Microbial species identification method
Patent Information
- Authority / Receiving Office
- JP · JP
- Patent Type
- Applications
- Filing Date
- 2023-09-01
- Publication Date
- 2026-07-03
Abstract
Description
Method for identifying microbial species
[0001] The present invention relates to a method for identifying microbial species.
[0002] A method for identifying bacterial species uses the base sequence of the 16S ribosomal RNA gene. However, using the base sequence of the 16S ribosomal RNA gene can sometimes make it difficult to distinguish and identify closely related bacterial species. In this regard, for example, Emerging Microbes & Infections, 2019, Vol. 8, pp. 1043-1053, proposes a method for identifying Mycobacterium tuberculosis subspecies by combining the results of long-read and short-read genome analysis.
[0003] An object of one aspect of the present invention is to provide a method for identifying microbial species based on genomic analysis of the microorganism.
[0004] A first aspect is a method for identifying a microbial species, comprising: directly obtaining a base sequence of each genomic DNA sample from a specimen containing genomic DNA samples derived from the genomic DNA of a microorganism; associating each genomic DNA sample with a specific microbial species based on the obtained base sequence; and identifying the microbial species based on the number of associated genomic DNA samples.
[0005] In one aspect, the method for identifying a microbial species may further include extracting genomic DNA from the microorganism and pre-treating the extracted genomic DNA to obtain a specimen containing the genomic DNA sample, and may further include randomly amplifying the genomic DNA.
[0006] In one embodiment, the microorganism may include at least one species selected from the group consisting of fungi and bacteria. The microorganism may be derived from at least one sample selected from the group consisting of food, alcoholic beverages, soft drinks, environmental samples, etc. The microorganism may be derived from a single colony. Furthermore, the base sequence of the genomic DNA sample may be obtained by at least one sequencing method selected from the group consisting of nanopore sequencing and single-molecule real-time sequencing.
[0007] According to one aspect of the present invention, a method for identifying a microbial species based on genome analysis of the microorganism can be provided.
[0008] As used herein, the term "step" refers not only to an independent step, but also to a step that cannot be clearly distinguished from other steps, as long as the intended purpose of the step is achieved. Furthermore, the content of each component in a composition refers to the total amount of the multiple substances present in the composition, unless otherwise specified, when multiple substances corresponding to each component are present in the composition. Furthermore, the upper and lower limits of the numerical ranges described herein can be arbitrarily selected and combined from the numerical values exemplified as numerical ranges. Hereinafter, embodiments of the present invention will be described in detail. However, the embodiments described below are merely examples of a method for identifying a microbial species to embody the technical concept of the present invention, and the present invention is not limited to the method for identifying a microbial species described below.
[0009] Method for Identifying Microbial Species The method for identifying microbial species may include a sequence acquisition step of directly obtaining the base sequence of each genomic DNA sample from a specimen containing a genomic DNA sample derived from the genomic DNA of the microorganism, an association step of associating each genomic DNA sample with a specific microbial species based on the acquired base sequence, and an identification step of identifying the microbial species of the microorganism contained in the specimen based on the number of associated genomic DNA samples.The method for identifying microbial species may further include an extraction step of extracting genomic DNA from the microorganism and a pretreatment step of pretreating the extracted genomic DNA to obtain a specimen containing a genomic DNA sample.Furthermore, the method for identifying microbial species may include an amplification step of randomly amplifying the extracted genomic DNA prior to the sequence acquisition step.Here, the method for identifying microbial species may be a method for identifying the species of a microorganism contained in a specimen.
[0010] For a specimen derived from a microorganism, the base sequence of a genomic DNA sample contained in the specimen is directly obtained without amplifying a specific DNA region using an amplification method such as PCR, and the species of the microorganism is identified based on the base sequence of the obtained genomic DNA sample, thereby making it possible to identify the species of the microorganism quickly and with high accuracy. Furthermore, since the method for identifying the species of the microorganism based on the base sequence of a genomic DNA sample analyzes the entire genomic DNA, it is highly accurate and versatile.
[0011] The method for identifying a microbial species may include an extraction step of extracting genomic DNA from a microorganism to be identified. The microorganism to be identified may include at least one species selected from the group consisting of fungi and bacteria, or at least one species selected from the group consisting of yeast and bacteria. Here, fungi include filamentous fungi, yeast, dimorphic fungi, etc., and bacteria include lactic acid bacteria, obligate anaerobic bacteria, spore-forming bacteria, etc.
[0012] The microorganism to be identified may be, for example, a microorganism contained in a sample to be tested. Examples of samples to be tested include samples derived from living organisms; materials present in the manufacturing process of food, alcoholic beverages, and soft drinks; materials discharged in the manufacturing process of food, alcoholic beverages, and soft drinks; intermediate products of food; and environmental samples. Examples of living organisms include mammals (e.g., humans, monkeys, mice, rats, rabbits, cows, pigs, horses, goats, and sheep), birds, and other animals, insects, mollusks, microorganisms, plants, and the like. The living organism from which the sample is derived is preferably a mammal, and may be a human or a non-human mammal. Examples of samples derived from living organisms include blood (e.g., whole blood, serum, plasma, etc.), sweat, saliva, urine, hair, breast milk, and the like.
[0013] The sample containing the microorganism may be subjected to an appropriate pretreatment depending on its origin. Examples of pretreatment include centrifugation, extraction, filtration, concentration, and purification. The sample may contain only one type of microorganism, or two or more types. The microorganism contained in the sample may be cultured in an appropriate medium under appropriate conditions depending on the microorganism to be identified, and then used as the target for identification. Furthermore, the microorganism to be identified may be derived from a single colony isolated by culture from a group of microorganisms contained in the sample.
[0014] The method for extracting genomic DNA from microorganisms can be appropriately selected from commonly used methods. For example, when the microorganism is a bacterium, the bacterium can be lysed with lysozyme, N-acetylmuramidase, or the like, treated with a protease as needed, extracted with chloroform / isoamyl alcohol, and then ethanol, isopropanol, or the like can be added to the aqueous phase and centrifuged to extract genomic DNA. Alternatively, genomic DNA can be extracted from microorganisms using a commercially available DNA extraction and purification kit according to the protocol included with the kit. Examples of commercially available DNA extraction and purification kits include the ZymoBIOMICS DNA Mini Kit and QuickDNA-DNA Fungal / Bacterial Microprep Kit (both manufactured by Zymo Research), and the DNeasy Blood & Tissue Kit (manufactured by QIAGEN).
[0015] The number of cells of the microorganism from which genomic DNA is extracted is, for example, 1 to 10 １１ It may be 10 cells or less, preferably 10 ２ Cells or more 10 １０ In one embodiment, when the method for identifying a microbial species does not include an amplification step, the number of cells of the microorganism from which genomic DNA is extracted may be, for example, 10 ７ Cells or more 10 １１ It may be 10 cells or less, preferably 10 ８ Cells or more 10 １０In one embodiment, when the method for identifying a microbial species includes an amplification step, the number of microorganisms from which genomic DNA is extracted may be, for example, 1 cell or more and 10 ７ The number of cells may be 10 or less, and preferably 100 or more and 10 ４ It may be less than cells.
[0016] The DNA concentration of the DNA extract obtained in the extraction step may be, for example, from 10 fg / μL to 3 μg / μL, and preferably from 20 ng / μL to 100 ng / μL. In one embodiment, when the method for identifying a microbial species does not include an amplification step, the DNA concentration of the DNA extract obtained in the extraction step may be, for example, from 10 ng / μL to 3 μg / μL, and preferably from 20 ng / μL to 100 ng / μL. In one embodiment, when the method for identifying a microbial species includes an amplification step, the DNA concentration of the DNA extract obtained in the extraction step may be, for example, from 10 fg / μL to 100 ng / μL, and preferably from 10 pg / μL to 10 ng / μL.
[0017] The method for identifying a microbial species may include an amplification step of randomly amplifying genomic DNA (hereinafter also referred to as genome amplification) to obtain an amplification product containing amplified genomic DNA fragments. By including the amplification step, the microbial species can be identified with high accuracy even when the amount of microorganisms contained in the sample to be tested is small. Furthermore, by randomly amplifying genomic DNA, the time required for amplification can be shortened. The amplification step may include replication of DNA fragments from random positions in the genomic DNA. Genomic DNA amplification may be performed using a commercially available kit in accordance with the protocol in the package insert.
[0018] Kits that can randomly amplify genomic DNA include Illustra TM Ready-To-Go TM GenomiPhi TM DNA Amplification Kit (cytiva), TruePrime Single Cell WGA Kit (4basebio), GenomePlex(R) Whole genome amplification (WGA) kit (Sigma-Aldrich), PicoPLEX (R) Examples include WGA Kit (manufactured by Clontech).
[0019] Random amplification of genomic DNA may be performed on genomic DNA extracted in the extraction step, or on fragmented genomic DNA after a DNA fragmentation treatment in which genomic DNA is randomly fragmented.
[0020] The DNA concentration contained in the amplification product obtained in the amplification step may be, for example, 10 ng / μL or more and 3 μg / μL or less, and preferably 20 ng / μL or more and 100 ng / μL or less.
[0021] The method for identifying a microbial species may include a pretreatment step in which the genomic DNA extracted in the extraction step or the genomic DNA amplified in the amplification step is pretreated to obtain a specimen containing a genomic DNA sample. Examples of genomic DNA pretreatment methods include DNA fragmentation treatments such as mechanical fragmentation and enzymatic fragmentation, and DNA end treatment in which an additional sequence or the like is added to the end of the genomic DNA. The pretreatment step may include at least a DNA fragmentation treatment, or may include a DNA fragmentation treatment in which the genomic DNA is randomly fragmented. Furthermore, the pretreatment step may include both a DNA fragmentation treatment and a DNA end treatment. Genomic DNA pretreatment may be performed using a commercially available kit according to the protocol in the package insert.
[0022] Examples of kits that can perform DNA fragmentation include Rapid Sequencing Kit (manufactured by Oxford NANOPORE Technologies) and Rapid Barcoding Kit (manufactured by Oxford NANOPORE Technologies).
[0023] The additional sequence or the like to be added to the end of the genomic DNA may be selected appropriately depending on the purpose, such as identification or sequencing. The genomic DNA to which the additional sequence or the like is added may be fragmented genomic DNA or genomic DNA extracted from a microorganism. The addition of the additional sequence or the like to the end of the genomic DNA can be carried out, for example, using a commercially available kit in accordance with the protocol in the accompanying instructions. Specific examples include the Ligation Sequencing Kit (Oxford NANOPORE Technologies) and the Rapid Sequencing Kit (Oxford NANOPORE Technologies). Note that some kits are capable of both fragmenting genomic DNA and adding the additional sequence or the like.
[0024] The size (number of bases) of the genomic DNA sample obtained by DNA fragmentation in the pretreatment step may be, for example, 200 bp to 100,000 bp, and preferably 500 bp to 50,000 bp. The amount of the genomic DNA sample obtained in the pretreatment step and subjected to the sequence acquisition step may be, for example, 10 ng to 1,000 ng, and preferably 100 ng to 400 ng.
[0025] In the sequence acquisition step, the base sequence of each genomic DNA sample contained in the specimen is directly acquired. Here, "directly acquiring the base sequence of the genomic DNA sample" means determining the base sequence of each genomic DNA sample by an appropriate base sequencing method without amplifying at least one of the specimens containing the genomic DNA of the microorganism and the genomic DNA sample obtained from the genomic DNA by an amplification method for amplifying a specific DNA region, such as PCR. Since there is no need to amplify the genomic DNA or a fragment thereof, rapid identification of the microbial species is possible.
[0026] Examples of the base sequence determination method for directly obtaining the base sequence of a genomic DNA sample include nanopore sequencing, single-molecule real-time sequencing, etc., and the method may include at least one base sequence determination method selected from the group consisting of these. The base sequence data obtained in the sequence obtaining step may include data corresponding to the base sequence of each genomic DNA sample.
[0027] Nanopore sequencing is a technique for deciphering the base sequence of a DNA sample as it passes through small holes (nanopores) embedded in a membrane. Unlike conventional sequencing methods, this method allows for direct reading of nucleotide sequences without relying on DNA synthesis or amplification. A nanopore sequencer used for nanopore sequencing has a membrane that separates a salt solution into two compartments, with numerous nanopores embedded in the membrane. When a voltage is applied to the membrane, ions flow, and the current is measured at each nanopore. When a DNA sample passes through the nanopore, the ion flow is partially obstructed, reducing the measured current. The amount of change in ion current corresponding to each of the four types of nucleic acid bases is different, allowing nucleic acid bases to be identified based on the change in current. Nanopore sequencing can be performed using commercially available instruments. Specifically, it can be performed using instruments such as the MinION, Frongle, GridION, and PromethION (Oxford NANOPORE Technologies).
[0028] Single-molecule real-time (SMRT) sequencing is a parallelized single-molecule DNA sequencing technique. SMRT sequencing utilizes a zero-mode waveguide (ZMW), in which DNA polymerases are immobilized at the bottom of the ZMW. The DNA polymerase then incorporates template DNA fragments one by one. The ZMW is designed to provide a microscopic fluorescent observation field, allowing for single-nucleotide observation of DNA fragments incorporated by the DNA polymerase. Each of the four nucleotides is attached to a different fluorescent dye. Upon incorporation of a nucleotide by the DNA polymerase, the fluorescent dye is cleaved, emitting fluorescence. The fluorescent dye rapidly diffuses away from the observation area of the ZMW, eliminating the fluorescence. A detector detects this transient fluorescent signal upon nucleotide incorporation and associates it with the corresponding base to determine the base sequence of the DNA fragment. SMRT sequencing can be performed using commercially available equipment. Specifically, for example, PacBio (R) This can be carried out using an apparatus such as the Sequel II / IIe system (PacBio).
[0029] The base sequence data obtained in the sequence obtaining step may be subjected to data processing such as quality check, if necessary. The base sequence data obtained in the sequence obtaining step may include one or more base sequences of a genomic DNA sample having a base number of 200 bp to 100,000 bp, preferably 10 or more, or 100 or more, and preferably 1,000 or less.
[0030] In the association step, each genomic DNA sample is associated with a specific microbial species based on the acquired base sequence. In the association step, for example, a genome database in which specific microbial species are associated with their genomic DNA base sequences is used to identify the microbial species corresponding to the acquired base sequence. Examples of genome databases that can be used include GenBank (http: / / www.ncbi.nlm.nih.gov / ), GenomeSync (http: / / genomesync.org / ), and Mifup (https: / / www.nite.go.jp / nbrc / mifup / ). The genome database may be an online database or an offline database. The association of the acquired base sequence with the microbial species can be performed, for example, by using a homology search tool to search the genome database for the acquired base sequence and selecting the microbial species that have the base sequence in their genomic DNA. Examples of homology search tools that can be used include BLAST (e.g., NCBI BLAST http: / / www.ncbi.nlm.nih.gov / BLAST / ) and the microbial identification system ENKI (https: / / www.tecsrg.co.jp / services / products-and-tec / enki / ).
[0031] In the identification step, the microbial species of the microorganism from which the genomic DNA sample was extracted is identified according to the number of associated genomic DNA samples. The identification of the microbial species can be performed, for example, by visualizing the relationship between the genomic DNA sample and the microbial species associated with its base sequence using a Krona Chart. Alternatively, for example, the microbial species associated with the genomic DNA samples contained in the specimen can be sorted in descending order of the number of associated genomic DNA samples, and the microbial species ranked at the top can be identified as the microorganism to be identified. Furthermore, for example, the microbial species associated with the genomic DNA samples contained in the specimen with the largest number of associated genomic DNA samples can be identified as the microorganism to be identified.
[0032] The method for identifying microbial species according to this embodiment can identify any microorganism, regardless of whether it is a fungus or a bacterium, at the species level, as long as its base sequence data is included in a genome database. Furthermore, since no amplification technique for amplifying a specific DNA region, such as PCR, is required, the method can identify the species using a unified protocol, regardless of whether it is a fungus or a bacterium, and is highly operable. Furthermore, since the target of analysis is the entire genomic DNA, rather than a specific gene, excellent identification accuracy can be achieved.
[0033] Therefore, the method for identifying a microbial species according to this embodiment is effective for distinguishing and identifying closely related bacterial species such as the Lactobacillus casei group, the Lactobacillus plantarum group, and the Bacillus cereus group. That is, the method for identifying a microbial species according to this embodiment may be a method for identifying a species in the Lactobacillus casei group, a method for identifying a species in the Lactobacillus plantarum group, or a method for identifying a species in the Bacillus cereus group. Furthermore, the method for identifying a microbial species can be applied to determining the risk of beer spoilage, determining whether or not a bacterium is a food poisoning bacterium, etc. That is, the method for identifying a microbial species according to this embodiment may be a method for determining the risk of beer spoilage, or a method for determining whether or not a bacterium is a food poisoning bacterium.
[0034] The present invention will be specifically described below with reference to examples, but the present invention is not limited to these examples.
[0035] Reference Example 1 DNA Extraction Lactobacillus casei, L. paracasei, L. rhamnosus, L. zeae, and L. chiayiensis were selected as test bacteria. Each of these strains was cultured on Merck MRS agar medium under anaerobic conditions at 25°C for 3 days. Each single colony obtained by agar culture was extracted to a sufficient degree to make the medium opaque (1 x 10 ８The cells were suspended in sterile water until the concentration reached 1000kJ / mL or more. DNA was extracted and purified from the bacterial suspension using a DNA extraction kit, ZymoBIOMICS DNA Mini Kit or QuickDNA-DNA Fungal / Bacterial Microprep Kit (both from Zymo Research), according to the protocol of each kit. The DNA concentration of the resulting DNA extract was measured using a NanoDrop (Thermo Fisher Scientific). The DNA concentration ranged from 75 ng / μL to 225 ng / μL.
[0036] Example 1 The DNA extract obtained in Reference Example 1 was pretreated using a Ligation Sequencing Kit (SQK-LSK109, Oxford NANOPORE Technologies (ONT)) or a Rapid Sequencing Kit (SQK-RAD004, ONT) according to the protocol of each kit, including the addition of adapter sequences, to obtain a pretreated DNA sample. A MinION flow cell (FLO-MIN106D, ONT) was returned to room temperature and connected to a MinION PC. Priming was performed using a Flow Cell Priming Kit (EXP-FLP002, ONT) according to the protocol included with the kit. The pretreated DNA sample obtained above was then added to the MinION flow cell, and nanopore sequencing was performed using the MinKNOW analysis software to obtain DNA sequence data (FAST5 format).
[0037] An external SSD containing the genome database GenomeSync (http: / / genomesync.org / ) and software related to the Genome Search Tool Kit (GSTK) was connected to the GSTK PC, and the DNA sequence data (FAST5 format) obtained above was stored in a designated folder on the PC. The Guppy-GPU software (ONT) was launched by entering a designated command, and the base sequence (FASTQ format) of each DNA sample was determined by base calling. The obtained base sequence data (FASTA format) of each DNA sample was compared with base sequences in the database using Minimap2 and visualized by displaying it as a Krona chart. The bacterial species with the highest number of base sequences assigned by the chart was used as the identification result. The identification results are shown in Table 1.
[0038] Comparative Example 1 PCR was performed under the following conditions, with reference to the method of LJH Ward et al. (Letters in Applied Microbiology 1999, 29, 90-92). Three primer sets, shown in Table 2, were used: one for detecting L. casei, one for detecting L. paracasei, and one for detecting L. rhamonosus. After PCR was performed with each primer set, the presence or absence of bands was confirmed by electrophoresis. The identification results are shown in Table 1.
[0039] Reaction solution composition: SapphireAmp Fast PCR Master Mix 25 μL, Forward primer (100 μmol / L) 0.2 μL, Reverse primer (100 μmol / L) 0.2 μL, Sterile water 19.6 μL, Pretreated DNA 5 μL
[0040] PCR conditions: (1) 94°C, 1 minute; (2) 30 cycles of 98°C, 5 seconds; 55°C, 5 seconds; 72°C, 10 seconds; (3) 4°C, stop
[0041] Comparative Example 2: A sample was prepared as follows according to the MALDI biotyper manual. Approximately one loopful of colonies obtained by agar culture was suspended in 300 μL of MilliQ water, 900 μL of 99.5% ethanol was added, and the mixture was vortexed and centrifuged at 15,000 rpm for 2 minutes to completely remove the supernatant. 5 μL to 50 μL of 70% formic acid was added to the resulting pellet, and the mixture was thoroughly mixed by pipetting or vortexing. An equal volume of acetonitrile to the formic acid was added and mixed thoroughly. After centrifugation at 15,000 rpm for 2 minutes, 4 μL of the supernatant was collected and evenly distributed over three spots on a target plate (approximately 1 μL per spot). After thorough drying, 4 μL of matrix solution was layered on top of the three dried spots (approximately 1 μL per spot). The calibration standards were spotted in the same manner as the samples, and calibration was performed using a flex control before MALDI-TOF mass spectrometry. Regarding the score indicating the accuracy of identification, a score of 2.0 or more was judged to be at the species level, a score of 1.7 to less than 2.0 was judged to be at the genus level, and a score of less than 1.7 was judged to be unidentifiable. The identification results are shown in Table 1.
[0042] Comparative Example 3: The pretreated DNA obtained above was used to amplify approximately 900 bp upstream of 16S ribosomal RNA under the same PCR conditions as in Comparative Example 1, using the universal primer for 16S rRNA shown in Table 3 as a primer. The PCR product was then purified using EXO-SAP IT (Thermo Fisher Scientific). After purification, the product was fluorescently labeled using cycle sequencing and purified again. The base sequence was obtained using a capillary DNA sequencer, and a homology search was performed using BLAST to identify the bacterial species. The identification results are shown in Table 1.
[0043]
[0044] As shown in Table 1, it can be seen that the identification method of Example 1 can identify five bacterial species belonging to the genus Lactobacillus with high accuracy.
[0045]
[0046]
[0047] Reference Example 2 DNA Extraction Lactobacillus casei was selected as the test bacterium. The strain was cultured on Merck's MRS agar medium under anaerobic conditions at 25°C for 3 days. Each single colony obtained by agar culture was extracted to a sufficient degree to become cloudy (1 x 10 ８ The bacterial suspensions were each suspended in sterile water to a concentration of 1000 cells / mL or more. DNA was extracted and purified from the bacterial suspensions using a DNA extraction kit, ZymoBIOMICS DNA Mini Kit or QuickDNA-DNA Fungal / Bacterial Microprep Kit (both from Zymo Research), according to the kit's protocol. The DNA concentration of the resulting DNA extracts was measured using a NanoDrop (Thermo Fisher Scientific).
[0048] Example 2 The DNA extract obtained in Reference Example 2 was diluted to DNA concentrations of 12 ng / μL, 1.2 ng / μL, 0.12 ng / μL, and 0.012 ng / μL, respectively, to prepare dilutions 1 to 4. 2.5 μL of each of Barcodes 02 to 06 from the Rapid Barcoding Kit (NANOPORE) was added to 7.5 μL of each dilution to fragment the DNA. 2 μL of each of the resulting solutions was placed in a tube, and 1 μL of Rapid Adapter (RAP) was added.
[0049] Illustrate 1 μL of the above DNA dilution. TM Ready-To-Go TM GenomiPhi TM Genome amplification was performed using a DNA Amplification Kit (Cytiva) according to the kit's protocol.
[0050] The DNA dilutions and each genome amplification reaction solution were pretreated using a Rapid Sequencing Kit (SQK-RAD004, ONT) following the kit's protocol, including adapter sequence addition (RAP addition), to obtain pretreated DNA samples. A MinION flow cell (FLO-MIN106D, ONT) was warmed to room temperature and connected to the MinION PC. Priming was performed using a Flow Cell Priming Kit (EXP-FLP002, ONT) according to the kit's protocol. The pretreated DNA samples were then loaded into the MinION flow cell, and nanopore sequencing was performed using the MinKNOW analysis software to obtain DNA sequence data (FAST5 format).
[0051] An external SSD containing the genome database GenomeSync (http: / / genomesync.org / ) and software related to the Genome Search Tool Kit (GSTK) was connected to the GSTK PC, and the DNA sequence data (FAST5 format) obtained above was stored in a designated folder on the PC. The Guppy-GPU software (ONT) was launched by entering a designated command, and the base sequence (FASTQ format) of each DNA sample was determined by base calling. The obtained base sequence data (FASTA format) of each DNA sample was compared with the base sequences in the database using Minimap2 and visualized by displaying it as a Krona chart. Table 4 shows the number of reads assigned to L. casei and their ratio to the total number of reads.
[0052]
[0053] Reference Example 3: DNA Extraction The test bacteria selected were Bacillus anthracis, B. cereus, B. thuringiensis, B. pacificus, B. mycoides, B. paranthracis, B. tropicus, B. mobilis, and B. luti. For B. anthracis and B. cereus, commercially available DNA was used. For other Bacillus cereus group species, DNA was extracted and DNA concentrations were measured in the same manner as in Reference Example 1 after culturing in STA medium at 37°C for several days. The DNA concentrations of the obtained DNA samples were between 20 ng / μL and 300 ng / μL.
[0054] Example 3 Using the DNA samples of Reference Example 3, the bacterial species of each DNA sample was identified in the same manner as in Example 1. The results are shown in Table 5. In Table 5, the case where the bacterial species was correctly identified is indicated as OK, the case where it was incorrectly identified is indicated as NG, and the case where it was not identified is indicated as -.
[0055] Comparative Examples 4 and 5: The bacterial species of each DNA sample was identified in the same manner as in Comparative Example 2 or Comparative Example 3, except that the DNA sample of Reference Example 3 was used. The results are shown in Table 5.
[0056]
[0057] As shown in Table 5, it can be seen that nine bacterial species belonging to the genus Bacillus can be identified with high accuracy by the identification method of Example 1. In particular, Bacillus cereus, a bacterium that causes food poisoning, can be identified quickly and with high accuracy.
Claims
1. This involves directly obtaining the base sequence of each genomic DNA sample from a sample containing genomic DNA derived from microbial genome DNA, and Based on the obtained base sequence, each genomic DNA sample is associated with a specific microbial species, A method for identifying microbial species, including identifying microbial species based on the number of associated genomic DNA samples.
2. Extracting genomic DNA from microorganisms, A method for identifying a microbial species according to claim 1, further comprising pre-treating the extracted genomic DNA to obtain a sample containing the genomic DNA sample.
3. The method for identifying a microbial species according to claim 2, further comprising randomly amplifying genomic DNA.
4. The method for identifying a microbial species according to any one of claims 1 to 3, wherein the microorganism comprises at least one selected from the group consisting of fungi and bacteria.
5. The method for identifying a microbial species according to any one of claims 1 to 3, wherein the microorganism is derived from at least one sample selected from the group consisting of food, alcoholic beverages, soft drinks, and environmental samples.
6. The method for identifying a microbial species according to any one of claims 1 to 3, wherein the microorganism is derived from a single colony.
7. The method for identifying a microbial species according to any one of claims 1 to 3, wherein the base sequence of the genomic DNA sample is obtained by at least one base sequencing method selected from the group consisting of nanopore sequencing and single-molecule real-time sequencing.