product. A method for identifying bacterial species.

10. The method for identifying bacterial species according to claim 9, wherein the nucleotide sequence of the amplification product is identified using a nanopore sequencer.

11. A method for identifying the fungal species 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 ITS gene of the fungi contained in the test sample; identifying the nucleotide sequence of the amplification product obtained by the recombinase polymerase amplification reaction; and identifying the fungal species contained in the test sample based on the nucleotide sequence of the obtained amplification product. A method for identifying fungal species.

12. The method for identifying fungal species according to claim 11, wherein the nucleotide sequence of the amplification product is identified using a nanopore sequencer.

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