JP7676121B2 - Method for extracting sRNA and method for determining the state of existence of organisms - Google Patents

Description

This disclosure relates to a method for extracting sRNA and a method for determining the state of existence of an organism.

In places where hygiene management is required, such as food manufacturing, medical care, welfare, and the home, it is important to control bacteria and other organisms that have undesirable effects on the human body. Most organisms in the environment are harmless, but some organisms can cause food poisoning, spoilage, or deterioration of products, and become a major hindrance to people's lives.

In recent years, attempts to detect organisms such as bacteria (so-called "visualization") by some method and use the detection results as an indicator for hygiene management of organisms are becoming more widespread. The most basic method for detecting bacteria is the culture method. The culture method is a method in which bacteria are cultivated on a medium and the bacteria are detected based on their morphology.

Methods for detecting organisms other than the culture method include methods that use antibodies to recognize surface antigens of organisms (antibody methods). Representative antibody methods include immunochromatography, latex agglutination, and ELISA.

Other methods for detecting organisms besides those mentioned above include methods that detect organisms based on the genes they contain (genetic methods).

One embodiment of the present disclosure aims to provide a method for efficiently extracting sRNA from an organism and a method for quickly determining the state of existence of the organism.

<1> A method for producing a method for producing a carrier coated with a coating material selected from the group consisting of a nonionic surfactant, a cationic surfactant, an anionic surfactant, an amino acid, an oligopeptide, and a protein, by causing an organism to be adsorbed onto the coated carrier in a first liquid; and, by immersing the coated carrier having the organism adsorbed thereon in a second liquid which may be the same as or different from the first liquid, extracting sRNA derived from the organism into the second liquid.
A method for extracting sRNA from an organism, comprising:
<2> The extraction method according to <1>, wherein the soaking is performed under heating.
<3> The extraction method according to <1> or <2>, wherein the organism is an organism having a cell wall.
<4> The extraction method according to <3>, wherein the second liquid is an aqueous solvent.
<5> A method for adsorbing an organism onto a carrier coated with a coating material selected from the group consisting of a nonionic surfactant, a cationic surfactant, an anionic surfactant, an amino acid, an oligopeptide, and a protein, by causing the organism to coexist in a first liquid;
immersing the coated carrier with the adsorbed organism in a second liquid, which may be the same as or different from the first liquid, thereby extracting sRNA derived from the organism into the second liquid;
Detecting the state of existence of the extracted sRNA; and determining the state of existence of the organism based on the state of existence of the obtained sRNA.
A method for determining the state of existence of a living organism, comprising:
<6> The method according to <5>, wherein the sRNA is one or more types of sRNA.
<7> The method according to <5> or <6>, wherein the immersion is performed under heating.
<8> The method according to any one of <5> to <7>, wherein the organism has a cell wall.
<9> The method according to <8>, wherein the second liquid is an aqueous solvent.
<10> The method according to <9>, wherein the aqueous solvent contains a nucleic acid amplification reagent.
<11> The method according to any one of <5> to <10>, wherein the immersion is performed at a temperature within a range of 0° C. to 50° C.
<12> The method according to any one of <5> to <11>, wherein the support is at least one selected from the group consisting of activated carbon, silica, zeolite, porous ceramics, carbon fiber, sand, and glass beads.
<13> The method according to any one of <5> to <12>, wherein the nonionic surfactant is at least one selected from the group consisting of polysorbate 20, polysorbate 80, and octylphenol ethoxylate.
<14> The method according to any one of <5> to <12>, wherein the cationic surfactant is at least one selected from the group consisting of benzalkonium chloride and cetyltrimethylammonium bromide.
<15> The method according to any one of <5> to <12>, wherein the anionic surfactant is at least one selected from the group consisting of sodium dodecyl sulfate and sodium 1-octanesulfonate.
<16> The method according to any one of <5> to <12>, wherein the amino acid is glycine.
<17> The method according to any one of <5> to <12>, wherein the protein is at least one selected from the group consisting of skim milk powder and bovine serum albumin.
<18> The method according to any one of <5> to <17>, wherein the sRNA is present in the organism.
<19> The method according to any one of <5> to <18>, wherein determining the existence state of the organism includes determining an overall existence state of two or more organisms.
<20> The method according to any one of <5> to <19>, wherein detecting the state of existence of the sRNA includes detecting the amount of the sRNA present.
<21> The method according to any one of <5> to <20>, wherein the organism includes at least one organism having a cell wall selected from the group consisting of Escherichia coli, Citrobacter freundii, and Salmonella gallinarum.
<22> The method according to any one of <5> to <21>, wherein the living thing includes a plant.
<23> The method according to any one of <5> to <22>, wherein the organism comprises a verotoxin-producing bacterium.
<24> The method according to any one of <5> to <23>, wherein the number of bases of each of the sRNAs is within the range of 5 to 500.
<25> The method according to any one of <5> to <24>, wherein the sRNA includes at least one selected from the group consisting of EC-5p-36 having a nucleotide sequence represented by SEQ ID NO: 1, EC-3p-40 having a nucleotide sequence represented by SEQ ID NO: 2, EC-5p-79 having a nucleotide sequence represented by SEQ ID NO: 3, EC-3p-393 having a nucleotide sequence represented by SEQ ID NO: 4, fox_milRNA_5 having a nucleotide sequence represented by SEQ ID NO: 5, miR156 having a nucleotide sequence represented by SEQ ID NO: 6, and miR716b having a nucleotide sequence represented by SEQ ID NO: 7.
<26> The method according to any one of <5> to <25>, wherein the detection of the state of the sRNA is carried out by PCR.

The present disclosure provides a method for efficiently extracting sRNA from an organism and a method for rapidly determining the state of existence of the organism.

The contents of the present disclosure will be described in detail below. The following description of the components may be based on a representative embodiment of the present disclosure, but the present disclosure is not limited to such an embodiment.
In the numerical ranges described in the present disclosure in stages, the upper or lower limit value described in one numerical range may be replaced with the upper or lower limit value of another numerical range described in stages. In addition, in the numerical ranges described in the present disclosure, the upper or lower limit value of the numerical range may be replaced with a value shown in the examples.

Furthermore, in this disclosure, when multiple substances corresponding to each component are contained, the content of each component refers to the total amount of the multiple substances contained, unless otherwise specified.

In the present disclosure, "mass %" and "weight %" are synonymous, and "parts by mass" and "parts by weight" are synonymous.
Furthermore, in this disclosure, multiple exemplary aspects described separately may be combined with each other to form a new aspect, unless they contradict each other.

The method for extracting sRNA from an organism according to the present disclosure includes causing an organism to coexist with a carrier coated with a coating material selected from the group consisting of nonionic surfactants, cationic surfactants, anionic surfactants, amino acids, oligopeptides, and proteins in a first liquid, thereby adsorbing the organism to the coated carrier, and immersing the coated carrier with the organism adsorbed in a second liquid, which may be the same as or different from the first liquid, thereby extracting sRNA derived from the organism into the second liquid (hereinafter, also referred to simply as the "extraction method according to the present disclosure").

The method for determining the state of existence of an organism according to the present disclosure includes the steps of: causing an organism to coexist with a carrier coated with a coating material selected from the group consisting of nonionic surfactants, cationic surfactants, anionic surfactants, amino acids, oligopeptides, and proteins in a first liquid, thereby adsorbing the organism to the coated carrier; immersing the coated carrier with the organism adsorbed thereon in a second liquid, which may be the same as or different from the first liquid, thereby extracting sRNA derived from the organism into the second liquid; detecting the state of existence of the extracted sRNA; and determining the state of existence of the organism based on the state of existence of the obtained sRNA (hereinafter also referred to simply as the "determination method according to the present disclosure").

As a result of intensive research, the inventors of the present application have found that sRNA can be efficiently extracted from organisms by a method for extracting sRNA from organisms, the method comprising: causing an organism to coexist with a carrier coated with a coating material selected from the group consisting of a nonionic surfactant, a cationic surfactant, an anionic surfactant, an amino acid, an oligopeptide, and a protein in a first liquid, thereby adsorbing the organism onto the coated carrier; and immersing the coated carrier having the organism adsorbed thereon in a second liquid, which may be the same as or different from the first liquid, thereby extracting sRNA derived from the organism into the second liquid.
Furthermore, the inventors have found that the existence state of an organism can be rapidly determined by a method for determining the existence state of an organism, the method comprising: causing an organism to coexist with a carrier coated with a coating material selected from the group consisting of a nonionic surfactant, a cationic surfactant, an anionic surfactant, an amino acid, an oligopeptide, and a protein in a first liquid, thereby adsorbing the organism to the coated carrier; immersing the coated carrier having the organism adsorbed thereon in a second liquid, which may be the same as or different from the first liquid, thereby extracting sRNA derived from the organism into the second liquid; detecting the existence state of the extracted sRNA; and determining the existence state of the organism based on the obtained existence state of the sRNA.

The cells of living organisms contain short RNA fragments called small RNAs. Small RNAs (hereafter referred to as "sRNAs") play an important role in controlling vital processes such as development, differentiation, transposon silencing, and virus defense.

The inventors of the present application have considered that, when collecting sRNA present in an organism, it may be beneficial to adsorb an organism (e.g., cells of an organism) to a carrier. For example, when collecting sRNA from an organism contained in a liquid, the organism can be separated from the solvent component by adsorbing the organism to a carrier. More specifically, by adding a carrier to the liquid to adsorb the organism, and then settling the carrier and removing the supernatant, the concentration of the organism contained in the liquid can be increased, that is, concentrated. Alternatively, by including a carrier in a filter and passing a liquid containing the organism through the filter, the organism can be adsorbed to the carrier in the filter and separated from the solvent component of the liquid. Furthermore, after removing the supernatant or passing the liquid through the filter, a different solvent can be added to the carrier adsorbing the organism, thereby performing solvent exchange. Even when the same type of solvent as the original liquid solvent is added to the carrier adsorbing the organism, there is an effect of removing impurity components that do not bind to the carrier among the components in the original liquid.
If the organisms can be concentrated, the efficiency of sRNA collection can be increased, and if solvent exchange is possible, a solvent suitable for sRNA extraction can be used. Furthermore, removal of contaminating components allows the purity of the collected sRNA to be improved.

The inventors of the present application considered that it would be useful to use such a carrier that adsorbs organisms, but found that in reality, problems arise due to the use of the carrier. Specifically, since the carrier adsorbs not only organisms but also sRNA, even if organisms are adsorbed on the carrier and immersed in liquid, the sRNA from the adsorbed organisms is adsorbed on the carrier and cannot be efficiently extracted into the solution.
Surprisingly, the inventors of the present application have found that by coating a carrier with a specific substance, the carrier can adsorb an organism while reducing the inhibition of extraction of sRNA into a liquid. More specifically, it has been found that by coexisting an organism with a carrier coated with a coating material selected from the group consisting of a nonionic surfactant, a cationic surfactant, an anionic surfactant, an amino acid, an oligopeptide, and a protein (hereinafter also referred to as a "specific coating material") in a first liquid and adsorbing the organism to the coated carrier, sRNA can be extracted more efficiently in a second liquid. It is presumed that by coating a part or the whole of the surface of the carrier with a specific coating material, the adsorption property of the carrier surface is changed, and while the organism can be adsorbed to the carrier, the adsorption of sRNA released from the organism to the carrier can be reduced.
Thus, when a carrier coated with a specific coating material and an organism are allowed to coexist in a first liquid, the organism is adsorbed to the carrier, and the organism can be captured on the carrier, and further, the adsorption of the released sRNA to the carrier coated with the specific coating material is reduced. Therefore, when the coated carrier on which the organism is adsorbed is immersed in a second liquid, sRNA can be extracted from the organism more efficiently than when the organism is adsorbed to an uncoated carrier.

The extracted sRNA can be used for any purpose, for example, as experimental nucleic acid. According to the present disclosure, a method for determining the state of existence of an organism is provided as an example of a method using the extracted sRNA. Since sRNA has an important function in cells, sRNA expressed in the same organism species has nucleotide sequence conservation, that is, the expression profile of various sRNAs is common among multiple cells of the same organism species. On the other hand, between different organism species, the expression profile of sRNA is differential according to the organism species. Therefore, like DNA and 16S rRNA, information on sRNA present in a measurement sample can be used to distinguish organism species. It was not previously known that organism species can be distinguished using sRNA.

For example, in a biological species (A), sRNA (A) having a nucleotide sequence (A) is expressed, but sRNA (B) having a nucleotide sequence (B) is not expressed, and in a biological species (B), sRNA (B) is expressed, but sRNA (A) is not expressed. If the presence of sRNA (A) is detected in the measurement sample but the presence of sRNA (B) is not detected, it can be determined that cells of biological species (A) are present but cells of biological species (B) are not present. However, if sRNA (A) is an sRNA that is also expressed in a biological species (C) other than biological species (A) and (B), if the presence of sRNA (A) is detected in the measurement sample but the presence of sRNA (B) is not detected, it can be determined that cells of one or more of biological species (A) and biological species (C) are present but cells of biological species (B) are not present.

Furthermore, since sRNA has a short chain length, it is less susceptible to attack by RNase compared to 16S rRNA, and can be analyzed more stably. In addition, the number of copies of sRNA within a cell can be several thousand to tens of thousands at most, so information on the state of existence of sRNA can be obtained with high sensitivity. In the determination method according to the present disclosure, the state of existence of sRNA extracted from an organism into a second solution is detected, and the state of existence of the organism is determined based on this.

<Biology>
The organism in the extraction method and the determination method according to the present disclosure is not particularly limited, and may be an organism having a cell wall or an organism not having a cell wall. Examples of the organism include animals, plants, microorganisms, etc.
In one embodiment, the organism is any organism having a cell wall. In general, cells other than animal cells, such as plant cells and microbial cells, have cell walls. That is, the organism having the cell wall may be a plant. The organism having the cell wall may be a fungus, such as yeast, slime mold, or mold, or a bacterium, such as Escherichia coli.
The object of extraction or determination does not have to be the whole organism, but may be a part of an organism, that is, a cell of an organism. In other words, the term "organism" in the extraction method according to the present disclosure and the determination method according to the present disclosure is not limited to the meaning of the whole organism, but also includes parts of an organism, such as cells of an organism. This is because sRNA is present in parts of organisms, and even detection of parts of organisms gives information about the existence of an organism. Examples of the part of an organism include parts of multicellular organisms, such as pollen of plants. However, it is preferable that the part of an organism is a part that maintains the shape of a cell. If the part of an organism does not maintain the shape of a cell, intracellular components have already flowed out into the surrounding medium before immersion in the second liquid, and even if the part of an organism is adsorbed on activated carbon, the part of the organism may not contain sRNA. As described above, the extraction method according to the present disclosure describes extracting sRNA from an organism, and the determination method according to the present disclosure describes determining the existence state of an organism, but the organism here represents a concept that includes parts of an organism. Therefore, it is sufficient that the extracted sRNA or the determined existence state gives information about the organism species or group of organism species. In fact, for example, in the case of detecting herbaceous plants, the sample will generally contain not the whole organism but a part of it, but the extraction method or determination method according to the present disclosure can provide an extraction result or determination result regarding the presence of a species or a group of species. Thus, the organism in the extraction method and determination method according to the present disclosure may be a plant, and in this case the component contained in the sample may be, for example, pollen.

The organism having the cell wall may be, for example, an organism whose presence may cause a problem in terms of hygiene management in a site where hygiene management is required (food production, medical care, welfare, home, etc.). Examples of such organisms include pathogenic microorganisms and putrefactive microorganisms. The pathogenic microorganism may be a pathogenic fungus, examples of which include tinea fungus, Candida, Aspergillus, etc. The pathogenic microorganism may be a pathogenic bacterium, examples of which include gram-positive bacteria (e.g., staphylococci, streptococci, pneumococci, enterococci, Corynebacterium diphtheriae, Mycobacterium tuberculosis, Mycobacterium leprae, Bacillus anthracis, Bacillus subtilis, Clostridium perfringens, Clostridium tetanus, Clostridium botulinum, etc.), gram-negative bacteria (Neisseria gonorrhoeae, Neisseria meningitidis, Salmonella, Escherichia coli, Pseudomonas aeruginosa, Shigella, Haemophilus influenzae, Bordetella pertussis, Vibrio cholerae, Vibrio parahaemolyticus, Acinetobacter, Campylobacter, Legionella, Helicobacter, etc.). The pathogenic bacteria may be verotoxin-producing bacteria. Examples of spoilage microorganisms include bacteria of the genera Pseudomonas, Micrococcus, Vibrio, and Flavobacterium in the case of seafood, bacteria of the genera Pseudomonas, Achromobacter, Micrococcus, and Flavobacterium in the case of meat, and bacteria of the genus Bacillus in the case of cooked rice and noodles.

In one embodiment, the organism having a cell wall preferably includes at least one selected from the group consisting of Escherichia coli, Citrobacter freundii, and Salmonella gallinarum. In another embodiment, the organism having a cell wall includes a plant, which may be a Japanese black pine. In another embodiment, the organism having a cell wall includes a verotoxin-producing bacterium.

In the extraction method and determination method disclosed herein, sRNA extracted from cells into the second liquid is used, so a treatment may be carried out in advance to remove substances that may interfere with such extraction.

<Sample>
In the extraction method or the determination method according to the present disclosure, a mixture containing an organism, a carrier coated with a coating material selected from the group consisting of a nonionic surfactant, a cationic surfactant, an anionic surfactant, an amino acid, an oligopeptide, and a protein, and a first liquid is also referred to as a sample. The sample in the extraction method according to the present disclosure is a sample used to extract the sRNA of the organism to be extracted, and the sample in the determination method according to the present disclosure is a sample used to determine the existence state of the organism to be detected. When an organism is present in an object containing an organism that is a source of sRNA or an object whose existence state is to be determined (hereinafter also simply referred to as an object), there is no particular limitation on the sample as long as the sample is prepared to contain the organism, and the sample may be a sample in which a carrier coated with a specific coating material is added to the object, or a sample in which a carrier coated with a specific coating material is added to a preparation prepared from the object by any treatment. The object may be, for example, a surface of an object such as a workbench, an object itself such as food, a liquid such as tap water, a gas such as air, a bacterial cell of unknown biological species, a bacterial cell culture liquid, etc. From the viewpoint of simplifying the operation, it is preferable that the sample is a sample in which a carrier coated with a specific coating material is simply added to the object.

For example, when it is desired to extract sRNA from an organism present in a liquid as a target, or when it is desired to determine the state of existence of the organism, a sample may be prepared by simply adding a carrier coated with a specific coating material to the liquid, or a sample may be prepared by diluting the liquid and then adding a carrier coated with a specific coating material. The dilution and the addition of the carrier coated with a specific coating material may be performed in any order, or may be performed simultaneously. For example, when it is desired to extract sRNA from an organism present on the surface of an object as a target (for example, the surface of a workbench), or when it is desired to analyze the organism, the surface may be wiped with a wipe, cotton swab, or the like, and the wipe, cotton swab, or the like, or a part thereof, may be immersed in a first liquid, and then the wipe, cotton swab, or the like, or a part thereof, may be removed, and a carrier coated with a specific coating material may be added to the first liquid to prepare a sample. The immersion and the addition of the carrier coated with a specific coating material may be performed in any order, or may be performed simultaneously. The operation of removing the wipe, swab, etc., or a portion thereof, may be performed before, after, or simultaneously with adding the carrier coated with the specific coating material to the first liquid.

When it is desired to extract sRNA from an organism present inside an object (e.g., inside food) as a target, or when it is desired to analyze the object for the organism, the object may be immersed in whole or in part in a first liquid, and then the object may be removed, and a carrier coated with a specific coating material may be added to the first liquid to obtain a sample. When it is desired to extract sRNA from an organism present in air as a target, or when it is desired to analyze the air for the organism, the object obtained by collecting airborne matter present in the air may be immersed in a first liquid, and a carrier coated with a specific coating material may be added to the first liquid to obtain a sample. Note that the immersion and the addition of the carrier coated with a specific coating material may be performed in any order, or simultaneously. The collection may be performed by a filter, a centrifugation (e.g., cyclone separation), or by simply leaving the container open and stationary (e.g., leaving the container open and stationary, such as a petri dish).

In this way, the sample can be, for example, a liquid as the subject; a diluted solution of the liquid; or a liquid obtained by immersing a surface wipe, cotton swab, or the like used to wipe a surface as the subject, matter collected on an air filter, or matter adhering to the inside of a container open to the surrounding atmosphere, in a first liquid to which a carrier coated with a specific coating material has been added.

The organisms contained in the sample preferably include organisms with cell walls and cell membranes that are not destroyed, or organisms without cell walls and cell membranes that are not destroyed. In other words, the sample preferably has not been subjected to cell disruption treatment, membrane destruction treatment, etc. This is because if the cell wall or cell membrane is destroyed, the organism may not contain sRNA even if it is adsorbed to a carrier. In the extraction method and determination method disclosed herein, it is preferable that the process up to the extraction of sRNA into the second liquid does not include treatment that destroys the cell wall and/or cell membrane of the organism (cell disruption treatment, membrane destruction treatment, etc.).

<Carrier>
The carrier in the extraction method and the determination method according to the present disclosure is not particularly limited as long as it is a carrier that is used by those skilled in the art as a carrier and can adsorb an organism, for example an organism having a cell wall.

The particle size of the carrier according to the present disclosure is not particularly limited as long as it is capable of adsorbing organisms, and may be, for example, a particle size that remains on a 4 mesh sieve and passes through a 400 mesh sieve (i.e., passes through a 4750 μm mesh sieve and remains on a 38 μm mesh sieve), remains on a 7 mesh sieve and passes through a 100 mesh sieve (i.e., passes through a 2800 μm mesh sieve and remains on a 150 μm mesh sieve), or remains on a 10 mesh sieve and passes through a 32 mesh sieve (i.e., passes through a 1700 μm mesh sieve and remains on a 500 μm mesh sieve).

The BET specific surface area of the carrier according to the present disclosure is not particularly limited as long as it is capable of adsorbing organisms, and may be, for example, a BET specific surface area of 500 ^{m 2} /g to 2,500 ^{m 2} /g, or 1,000 m ² /g to 2,000 m ² /g.
The BET specific surface area can be measured using an automatic flow type specific surface area measuring device (Shimadzu Corporation's "Flowsorb III2305") in accordance with the device's instruction manual. The support to be measured may be pretreated by heating at 200°C for 15 minutes.

The average pore size of the carrier according to the present disclosure is not particularly limited as long as it is capable of adsorbing organisms, and may be, for example, an average pore size of 20 angstroms or less, 10 angstroms to 200 angstroms, 20 angstroms to 500 angstroms, or 500 angstroms or more.
The average pore size can be calculated by observing the carrier from above with a scanning electron microscope (SEM), measuring the pore sizes of 50 randomly selected pores, and averaging the measurements.

The concentration of the carrier in the first liquid according to the present disclosure is not particularly limited as long as it is capable of adsorbing the organism, and may be, for example, a concentration of 0.01 g/ml to 100 g/ml, 0.1 g/ml to 70 g/ml, 1 g/ml to 50 g/ml, or 5 g/ml to 40 g/ml.

Examples of the carrier include at least one selected from the group consisting of activated carbon, silica, zeolite, porous ceramics, carbon fiber, sand, and glass beads.
Examples of the activated carbon include activated carbon produced using coconut shells or coal as raw materials.
The carrier may be a granular carrier, or may be a carrier in a state where the granular carrier is set inside a filter, a column, or the like.

<Coating material>
In the extraction method and the determination method according to the present disclosure, the carrier is coated with a coating material selected from the group consisting of a nonionic surfactant, a cationic surfactant, an anionic surfactant, an amino acid, an oligopeptide, and a protein.

The nonionic surfactant refers to a compound that has a hydrophilic group and a hydrophobic group in one molecule and has no groups that dissociate into ions in water.
The nonionic surfactant according to the present disclosure may have a hydroxy group, and may be a polyol ester-based nonionic surfactant, an alkanolamide-type nonionic surfactant, an alkyl glucoside-type nonionic surfactant, an alkoxylate-type nonionic surfactant, or a fatty acid alkyl ester-type nonionic surfactant. Specifically, the nonionic surfactant may be at least one selected from the group consisting of polysorbate 20 (e.g., Tween 20), polysorbate 80 (e.g., Tween 80), and octylphenol ethoxylate (e.g., Triton X-100).

The structural formula of polysorbate 20 is shown below. In the formula, w, x, y, and z represent integers, and w+x+y+z=20.

The structural formula of polysorbate 80 is shown below. In the formula, w, x, y, and z represent integers, and w+x+y+z=20.

The structural formula of octylphenol ethoxylate is shown below. In the formula, x represents the average degree of polymerization, which is approximately 9.5 for Triton X-100.

The cationic surfactant refers to a compound that has a hydrophilic group and a hydrophobic group in one molecule and ionizes in water to become a cation.
The cationic surfactant according to the present disclosure may be an amine salt type cationic surfactant or a quaternary ammonium salt type cationic surfactant. Specifically, the cationic surfactant may be at least one selected from the group consisting of benzalkonium chloride (e.g., Osvan) and cetyltrimethylammonium bromide (CTAB).

The structural formula of benzalkonium chloride is shown below, where R represents a hydrocarbon group of C ₈ H ₁₇ to C ₁₈ H ₃₇ .

The anionic surfactant refers to a compound that has a hydrophilic group and a hydrophobic group in one molecule and ionizes in water to become an anion.
The anionic surfactant according to the present disclosure may be a sulfosuccinate-type anionic surfactant, a sulfonate-type anionic surfactant, a sulfate ester-type anionic surfactant, or a fatty acid salt-type anionic surfactant. Specifically, the anionic surfactant may be at least one selected from the group consisting of sodium dodecyl sulfate (SDS) and sodium 1-octanesulfonate.

An amino acid refers to a compound having an amino group (-NH ₂ ) and a carboxy group (-COOH) in one molecule.
The amino acids according to the present disclosure may be hydrophobic or hydrophilic amino acids, or may be acidic, basic, or neutral amino acids. In the present disclosure, the hydrophobic amino acids refer to amino acids selected from the group consisting of leucine, isoleucine, tryptophan, phenylalanine, valine, alanine, glycine, proline, and methionine. The hydrophilic amino acids refer to amino acids selected from the group consisting of serine, threonine, aspartic acid, glutamic acid, lysine, arginine, histidine, asparagine, glutamine, tyrosine, and cysteine. The acidic amino acids refer to aspartic acid or glutamic acid. The basic amino acids refer to amino acids selected from the group consisting of lysine, arginine, and histidine. The neutral amino acids refer to amino acids selected from the group consisting of asparagine, glutamine, serine, threonine, tyrosine, cysteine, and the amino acids described in the hydrophobic amino acids. The amino acids may be α-amino acids, β-amino acids, or other amino acids. The amino acid according to the present disclosure can specifically be, for example, glycine.

An oligopeptide refers to a polymer in which about 2 to 20 amino acid residues are linked in a chain form in one molecule.
The oligopeptide according to the present disclosure may be a polymer in which approximately 2 to 20 amino acid residues are linked in a chain form in one molecule, or approximately 5 to 15 amino acid residues are linked in a chain form in one molecule, or approximately 7 to 13 amino acid residues are linked in a chain form in one molecule.

A protein refers to a compound formed by linking a large number of amino acids (for example, 21 or more residues) in a chain in one molecule.
The protein according to the present disclosure may be a dairy product, milk powder, gelatin, hemoglobin, albumin, serum or plasma. Specifically, the protein may be at least one selected from the group consisting of nonfat milk powder (e.g., skim milk) and bovine serum albumin (BSA).

The method of coating the carrier with the specific coating material is not particularly limited as long as the carrier is brought into contact with the specific coating material and the carrier is partially or entirely coated. The coating can be performed by bringing the carrier into contact with the specific coating material, but such contact may be performed, for example, by having the carrier and the specific coating material coexist in a liquid. When the specific coating material comes into contact with the surface of the carrier, it is adsorbed to the surface of the carrier. In coating, it is not necessary to coat the entire surface of the carrier, and if only a portion of the carrier is coated, it is possible to adsorb the organism to the carrier and reduce the adsorption of sRNA released from the organism to the carrier.

In the coating, the mass of the specific coating material relative to the mass of the carrier may be 0.0001 to 10 times, 0.001 to 5 times, 0.01 to 2 times, or the same mass.
The specific coating material may be contacted with the carrier as a specific coating material solution in which the specific coating material is dissolved in a solvent. In this case, the mass of the specific coating material solution relative to the mass of the carrier may be 0.1 to 1000 times, 0.5 to 100 times, 1 to 10 times, or the same mass. In this case, the mass of the specific coating material in the specific coating material solution relative to the mass of the carrier may be 0.0001 to 10 times, 0.001 to 5 times, 0.01 to 2 times, or the same mass.

The temperature at which the carrier and the specific coating material are brought into contact may be room temperature or may be heated or cooled. In order to minimize denaturation of the carrier or the specific coating material, the contact is preferably carried out at a temperature within the range of 0°C to 50°C, more preferably at a temperature within the range of 4°C to 40°C, even more preferably at a temperature within the range of 10°C to 40°C, even more preferably at a temperature within the range of 20°C to 40°C, even more preferably at a temperature within the range of 25°C to 40°C, and even more preferably at a temperature within the range of 30°C to 37°C. The contact may be carried out at room temperature. The contact may be carried out under heating.

The contact time between the carrier and the specific coating material is not particularly limited as long as a part or the whole of the carrier is coated with the specific coating material. The contact time is, for example, 10 seconds to 30 hours, and may be 10 seconds to 10 hours, 1.0 minute to 5.0 hours, or 5.0 minutes to 1.0 hour. The contact time may alternatively be 0.50 hours to 6.0 hours, 0.70 hours to 4.5 hours, or 0.80 hours to 2.0 hours. The state when the carrier is contacted with the specific coating material may be static, shaking, or stirring.
The carrier contacted with the specific coating material may be further washed with water. For example, the method of washing with water may be a series of operations of centrifuging a suspension containing the carrier contacted with the specific coating material, removing the supernatant, and adding water, which may be repeated once or several times. The water used for washing with water may be sterilized water, a buffer solution, tap water, physiological saline, or an aqueous solvent described later.

The method of coating the carrier with the specific coating material may be, for example, a method of mixing 50 mg of coconut shell activated carbon as the carrier and 100 μL of a 2% solution of polysorbate 20 (Tween 20) as the specific coating material, and leaving the mixture at room temperature for 10 minutes or more. The activated carbon coated with polysorbate 20 thus obtained may be further washed with water. Specifically, after leaving the mixture at rest, the suspension containing the carrier contacted with the specific coating material may be centrifuged at 14,000 rpm (revolutions per minute) for 2 minutes, the supernatant may be removed, and then 300 μL of sterilized water may be added, centrifuged at 14,000 rpm for 2 minutes, and the supernatant may be removed to obtain a coated carrier that has been washed with water.

<First Liquid>
The first liquid in the extraction method and the determination method according to the present disclosure is a liquid that exists when an organism and a carrier coated with a specific coating material selected from the group consisting of nonionic surfactants, cationic surfactants, anionic surfactants, amino acids, oligopeptides, and proteins coexist. The first liquid in the extraction method and the determination method according to the present disclosure is not particularly limited, but may be, for example, sterile water, buffer solution, physiological saline, a liquid medium for culturing an organism, or an aqueous solvent described below. The first liquid is determined by the preparation process of the sample, and for example, when an object containing an organism that is a source of sRNA or an object whose existence state of an organism is to be determined is a liquid and a carrier coated with a specific coating material is added and used, it is the liquid part of the object. In addition, when the object is a solid and is immersed in the first liquid and a carrier coated with a specific coating material is added before, after, or simultaneously therewith to prepare a sample, a desired liquid can be used as the first liquid. From the viewpoint of efficiently adsorbing an organism containing sRNA, it is preferable that the first liquid is a liquid that does not destroy the cell wall and cell membrane of the organism (in the case of an organism having a cell wall) or the cell membrane of the organism (in the case of an organism not having a cell wall).

As described above, if the object is in a liquid state from the beginning, a carrier coated with a specific coating material can be added to the object to make a sample, in which case the liquid part of the object is the first liquid. For example, by adding a carrier coated with a specific coating material to tap water, it is possible to extract sRNA from organisms in the tap water, or to determine the presence of the organisms in the tap water. Also, for example, by adding a carrier coated with a specific coating material to a culture solution in which an organism is cultured, it is possible to extract sRNA from organisms in the culture solution, or to determine the presence of the organisms in the culture solution. When the object contains an organism, the above sample contains the organism in the first liquid.

If the subject is a liquid but requires pretreatment due to a large amount of impurities, the impurities may be at least partially removed by filtration, centrifugation, dialysis, or other methods, and a carrier coated with a specific coating material may be added to prepare a sample. For example, when extracting sRNA from organisms in muddy water or inspecting the presence of organisms in muddy water, the mud in the muddy water is removed by filtration or other methods, and then a carrier coated with a specific coating material is added to the filtrate (containing organisms) to prepare a sample, and sRNA can be extracted from the organisms or the presence of the organisms can be determined.

If the organisms and the coated carrier are kept in contact with each other in the first liquid for a certain period of time (for example, the period of time given as an example of the immersion time described below), the organisms will be adsorbed onto the coated carrier.

As long as the operation involves contacting the coated carrier with the organism in the first liquid, the operation is included in the aforementioned operation of causing the coated carrier and the organism to coexist in the first liquid. The operation of causing the coated carrier and the organism to coexist in the first liquid may be an operation of artificially allowing time for the organism to adsorb to the coated carrier by leaving the coated carrier and the organism stationary or stirring them in the first liquid.

Adsorption of the organism onto the coated carrier by allowing the organism and the carrier coated with a specific coating material to coexist in a first liquid may include partially or completely removing the first liquid from the coated carrier onto which the organism is adsorbed. For example, the first liquid may be entirely or partially removed after allowing the organism and the carrier coated with a specific coating material to coexist in the first liquid for a predetermined time.
When the coated carrier is particulate, removing all or a portion of the first liquid may be, for example, a procedure of spinning down or low-speed centrifugation at 500 rpm (revolutions per minute) for about 3 minutes, followed by removal of the supernatant.
When the coated carrier is set inside the device as a filter or a column, passing the sample through the filter itself includes partially or completely removing the first liquid. The coated carrier may be removed from inside the device after passing the sample. For example, the coated carrier may be removed alone, or a filter or column including the coated carrier may be removed.
By partially or completely removing the first liquid, the liquid volume can be reduced, in other words the concentration of the organism can be increased. At the same time, substances that are not adsorbed to the coated carrier, such as contaminants or the part of the total amount of the specific coating material that is not adsorbed, can be removed. This can make it easier to extract, for example, high-purity sRNA from the organism.

<Second Liquid>
The second liquid in the extraction method and the determination method according to the present disclosure is used to immerse the coated carrier adsorbing the organism and extract the sRNA derived from the organism into the second liquid. The second liquid in the extraction method and the determination method according to the present disclosure is not particularly limited, but may be a general nucleic acid extraction solvent or an aqueous solvent described below. It is also possible to use a strong denaturant such as phenol, guanidine salt, or sodium hydroxide in order to dissolve the cell membrane and quickly release the nucleic acid in the cell. However, when such a denaturant is used, the trouble of solvent exchange is required when performing a nucleic acid amplification process or the like afterwards.

The second liquid in the extraction method according to the present disclosure and the determination method according to the present disclosure may be the same as or different from the first liquid described above. That is, after using the first liquid for coexistence, the first liquid may not be removed or may be partially removed, and the first liquid may be used as the second liquid as it is (i.e., without solvent exchange) for immersion. Alternatively, after using the first liquid for coexistence, the first liquid may be partially or entirely removed, and a newly prepared first liquid may be added and used for immersion. Alternatively, after using the first liquid for coexistence, the first liquid may be partially or entirely removed, and a second liquid having a composition different from that of the first liquid may be added and used for immersion. When removing the first liquid, it is preferable to remove the entire amount from the viewpoint of solvent exchange. However, since it is difficult to completely remove the entire amount of the first liquid in terms of operation, the "total amount" referred to here is a concept that allows a small amount of the first liquid that cannot be removed due to technical constraints of the operation to remain.
Coexistence of the organism and the carrier coated with a specific coating material in a first liquid and immersion of the coated carrier adsorbing the organism in a second liquid, which may be the same as or different from the first liquid, may occur in the same process or may be performed as separate processes. After coexistence and adsorption in the first liquid in the extraction method and the determination method according to the present disclosure, it is preferable to remove part or all of the first liquid, and then immerse and extract with a second liquid, which may be the same or different in composition as the first liquid, from the viewpoint of removing impurities and expanding the options of solvents for sRNA extraction. The method of removing part or all of the first liquid may be, for example, a method of removing the supernatant after centrifugation or natural sedimentation.
Furthermore, in the determination method according to the present disclosure, after extraction with the second liquid, it is preferable to remove a part or all of the coated carrier adsorbing the organism, and then detect the presence state of sRNA using the second liquid. This is because, in detecting the presence state of sRNA, the coated carrier adsorbing the organism may become an impurity in the reaction system for detection. The method for removing a part or all of the coated carrier adsorbing the organism may be, for example, a method such as filtration.

It is also a preferred embodiment that the second liquid does not contain any coexisting components other than the coated carrier to which the above-mentioned organisms are adsorbed. If the organisms are allowed to contact the second liquid for a certain period of time (for example, the period of time exemplified as an example of the immersion time described below), sRNA will leak from the organisms, and the sRNA will be extracted into the second liquid, similar to the case of immersion described below.

As long as the operation involves contacting the coated carrier with the adsorbed organism with a second liquid, the operation is included in the above-mentioned immersion of the coated carrier with the adsorbed organism in the second liquid. When the coated carrier with the adsorbed organism is immersed in the second liquid, the operation may be an operation in which the second liquid in which the coated carrier with the adsorbed organism is immersed is left to stand or stirred, etc., and artificially allowed to age for the extraction of sRNA.

<Aqueous Solvent>
The second liquid in the extraction method according to the present disclosure and the determination method according to the present disclosure may be an aqueous solvent.
The aqueous solvent in the extraction method and the determination method according to the present disclosure is a liquid mainly composed of water, and the aqueous solvent may be pure water itself, or may be an aqueous solution containing other components in addition to water (hereinafter also referred to as "coexisting components") as long as it does not cause denaturation of the cell membrane. The content of solvent components other than water in the aqueous solvent is preferably as low as possible, and the amount of solvent components other than water may be 1% by mass or less, 0.1% by mass or less, 0.01% by mass or less, 0.001% by mass or less, or 0% by mass (i.e., the solvent component may only contain water) relative to the amount of water. In addition, in the present disclosure, the "aqueous solvent" does not contain components that denature the cell membrane and destroy the membrane, or contains only a small amount that does not cause denaturation of the cell membrane. In other words, the aqueous solvent has a basic property in common with water in that it does not cause denaturation of the cell membrane, and is a solvent that can contain coexisting components as long as this basic property is not impaired. Therefore, the aqueous solvent according to the present disclosure does not include an extraction solution for destroying cell membranes and extracting intracellular components (for example, the cell component extract EXTRAGEN (manufactured by Toso Corporation) mentioned in Publication No. 2013-93).

However, the aqueous solvent may contain a total amount of 65% by mass or less of monovalent or divalent linear, branched, or alicyclic C2-C8 alcohols and acetone. The addition of a specific type of alcohol in such an amount may increase the efficiency of extraction of sRNA into the aqueous solvent. Specifically, the content of each of the monovalent or divalent linear, branched, or alicyclic C2-C8 alcohols and acetone is 65% by mass or less, 60% by mass or less, 55% by mass or less, 50% by mass or less, 40% by mass or less, 30% by mass or less, 20% by mass or less, 10% by mass or less, or 5.0% by mass or less, based on the total amount of the aqueous solvent.
The lower limit of the range of the content of the monovalent or divalent linear, branched, or alicyclic C2 to C8 alcohol in the aqueous solvent is not particularly limited, and may be 0% by mass (non-content). The content of the monovalent or divalent linear, branched, or alicyclic C2 to C8 alcohol in the aqueous solvent may be 0.5% by mass or more, 1.0% by mass or more, 5.0% by mass or more, 10% by mass or more, 20% by mass or more, or 40% by mass or more. The above upper and lower limit values may be arbitrarily combined to form a numerical range.
When the aqueous solvent contains two or more of a monovalent or divalent linear, branched or alicyclic C2 to C8 alcohol, and acetone, it is preferable that the total amount of these is also within the above range.

The monohydric linear, branched or alicyclic C2 to C8 alcohol is preferably a monohydric linear, branched or alicyclic C2 to C5 alcohol, more preferably a monohydric linear, branched or alicyclic C2 to C4 alcohol. Examples of the monohydric linear, branched or alicyclic C2 to C8 alcohol include ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, 2-methyl-2-propanol, 1-pentanol, 2-pentanol, 3-pentanol, 2-methyl-1-butanol, 2-methyl-2-butanol, 3-methyl-1-butanol, 3-methyl-2-butanol, and 2,2-dimethyl-1-propanol.
The divalent linear, branched or alicyclic C2-C8 alcohol is preferably a divalent linear, branched or alicyclic C2-C6 alcohol, more preferably a divalent linear, branched or alicyclic C2-C4 alcohol. Examples of divalent linear, branched or alicyclic C2-C8 alcohols include 1,2-ethanediol, 1,2-propanediol, 1,3-propanediol, 1,2-butanediol, 1,3-butanediol, 1,4-butanediol and 2,3-butanediol.
One or more of these monovalent or divalent linear, branched or alicyclic C2 to C8 alcohols can be arbitrarily selected and used.

According to the present disclosure, even when the organism has a cell wall and the second liquid is an aqueous solvent, extraction of sRNA is possible.
sRNA is confined inside the cell by the cell membrane of the microorganism, and it was thought that it was difficult to measure directly. In order to analyze sRNA, it was thought that it was necessary to denature the cell membrane with a strong denaturant (a denaturant strong enough to denature the cell membrane) such as phenol, guanidine salt, sodium hydroxide, etc., to extract the nucleic acid, and then to remove or neutralize the denaturant. This is because, in fact, in order to extract the DNA and 16S rRNA of the microorganism from the cell, it is necessary to denature the cell membrane with a strong denaturant (a denaturant strong enough to denature the cell membrane) such as sodium hydroxide, and it was not possible to extract the DNA and 16S rRNA without using a strong denaturant. Thus, in the conventional nucleic acid extraction method, the operation of treating with a denaturant and the operation of removing or neutralizing the denaturant are time-consuming, making the operation complicated and not simple.

Considering the above situation, the inventors of the present application have conducted intensive research and found that sRNA in cells can be extracted by immersing cells of an organism having a cell wall in water or an aqueous solution containing additional components in addition to water to an extent that does not significantly increase the cell degeneration effect. This finding is surprising. For cells of an organism that does not have a cell wall, when the cells are immersed in a hypotonic solution, the cell membrane is ruptured by osmotic pressure, and it is possible to extract DNA and 16S rRNA into the surrounding environment without using the above-mentioned denaturing agent, whereas such a method cannot be applied to cells of an organism having a cell wall according to the conventional technical common sense. More specifically, cells of an organism having a cell wall are not easily lysed because the structure is maintained by the cell wall, and therefore the cell membrane does not rupture due to changes in the osmotic pressure of the surrounding environment. For this reason, according to the conventional technical common sense, it is considered difficult to release nucleic acids present in the cells of an organism having a cell wall into the surrounding environment without the above-mentioned cell membrane denaturation operation. For this reason, it is surprising that sRNA can be extracted outside the cells of organisms that have cell walls by immersion in water.

Thus, although the existence of sRNA itself has been known for some time, it was thought that sRNA could not be extracted from cells having cell walls without the use of strong denaturants. However, in the present disclosure, it has surprisingly been found that when cells having cell walls are placed in an aqueous solvent, sRNA within the cells is extracted into the aqueous solvent surrounding the cells (i.e., leaks into the aqueous solvent) without destroying the cell membrane with a strong denaturant or the like. This extraction is possible even at room temperature. The sRNA extracted into the aqueous solvent can be used to detect the presence of a specific organism of interest or to identify the type of organism present in a measurement sample. Therefore, by using extraction of sRNA with an aqueous solvent, sRNA can be detected from an organism having a cell wall of interest easily and without much effort.

In order to prevent the denaturation of cell membranes under strongly acidic or strongly alkaline conditions, the pH of the aqueous solvent is preferably in the range of pH 5 to pH 9, more preferably in the range of pH 6 to pH 8, and even more preferably in the range of pH 6.5 to pH 7.5. The aqueous solvent may contain coexisting components other than water as described above. The total content of the coexisting components may be 30% by mass or less, 10% by mass or less, 1% by mass or less, 0.1% by mass or less, 0.01% by mass or less, or 0.001% by mass or less, based on the total amount of the aqueous solvent. Examples of coexisting components that may be contained in the aqueous solvent include salts, buffers, surfactants, DTT, RNase inhibitors, and the like. Since sRNA has a short chain length, it is less susceptible to attack by RNase compared to longer RNAs such as 16S rRNA, but sRNA can be further stabilized by the coexistence of an RNA stabilizer such as DTT or an RNase inhibitor.

The aqueous solvent does not contain coexisting components in an amount that would denature and destroy the cell membrane. In addition, from the viewpoint of preventing denaturation of the cell membrane, it is preferable that the aqueous solvent does not contain phenol, guanidine, alcohols other than the above-mentioned monovalent or divalent linear, branched or alicyclic C2-C8 alcohols, ionic surfactants, and strong alkalis such as NaOH. When the aqueous solvent contains denaturing components such as phenol, guanidine, alcohols other than the above-mentioned monovalent or divalent linear, branched or alicyclic C2-C8 alcohols, ionic surfactants, and strong alkalis such as NaOH, the total content of the components is preferably 0.1% by mass or less, more preferably 0.01% by mass or less, and even more preferably 0.001% by mass or less, based on the total amount of the aqueous solvent. When the aqueous solvent does not contain denaturing components such as strong alkalis, or contains denaturing components but the content is within the above range, it is not necessary to perform additional processing to remove these denaturing components for further processing after immersion in the second liquid, and extraction of sRNA or determination of the state of existence of the organism can be performed more easily. From this perspective, it is preferable that the product does not contain any denaturing components, or that even if it does contain them, the content is within the above range.

Depending on the type and amount of surfactant, it may denature or destroy cell membranes, so if the aqueous solvent contains a surfactant, the type and amount must be limited. The surface tension of the aqueous solvent is preferably 50 mN/m to 72.8 mN/m (surface tension of water) at 20°C, more preferably 60 mN/m to 72.8 mN/m, even more preferably 65 mN/m to 72.8 mN/m, and even more preferably 70 mN/m to 72.8 mN/m. If the aqueous solvent contains a surfactant, the surfactant is preferably a nonionic surfactant, and examples of nonionic surfactants include polysorbate 20 or polysorbate 80 (e.g., Tween series surfactants such as Tween 20 or Tween 80), NP-40 (nonylphenol ethoxylate), and octylphenol ethoxylate (e.g., Triton series surfactants such as Triton X-100).

From the viewpoint of making the properties of the aqueous solvent similar to those of pure water, the salt concentration in the aqueous solvent is preferably 0 mol/L to 0.2 mol/L, more preferably 0 mol/L to 0.1 mol/L, and even more preferably 0 mol/L to 0.05 mol/L.

The coexisting components do not need to be solvent components, and may be fine particles suspended in water, water-soluble substances, etc. The aqueous solvent may also contain a nucleic acid amplification reagent as a coexisting component. The nucleic acid amplification reagent is a reagent necessary for amplifying nucleic acids, including polymerase, nucleotide triphosphates (a mixture of dNTPs), primers, Mg ²⁺ ions, etc. It is preferable that the nucleic acid amplification reagent contains a polymerase having an activity (reverse transcription activity) capable of synthesizing a DNA chain using RNA as a template. The nucleic acid amplification reagent may contain a weak surfactant (particularly a nonionic surfactant) such as Triton X-100, but the concentration of the nucleic acid amplification reagent does not denature cells and destroy membranes, so the surfactant in the nucleic acid amplification reagent may be contained in the aqueous solvent as it is. For example, when the aqueous solvent contains Triton X-100, the content of Triton X-100 is preferably 0.01% by mass to 5% by mass, more preferably 0.05% by mass to 3% by mass, and even more preferably 0.1% by mass to 2% by mass, based on the total amount of the aqueous solvent, from the viewpoint of preventing denaturation of the cell membrane and efficient nucleic acid amplification. The nucleic acid amplification reagent may be, for example, a PCR reagent, a reagent for isothermal gene amplification, or the like. By including the nucleic acid amplification reagent in the aqueous solvent, it becomes possible to directly carry out nucleic acid amplification using the second liquid containing sRNA leaked from the cells without carrying out a reagent addition operation or a temperature cycle operation.
Although the use of the aqueous solvent has been described above mainly in cases where the organism has a cell wall, the aqueous solvent may also be used to extract sRNA from organisms that do not have a cell wall.

Examples of coexisting components other than the solvent in the aqueous solvent include a nonionic surfactant at 5.0% by mass or less based on the total amount of the aqueous solvent, an antimicrobial peptide at 1.0% by mass or less based on the total amount of the aqueous solvent, and a reducing agent at 1.0 M or less. A nonionic surfactant is a compound that has a hydrophilic group and a hydrophobic group in one molecule and does not have a group that dissociates into ions in water. The nonionic surfactant may have a hydroxy group, and may be, for example, a compound having a hydrophilic polyoxyalkylene (polyoxypropylene, polyoxyethylene, etc.) chain portion and a hydrophobic portion such as an alkyl, aryl, alkylene, or arylene. Alternatively, the nonionic surfactant may be a polyol ester type, an alkanolamide type, an alkyl glucoside, an alkoxylate type, or a fatty acid alkyl ester type. More specifically, it may be a polyoxyethylene alkyl ether type, a polyoxyethylene alkyl phenyl ether type, or the like. Specifically, the nonionic surfactant may include at least one selected from the group consisting of polysorbate 20 (e.g., Tween 20), polysorbate 80 (e.g., Tween 80), nonylphenol ethoxylate (NP-40), octylphenol ethoxylate (e.g., Triton X-100), and poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol) (e.g., Synperiinc F108).

The content of the nonionic surfactant in the aqueous solvent is 5.0% by mass or less relative to the total amount of the aqueous solvent from the viewpoint of reducing damage to the cell membrane of an organism having a cell wall. The content of the nonionic surfactant in the aqueous solvent may be 4.0% by mass or less, 3.0% by mass or less, 2.0% by mass or less, or 1.5% by mass or less relative to the total amount of the aqueous solvent.
The lower limit of the range of the content of the nonionic surfactant in the aqueous solvent is not particularly limited, and may be 0 mass% (non-content). The content of the nonionic surfactant in the aqueous solvent may be 0.01 mass% or more, 0.1 mass% or more, 0.3 mass% or more, 0.5 mass% or more, or 0.7 mass% or more. The upper limit and lower limit values may be arbitrarily combined to form a numerical range.

The antimicrobial peptide is not particularly limited as long as it is an antimicrobial peptide known in the art. The antimicrobial peptide is basically an amphipathic peptide having a hydrophilic face and a hydrophobic face, which allows it to exist in the cell membrane. Examples of antimicrobial peptides include anionic peptides such as maximin H5 and dermcidin; linear cationic α-helical peptides that do not contain cysteine residues, such as cecropin, andropin, moricin, ceratotoxin, melittin, magainin (magainin I and magainin II), dermaseptin, bombinin, brevinin-1, esculetin, buforin II, CAP18, and LL37; cationic peptides rich in one or more of proline, arginine, phenylalanine, and tryptophan, such as abaecin, apidaecin, prophenin, and indolicidin; and disulfide bond-containing brevinin, protegrin, tachyplesin, defensin, and drosomycin. One or more types of antimicrobial peptides known in the art can be selected and used as desired.

The content of the antimicrobial peptide in the aqueous solvent is 1.0% by mass or less based on the total amount of the aqueous solvent from the viewpoint of reducing damage to the cell membrane of an organism having a cell wall. The content of the antimicrobial peptide in the aqueous solvent may be 0.8% by mass or less, 0.5% by mass or less, 0.2% by mass or less, or 0.15% by mass or less based on the total amount of the aqueous solvent.
The lower limit of the range of the content of the antimicrobial peptide in the aqueous solvent is not particularly limited, and may be 0% by mass (non-content). The content of the antimicrobial peptide in the aqueous solvent may be 0.001% by mass or more, 0.005% by mass or more, 0.01% by mass or more, 0.05% by mass or more, or 0.07% by mass or more. The upper and lower limit values may be arbitrarily combined to form a numerical range.

The reducing agent is not particularly limited as long as it is a reducing agent known in the art. Examples of reducing agents include oxalic acid, formic acid, gallic acid, ascorbic acid, β-mercaptoethanol, and dithiothreitol. The reducing agent is preferably a reducing agent having a mercapto group, and may be, for example, β-mercaptoethanol or dithiothreitol. One or more types of reducing agents known in the art may be arbitrarily selected and used as the reducing agent.

From the viewpoint of reducing damage to the cell membrane of an organism having a cell wall, the concentration of the reducing agent in the aqueous solvent is 1.0 M or less. The concentration of the reducing agent in the aqueous solvent may be 0.8 M or less, 0.5 M or less, 0.2 M or less, or 0.15 M or less.
The lower limit of the range of the concentration of the reducing agent in the aqueous solvent is not particularly limited, and may be 0 M (not contained). The concentration of the reducing agent in the aqueous solvent may be 0.001 M or more, 0.005 M or more, 0.01 M or more, 0.05 M or more, or 0.07 M or more. The above upper and lower limit values may be arbitrarily combined to form a numerical range.

<Coexistence>
In the extraction method and the determination method according to the present disclosure, the organism and the carrier coated with a specific coating material may be coexisted in the first liquid at room temperature or with heating or cooling. In order to minimize denaturation of the cell membrane, the coexistence is preferably performed at a temperature within a range of 0°C to 50°C, more preferably at a temperature within a range of 4°C to 40°C, even more preferably at a temperature within a range of 10°C to 40°C, even more preferably at a temperature within a range of 20°C to 40°C, even more preferably at a temperature within a range of 25°C to 40°C, and even more preferably at a temperature within a range of 30°C to 37°C. The organism and the carrier coated with a specific coating material may be coexisted in the first liquid at room temperature. The organism and the carrier coated with a specific coating material may be coexisted in the first liquid under heating.
The time for which the organism and the carrier coated with the specific coating material are allowed to coexist in the first liquid is not particularly limited, as long as a sufficient amount of the organism is adsorbed to the carrier coated with the specific coating material. The time for which the organism and the carrier coated with the specific coating material are allowed to coexist in the first liquid is, for example, 10 seconds to 30 hours, may be 10 seconds to 10 hours, may be 1.0 minutes to 5.0 hours, or may be 5.0 minutes to 1.0 hours. The time for which the organism and the carrier coated with the specific coating material are allowed to coexist in the first liquid may alternatively be 0.50 hours to 6.0 hours, may be 0.70 hours to 4.5 hours, or may be 0.80 hours to 2.0 hours.

The method of coexisting the organism and the carrier coated with the specific coating material in the first liquid is not particularly limited, and may be any treatment in which the organism comes into contact with the carrier coated with the specific coating material. The coexistence may be achieved, for example, by coexisting the organism and the coated carrier in the first liquid stored in a container. In this case, the first liquid may be left stationary or stirred.
By causing the organism and a carrier coated with a specific coating material to coexist in a first liquid, adsorption of the organism to the carrier coated with the specific coating material occurs, and a sample containing the coated carrier with the organism adsorbed thereon is obtained in the first liquid.

<Soaking>
The immersion in the extraction method and the determination method according to the present disclosure may be performed at room temperature or with heating or cooling. From the viewpoint of not requiring equipment, it is preferable to perform the immersion at room temperature. From the viewpoint of minimizing denaturation of the cell membrane and simplifying the operation, it is preferable to perform the immersion at a temperature within the temperature range of 0°C to 50°C, more preferably at a temperature within the temperature range of 4°C to 40°C, even more preferably at a temperature within the temperature range of 10°C to 40°C, even more preferably at a temperature within the temperature range of 20°C to 40°C, even more preferably at a temperature within the temperature range of 25°C to 40°C, and even more preferably at a temperature within the temperature range of 30°C to 37°C. The immersion may be performed at room temperature. On the other hand, from the viewpoint of increasing the extraction rate of sRNA, the immersion may be performed under heating.
The immersion time is not particularly limited as long as a sufficient amount of sRNA is leaked outside the cell. The immersion time is, for example, 10 seconds to 30 hours, and may be 10 seconds to 10 hours, 1.0 minutes to 5.0 hours, or 5.0 minutes to 1.0 hours. Alternatively, the immersion time may be 0.50 hours to 6.0 hours, 0.70 hours to 4.5 hours, or 0.80 hours to 2.0 hours.

When immersion is performed under heating, the immersion temperature and time may be, for example, 10 seconds to 30 hours at a temperature within the range of 50°C to 100°C, 1.0 minute to 10 hours at a temperature within the range of 70°C to 100°C, 3.0 minutes to 1 hour at a temperature within the range of 80°C to 100°C, or 5.0 minutes at a temperature of 95°C.

The method of immersion is not particularly limited, and may be any treatment in which the coated carrier having the adsorbed organisms comes into contact with a second liquid. The immersion may be performed, for example, by immersing the coated carrier having the adsorbed organisms in the second liquid stored in a container.
By soaking, the sRNA is leaked out of the cells, and a sample containing the sRNA in the second liquid is obtained.

Surprisingly, the leakage of sRNA from cells with cell walls, as described in the "aqueous solvent" section above, is not a phenomenon that occurs with nucleic acids in general, but is a phenomenon that occurs specifically with sRNA. For this reason, even if a cell with a cell wall is immersed in an aqueous solvent in an attempt to leak 16S rRNA, which has traditionally been used to distinguish biological species, into an aqueous solvent, 16S rRNA does not leak from the cell with a cell wall at all, or leaks only in small amounts, if any. This difference is thought to be due in part to the difference in length between 16S rRNA (about 1600 bases) and sRNA. In other words, sRNA, which has a shorter chain length than mRNA and 16S rRNA, surprisingly leaks into the aqueous solvent outside the cell with a cell wall without destroying the cell membrane. On the other hand, larger molecules such as mRNA and 16S rRNA do not substantially exhibit such leakage, and cannot be taken out of the cell with a cell wall unless an extraction operation involving denaturation of the cell membrane is performed.

<sRNA>
In the extraction method and the determination method according to the present disclosure, sRNA is also called small RNA and means short-chain RNA contained in cells. The specific chain length of sRNA is preferably 5 to 500 bases, more preferably 8 to 500 bases, even more preferably 10 to 200 bases, still more preferably 12 to 100 bases, and particularly preferably 15 to 30 bases.
In addition, sRNA includes microRNA, which is an RNA of about 20 to 30 bases contained in eukaryotic cells. In prokaryotes, particularly short sRNA of a similar length is sometimes called microRNA-size small RNA.

The sRNA in the extraction method and the determination method according to the present disclosure is preferably present in the organism from the viewpoint of extracting or detecting the presence of sRNA released extracellularly by the organism. In other words, the sRNA is preferably contained within the cells of the organism.

Research into sRNAs present in living organisms is ongoing, and sequence information for sRNAs found in each organism is stored in databases such as Rfam (EMBL EBI), Small RNA Database (MD Anderson Cancer Center), miRBase (Griffiths-Jones lab at the Faculty of Biology, Medicine and Health, University of Manchester), and the National Center for Biotechnology Information (NCBI) database, and has also been reported in various academic papers. For this reason, information on sRNAs contained in various organisms can be obtained by known methods, including database searches (see Kyorin Medical Journal, Vol. 41, No. 1, pp. 13-18, April 2010). For example, the nucleic acid sequence contained in the National Center for Biotechnology Information (NCBI) database and the nucleic acid sequence to be searched can be compared and searched for identical sequences (including information on the organism from which the identical sequence originates) using Nucleotide BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi).

sRNAs are differentially expressed depending on the type of organism. For example, by referring to sequence databases such as Rfam, Small RNA Database, miRBase, and the National Center for Biotechnology Information (NCBI) database, it is possible to search for which organism or organisms a particular sRNA is expressed in, and also to search for which organism or organisms a particular sRNA is expressed in.

In the extraction method and the determination method disclosed herein, the sRNA may be one or more types of sRNA.

In the method of the present disclosure, the sRNA is preferably one that exhibits a differential expression state in a particular organism of interest, so that the state of the organism can be better determined from the state of the sRNA.
Here, the term "differential expression state" does not necessarily refer to an sRNA that is differentially expressed only in the specific organism, but also refers to a concept that includes an sRNA that is not completely differential only to the specific organism, such as an sRNA that is differentially expressed only in a specific group of organisms including the specific organism.

In the extraction method according to the present disclosure, it is preferable that the sRNA to be extracted exhibits differential expression states depending on the organism.

As described above, when the second liquid is an aqueous solvent and the organism has a cell wall, the extraction method and determination method disclosed herein can cause sRNA to leak into the second liquid outside the cell having a cell wall without denaturing the cell membrane with strong alkali treatment, guanidine treatment, phenol, monovalent or divalent straight-chain, branched-chain or alicyclic alcohol other than C2-C8 alcohol, ionic surfactant, or the like. This eliminates the need to use a denaturant and the time required for treatment with a denaturant, and the second liquid from which sRNA has leaked can be subjected to subsequent treatment (e.g., nucleic acid amplification treatment for sRNA detection) as is.

<State of Existence>
In the determination method according to the present disclosure, the "state of existence" may simply refer to the presence or absence, or may refer to the amount of existence. In other words, "detecting the state of existence" may be obtaining binary information of existence, or may be obtaining information on the amount of existence in addition to the information on existence. The information on the amount of existence also contains information on the presence or absence. Similarly, "determining the state of existence" may be performing a binary determination of existence, or may be performing a determination of the amount of existence in addition to the information on existence. Here, the amount of existence is not limited to the absolute amount of existence, and may be a relative amount of existence with respect to a comparison target such as a negative control or a positive control.

<Detection of Presence State>
In the determination method according to the present disclosure, the method for detecting the state of existence of sRNA is not particularly limited. Methods for detecting the state of existence of sRNA include hybridization with a labeled nucleic acid probe (including Northern blotting) and nucleic acid amplification. Nucleic acid amplification may be any method that amplifies DNA or RNA, and examples thereof include the Polymerase Chain Reaction (PCR) method, the Reverse Transcription-PCR (RT-PCR) method, the Ligase Chain Reaction (LCR) method, the Strand Displacement Amplification (SDA) method, the Nucleic Acid Sequence-based Amplification (NASBA) method, the Transcription Reverse-transcription Concerted Reaction (TRC) method, and the Loop-mediated Amplification (LAMP) method. Isothermal Amplification) method, RT-LAMP (Reverse Transcription-LAMP) method, ICAN (Isothermal and Chimeric Primer-initiated) Amplification of Nucleic Acids) method, RCA (Rolling Cycle Amplification) method, Smart Amp (Smart Amplification) method process) method, TMA (transcription-mediated amplification) method, TAS (transcription amplification) method Examples of amplification methods include the Self-sustained Sequence Replication System (3SR) method and the like. In the case of a method that cannot directly amplify RNA, the sRNA may be first reverse transcribed with a reverse transcriptase to convert it to DNA, and then nucleic acid amplification may be performed. In an embodiment, the presence of the sRNA is detected by isothermal gene amplification or PCR.

The nucleic acid amplification used to detect the presence of sRNA is preferably an isothermal gene amplification method, LAMP method or RCA method, in that it does not require equipment for temperature cycling. As the RCA method, in particular, the SATIC (termed signal amplification by ternary initiation complexes) method can be mentioned. Isothermal gene amplification is preferable in that it does not require the preparation of an equipment for amplification (an equipment that changes the temperature according to the temperature cycle). Isothermal gene amplification can be performed at a temperature within a range of, for example, 10°C to 40°C, so it can be performed at room temperature. In this disclosure, room temperature may be a temperature within a range of 20°C to 40°C, except as otherwise specified in the examples, etc.
In addition, the above-mentioned PCR may be qPCR (quantitative PCR), and the qPCR may be real-time PCR. As the real-time PCR, in particular, a method using Taqman (registered trademark) miRNA assay (manufactured by Thermo Scientific) can be mentioned. By using qPCR, it becomes easy to quantitatively analyze the state of existence of sRNA.

Reagents such as probes or primers used for detecting sRNA (hereinafter also referred to as sRNA detection reagents) may be added to the second liquid after immersion is complete, or may be pre-contained in the second liquid before immersion. If the sRNA detection reagent is pre-contained in the second liquid before immersion, this is preferable in that no addition operation is required after immersion is complete. In addition, in the case of an aqueous solvent sample, when the aqueous solvent is used as the second liquid without solvent exchange, the sRNA detection reagent may be added to the second liquid, or, if there is a process for preparing the second liquid, the sRNA detection reagent may be added during the preparation process.

The amplified nucleic acid can be detected by using existing amplified nucleic acid detection methods such as attaching a fluorescent dye to a primer, visually checking a precipitate, using a nucleic acid staining dye, hybridizing with a fluorescent probe, or subjecting to nucleic acid chromatography. The fluorescent probe may be arranged on a microarray. By using a microarray, the presence information of multiple types of sRNA can be obtained at once.
For example, by using a Taqman (registered trademark) probe, SYBR Green dye, or the like, the amount of nucleic acid amplification can be measured by a fluorescent signal, and the state of sRNA can be known based on that information; this method is particularly effective in qPCR.

As described above, nucleic acid amplification is not essential for detecting sRNA, and sRNA may be directly detected by hybridizing a fluorescent probe with sRNA without nucleic acid amplification.
In the above-mentioned detection procedures such as nucleic acid amplification or hybridization with a probe, the entire length of the sRNA may be amplified and/or a probe that hybridizes with the entire length of the sRNA may be used. However, it is not essential to amplify the entire length of the sRNA and/or to use a probe that hybridizes with the entire length of the sRNA. Nucleic acid amplification or hybridization may be performed on a partial region of the sRNA, for example, about 10 to 30 bases in the 3'-terminal region of the sRNA.

The detection of the state of the sRNA may be performed simultaneously with the extraction of the sRNA from the cells. For example, the coated carrier with the adsorbed organism may be directly immersed or added to the reaction solution of an isothermal gene amplification method such as the PCR method or the SATIC method, so that leakage and detection of the sRNA can be performed simultaneously. In the case of an isothermal gene amplification method, since there is no temperature cycle, nucleic acid amplification can be started at any time as long as the reagents necessary for nucleic acid amplification are present in the second liquid.

Detecting the state of existence of the sRNA according to the present disclosure may also include detecting the amount of the sRNA present.

<Determination of the state of existence of living organisms>
Detection of sRNA differential to an organism in a sample indicates the presence of that organism in the sample. Information on the abundance of sRNA differential to an organism in a sample can also provide information about the abundance of that organism in the sample.

Among the methods for detecting living organisms, the culture method is highly sensitive and reliable. However, there are problems with the culture method. For example, it takes a long time to obtain results because it takes one to several days to culture, it is difficult to apply to bacteria that are difficult to culture, special techniques are required to identify bacteria after culture, and sterilization procedures such as autoclaving are required to dispose of cultured bacteria, which is bulky, resulting in waste disposal that is time-consuming and costly.

In addition, antibody methods, such as immunochromatography and latex agglutination, have the advantage of being easy to operate, since microorganisms can be detected simply by contacting the sample. However, immunochromatography and latex agglutination have the problem that they have low detection sensitivity and cannot detect microorganisms unless a high concentration of microbial cells is present. The ELISA method is a more sensitive method than immunochromatography, but is time-consuming because it requires washing and coloring procedures.

Unlike the culture method, the method for determining the presence of an organism based on the detection of sRNA does not require culturing organisms that may be contained in the measurement sample. This makes it possible to determine or identify the organism in a short period of time.

It can be known in which organisms the sRNA is expressed, for example, as follows. Using the nucleic acid sequence of the sRNA as a reference sequence, nucleic acid sequences similar to the reference sequence are compared using Nucleotide BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi) from the database of the National Center for Biotechnology Information (NCBI). From the comparison results obtained, an organism containing an sRNA whose nucleic acid sequence is 100% identical to the sRNA used as the reference sequence is determined to be an organism that expresses the sRNA.

Before detecting the presence of sRNA, the type of sRNA extracted outside the cell is unknown, but if a specific sRNA is detected outside the cell, it can be determined that an organism known to have that specific sRNA is present.

The organism in the determination method according to the present disclosure refers to an organism to be detected, and may be any organism to be detected. The existence state of the organism can be determined by detecting the existence state of sRNA that is differentially expressed in the organism compared to other organisms. Detection of sRNA that is differentially expressed in the organism suggests the presence of the organism, and non-detection of the sRNA suggests the absence of the organism. In the determination method according to the present disclosure, it is preferable to select an sRNA that shows a differential expression state in the organism as the sRNA for detecting the existence state according to the type of organism whose existence state is to be detected in the sample. The differential expression state of the sRNA may be differentially expressed in the organism whose existence state is to be detected, or may be differentially expressed in the organism whose existence state is to be detected. However, it is preferable that at least one of the one or more types of sRNA whose existence state is to be detected is an sRNA that is differentially expressed in the organism whose existence state is to be detected.

Note that one type of sRNA is not necessarily completely differential to one type of organism, and there may be multiple types of organisms expressing the same sRNA. However, since the expression profile of sRNA in one type of organism is different for each sRNA, the types of organisms that may exist can be further narrowed down based on the existence states of each of the multiple types of sRNA. For this reason, in the determination method according to the present disclosure, the existence state of an organism may be determined based on the existence states of each of the multiple types of sRNA. In this determination, the expression state of sRNA that is differentially less expressed in the organism can also be used. The detection or non-detection of sRNA that is differentially less expressed in the organism does not alone suggest the existence state of the organism, but by combining it with information about the existence state of sRNA that is differentially expressed in the organism, it is possible to further narrow down candidates for organisms that show the expression profile of those sRNAs.

As the sRNA for detecting the presence state in the determination method according to the present disclosure, it is preferable to select an sRNA that can better distinguish a particular organism of interest from other organisms based on its expression profile.

However, it is not essential in the determination method according to the present disclosure to limit the number of candidates for the organisms present to one type, and it is sufficient to simply identify a group of candidates consisting of multiple organisms. For this reason, the determination method according to the present disclosure may determine the state of existence of multiple organisms, in which case the determination method does not determine the state of existence of each of the multiple organisms individually, but determines the state of existence of the multiple organisms as a whole. In other words, even if the determination method cannot determine which of the multiple organisms actually exists, it is sufficient if it can determine that at least one of the multiple organisms constituting the candidate group (group) is present.

In other words, in the determination method according to the present disclosure, determining the state of existence of organisms may include determining the overall state of existence of two or more organisms. When the determination method according to the present disclosure includes determining the overall state of existence of two or more organisms, determining the overall state of existence of two or more organisms may include determining whether none of the two or more organisms are present, or whether at least one of the two or more organisms is present, and when it is determined that at least one of the two or more organisms is present, may further include determining the abundance of at least one of the two or more organisms.

In this case, the presence state of one organism of interest among two or more organisms cannot be completely determined, but the possibility of the presence of the organism of interest can be determined, and further testing may be performed based on this determination result, or the object from which the sample was obtained may be cleaned based on this determination result. In other words, determining the overall presence state of two or more organisms may include determining the possibility of the presence of a particular organism included in the two or more organisms, and may further include determining the estimated abundance of the particular organism.

When detecting the state of sRNA, the amount of sRNA present can also be measured, making it possible to determine the amount of the organisms present. The amount of sRNA present can be detected by methods commonly used in the industry, such as measuring the amount of fluorescence emitted from a fluorescently labeled probe hybridized to the sRNA, using a fluorescently labeled primer during nucleic acid amplification from the sRNA and measuring the amount of fluorescence emitted from the primer, or using qPCR.

Identification of organisms present in a sample
The present disclosure also provides an identification method for identifying one or more organisms having an sRNA expression profile that matches the sRNA state from the sRNA state. The sRNA is preferably an sRNA that exhibits a differential expression state depending on the organism. Detecting the sRNA state in the second liquid may include comprehensively detecting the sRNA present in the second liquid, but may also include measuring the state of one or more sRNAs selected in advance in consideration of the type of organism that may be present in the sample.

A specific state of existence of one type of sRNA is not necessarily completely different from the sRNA expression profile of one type of organism, and there may be multiple organisms that have sRNA expression profiles that match a specific state of existence of sRNA. However, since the sRNA expression profile of each organism is different for each sRNA, the types of organisms present can be further narrowed down based on information on the state of existence of each of the multiple types of sRNA. For this reason, in the identification method, organisms present in a sample may be identified based on the state of existence of each of the multiple types of sRNA. However, it is not essential for the identification method to narrow down the candidates for the existing organism species to one type, and it is sufficient to simply identify a group of candidates consisting of multiple types of organisms.

For this reason, the identification method may identify multiple species of organisms, in which case the identification method may not detect the presence of each of the multiple species individually, but may detect the presence of the multiple species as a whole. In other words, it is sufficient for the identification method to detect the presence of at least one of the multiple species, even if it cannot detect which of the multiple species are actually present.

That is, in the identification method, identifying the organisms present in the sample may include identifying one or more of two or more candidate organisms as being present. When at least one of the two or more organisms is identified as being present, it may further include identifying the abundance of at least one of the two or more candidate organisms. That is, in the identification method, when identifying the organisms present in the sample includes identifying one or more of the two or more candidate organisms as being present, it is not possible to accurately identify which of the two or more candidate organisms is present, but further testing may be performed based on this identification result, or the object from which the sample was obtained may be cleaned (appropriate cleaning according to the type of candidate organism) based on this identification result.

That is, identifying one or more of two or more candidate organisms as being present may include identifying a specific organism among the two or more candidate organisms as an organism that may be present, and may further include identifying the estimated abundance of the specific organism.

When detecting the state of sRNA, the amount of sRNA present can also be measured, making it possible to identify the amount of the organisms present. This is because the amount of sRNA present in the second liquid is thought to reflect the amount of organisms expressing the sRNA (in the simplest case, proportional to the amount of organisms expressing the sRNA). The amount of sRNA present can be detected by methods commonly used in the industry, such as measuring the amount of fluorescence emitted from a fluorescently labeled probe hybridized to the sRNA, using a fluorescently labeled primer during nucleic acid amplification from the sRNA, or using qPCR (e.g., determining the Ct value in real-time PCR).

An example of identification of an organism based on the presence state of one or more types of sRNA in the second liquid in an identification method that may be included in the determination method according to the present disclosure will be described below. According to a Nucleotide BLAST search, the sRNA EC-5p-36 (SEQ ID NO: 1; 5'-UGUGGGCACUCGAAGAUACGGAU-3', see Curr Microbiol. 2013 Nov; 67(5):609-13) is commonly expressed in bacteria of the genera Escherichia, Shigella, Salmonella, and Citrobacter. From this, when EC-5p-36 is detected to be present in the second liquid, the present organisms can be identified as one or more of Escherichia, Shigella, Salmonella, and Citrobacter. Furthermore, according to a Nucleotide BLAST search, EC-3p-40, which is an sRNA, is commonly expressed in Klebsilia bacteria in addition to Shigella, Salmonella, Escherichia, and Citrobacter bacteria. Therefore, when EC-3p-40 (SEQ ID NO: 2; 5'-GUUGUGAGGUUAAGCGACU-3') is detected to be present in the second liquid, the organisms present can be identified as one or more of Escherichia, Shigella, Salmonella, Citrobacter, and Klebsilla bacteria. Identification may also be performed by combining these results. For example, since EC-5p-36 is not expressed in Klebsilla bacteria, but EC-3p-40 is expressed in Klebsilla bacteria, when EC-3p-40 but not EC-5p-36 is detected to be present in the second liquid, the organisms present can be identified as Klebsilla bacteria.

Organisms may also be identified based on the presence of sRNA with a specific function. For example, 24B_1 (SEQ ID NO: 8; 5'-UAACGUUAAGUUGACUCGGG-3', see Scientific Reports volume 5, Article number: 10080 (2015)), an sRNA involved in the toxin (verotoxin, also called ciguatoxin) of enterohemorrhagic Escherichia coli (O-157, etc.), is commonly expressed in verotoxin (ciguatoxin)-producing bacteria according to a Nucleotide Blast search. Therefore, if the presence of 24B_1 is detected in the second liquid, the organism can be identified as a verotoxin-carrying bacterium.

In addition, in the determination method according to the present disclosure, when it is desired to determine the overall presence state of bacteria of the genus Escherichia, Shigella, Salmonella, and Citrobacter (whether none of them are present, or whether one or more of them are present, etc.), it is sufficient to detect the presence state of EC-5p-36. In the determination method according to the present disclosure, when it is desired to determine the presence state of bacteria of the genus Klebsilla, it is sufficient to detect the presence states of EC-3p-40 and EC-5p-36.

In addition, the sRNAs EC-5p-79 (SEQ ID NO: 3; 5'-UUUGCUCUUUAAAAAAUC-3') and EC-3p-393 (SEQ ID NO: 4; 5'-CUCGAAGAUACGGAUUCUUAAC-3') are expressed in Escherichia coli, Citrobacter freundii, and Salmonella gallinarum. From the above, the correspondence between sRNA and biological species is as follows: a combination of at least one selected from the group consisting of EC-5p-36, EC-3p-40, EC-5p-79, and EC-3p-393 with at least one selected from the group consisting of Escherichia coli, Citrobacter freundii, and Salmonella gallinarum; and a combination of fox_milRNA_5 having a nucleotide sequence represented by SEQ ID NO: 5 (SEQ ID NO: 5; 5'-UCCGGUAUGGUGUAGUGGC-3', see PLoS One. 2014 Aug 20; 9(8): e104956.) with Fusarium. oxysporum; miR156 having the nucleotide sequence represented by SEQ ID NO:6 (SEQ ID NO:6; 5'-CAGAAGAUAGAGAGCACAUC-3'; see http://www.mirbase.org/; pta-miR156a) and Pinus thunbergii; and miR716b having the nucleotide sequence represented by SEQ ID NO:7 (SEQ ID NO:7; 5'-GAGAUCUUGGUGGUAGUAGCAAAUA-3'; see Sci. Rep., 2015,5,7763) and Saccharomyces cerevisiae.

In addition, the sRNAs listed in Tables 1 and 2 below have been found to be expressed in bacteria of the Enterobacteriaceae family, such as Escherichia coli. In particular, sRNA13 to sRNA32 are tRNA sequences. The sRNA extracted by the extraction method according to the present disclosure may be selected from these sRNAs. Furthermore, in the extraction method according to the present disclosure and the determination method according to the present disclosure, the amount of sRNA selected from these sRNAs may be measured.

In addition, for sRNA9, 10, 13-15, 17-19, 21-24, 26, and 27, the organisms in which their expression has been confirmed are shown by genus name (species name for the genus Candida) in Tables 3 and 4 below. In Tables 3 and 4, "Yes" indicates that the sRNA is expressed in that organism.

The sRNAs listed in Tables 1 and 2 can also be extracted in the extraction method according to the present disclosure, and can be used in the extraction method according to the present disclosure and the determination method according to the present disclosure. The sRNA extracted in the extraction method according to the present disclosure preferably contains at least one type of sRNA represented by SEQ ID NOs: 1 to 48, and more preferably contains at least one type of sRNA represented by SEQ ID NOs: 1 to 8, 13 to 15, and 34. In the extraction method according to the present disclosure and the determination method according to the present disclosure, the sRNA preferably contains at least one type of sRNA represented by SEQ ID NOs: 1 to 48, and more preferably contains at least one type of sRNA represented by SEQ ID NOs: 1 to 8, 13 to 15, and 34, and even more preferably contains at least one type of sRNA represented by SEQ ID NOs: 1 to 8.

For example, the sRNA to be extracted may include at least one type of sRNA represented by SEQ ID NOs: 9 to 48 (i.e., at least one type of sRNA9 to sRNA48).

For example, the sRNA to be extracted may include at least one type of sRNA represented by SEQ ID NOs: 9 to 48 (preferably at least one type of sRNA represented by SEQ ID NOs: 13 to 15 and 34) and at least one type of sRNA represented by SEQ ID NOs: 1 to 8.

The sRNA to be detected is preferably at least one selected from the group consisting of EC-5p-36, EC-3p-40, EC-5p-79, EC-3p-393, fox_milRNA_5, miR156, and miR716b, which are used in the tests described in the Examples.

Although several examples of sRNAs have been mentioned above, the extraction method and determination method according to the present disclosure can be similarly performed when detecting other sRNAs. The correspondence between the sRNAs required for extraction or determination and the organisms can be easily obtained from the above-mentioned database.

According to the extraction method of the present disclosure, sRNA can be extracted from organisms more efficiently than when organisms are adsorbed onto a carrier that is not coated with a specific coating material. Furthermore, according to the determination method of the present disclosure, unlike the culture method, determination can be made in a short time, and high sensitivity can be achieved by using nucleic acid amplification or the like. Furthermore, the extraction method of the present disclosure and the determination method of the present disclosure can be applied to bacteria that are difficult to culture. Furthermore, unlike the culture method, the determination method of the present disclosure allows for rapid determination, requires a small amount of sample, and is easy to dispose of materials used in processing. The determination method of the present disclosure maintains the advantages of the genetic method, while still allowing the determination of the state of existence of organisms to be made more quickly and with simpler operations than conventional genetic methods.

The extraction method according to the present disclosure can be used, for example, for the research of sRNA possessed by organisms and the production of sRNA possessed by organisms. Examples of such research include basic research of species-specific sRNA, the production of sRNA as a nucleic acid drug, and the production of sRNA as a detection agent. Furthermore, the extraction method according to the present disclosure can also be used in the determination method according to the present disclosure.
The determination method according to the present disclosure can be used, for example, for rapid and simple microbial testing in sites where hygiene control is required (food production, medical care, welfare, home, etc.). Examples include hygiene control of products by testing for spoilage bacteria in the food production process, prompt identification of the cause of a food poisoning accident, prevention of secondary infection by detecting food poisoning bacteria in beds and waiting rooms of medical facilities, prevention of secondary infection by food poisoning bacteria in child welfare facilities and elderly welfare facilities, and prevention of secondary infection by detecting food poisoning bacteria when there is a food poisoning patient in the home.

The following examples further illustrate the embodiments, but the present disclosure is not limited to these examples. In addition, the percentages indicating the amounts of components contained in the compositions of the examples are based on mass unless otherwise specified. In the following examples, "room temperature" was approximately 30°C.

<Method for quantification of sRNA by real-time PCR method>
In Examples 1 to 24, Comparative Examples 1 to 5, and Reference Examples 1 to 13, sRNA to be extracted or the presence state to be detected was quantified by the method described below.
The sRNA in the reaction solution in each of Examples 1 to 24, Comparative Examples 1 to 5, and Reference Examples 1 to 13 was quantified by real-time PCR. A quantitative reagent (Taqman (registered trademark) microRNA Assays, Applied Biosystems) and reverse transcriptase (Taqman (registered trademark) microRNA RT kit, Applied Biosystems) were custom-synthesized according to the sRNA to be quantified, and a reverse transcription reaction solution containing DNA formed by reverse transcription was obtained according to the manufacturer's instructions. Specifically, in the reverse transcription reaction, 1 μL of RNA sample, 1.5 μL of 10× RT buffer, 0.15 μL of dNTP mix, 0.19 μL of RNase inhibitor, a first solution of custom-synthesized Taqman assays (e.g., 0.75 μL for 20× Taqman assays) with a final concentration of 1×, 1 μL of Multiscribe RT enzyme, and the volume of the solution was adjusted to 15 μL with pure water. The temperature profile of the reverse transcription reaction included steps consisting of (1) 30 minutes at 16°C, followed by (2) 30 minutes at 42°C, and (3) 5 minutes at 85°C.

Next, real-time PCR reaction was carried out using the obtained reverse transcription reaction solution. A quantification reagent (Taqman (registered trademark), microRNA Assays, Applied Biosystems) custom-synthesized according to the sRNA to be quantified, and a master mix for real-time PCR (TaqMan (registered trademark) Universal PCR Master Mix II with UNG, Applied Biosystems) were used to prepare a reaction solution according to the manufacturer's instructions. Specifically, 2 μL of reverse transcription reaction solution, 10 μL of Universal PCR Master Mix II with UNG, and a custom-synthesized Taqman assay (e.g., 20× Taqman) in an amount to give a final concentration of 1× were used. The reaction solution was prepared by using 1 μL of the second solution (1 μL for Taqman assays) and adjusting the volume of the solution to 20 μL with pure water. The prepared reaction solution was subjected to real-time PCR using a StepOnePlus (registered trademark) real-time PCR system (manufactured by Applied Biosystems) with a temperature profile including a step of 10 minutes at 95°C, followed by 40 cycles of 15 seconds at 95°C and 1 minute at 60°C. At that time, a StepOnePlus (registered trademark) real-time PCR system (manufactured by Applied Biosystems) was used, and reagents = "Taqman reagents", ramp speed Ct values were calculated by automatic calculation using StepOnePlus software (default settings except for the above settings) under the condition of "Standard". When quantifying by comparison with a standard, the sRNA sequence to be quantified was synthesized (RNA primer synthesis by Eurofins Genomics, HPLC purification grade), and a dilution series was prepared in the range of 10 ^-10 M to 10 ^-15 M. Real-time PCR was also performed on the dilution series in parallel with the measurement of the sample, and the amount of sRNA was quantified by comparing the Ct values.

Example 1
-Coating of carrier with nonionic surfactant (Tween 20)-
50 mg of coconut shell activated carbon (10-32 mesh; manufactured by Nacalai Tesque) was weighed out into a 1.5 mL tube. 100 μL of a 2% solution of Tween 20 (manufactured by Nacalai Tesque) (stock concentration of Tween 20 is 2%) was added to the weighed coconut shell activated carbon, and the mixture was allowed to stand at room temperature for 10 minutes or more. The coconut shell activated carbon that had been allowed to stand was then centrifuged at 14,000 rpm (revolutions per minute) for 2 minutes, the supernatant was removed, 300 μL of sterilized water was added for washing, and the mixture was centrifuged at 14,000 rpm for 2 minutes, and the supernatant was removed to obtain water-washed activated carbon coated with Tween 20.

-Biology-
Escherichia coli W3110 (hereinafter simply referred to as "E. coli W3110"), an organism having a cell wall, was used.
E. coli W3110 was applied to an LB plate and cultured, and the colonies were suspended in 2 mL of LB medium and cultured at 37 ° C. and 180 rpm for 24 hours with shaking. Then, 40 μL of the culture solution was collected and inoculated into 4 mL of LB medium, and cultured at 37 ° C. and 180 rpm for 5 hours with shaking. Then, the culture solution was centrifuged at 3000 g (centrifugal acceleration) for 3 minutes, the supernatant was aspirated, and sterile water was added to obtain an E. coli suspension. After suspension, OD600 (optical density (OD) of the sample measured at a wavelength of 600 nm) was measured, and the concentration of the E. coli suspension was adjusted with sterile water so that OD600 = 1.0.

- Adsorption of E. coli W3110 and extraction of sRNA -
To the water-washed activated carbon coated with the nonionic surfactant (Tween 20), 30 μL of a suspension of E. coli W3110 adjusted to an OD600 of 1.0 was added, and the mixture was left to stand at room temperature for 10 minutes to adsorb E. coli W3110 to the coated activated carbon. Next, the suspension containing the activated carbon adsorbed with E. coli W3110 was spun down to remove the supernatant, and then 1 mL of sterilized water was added, and the mixture was spun down again to remove the supernatant, thereby obtaining water-washed activated carbon adsorbed with E. coli W3110.
50 μL of sterilized water containing 1% Triton X-100 was added to the water-washed activated carbon to which the E. coli W3110 had been adsorbed, and the mixture was allowed to stand at 37° C. for 1 hour to extract a sample containing sRNA from the E. coli W3110.

-Quantification of sRNA-
An attempt was made to quantify sRNA from E. coli W3110, focusing on EC-5p-36, a type of sRNA contained in E. coli W3110.
The samples were quantified for EC-5p-36 by real-time PCR. Specifically, after spinning down, 1 μL of the supernatant was collected and real-time PCR was carried out according to the above-mentioned <Method for quantification of sRNA by real-time PCR method>. The primer used was a Taqman® Assay primer having a nucleotide sequence corresponding to EC-5p-36. The results of the Ct values obtained using a StepOnePlus® Real-Time PCR System (Applied Biosystems) are shown in Table 5.

(Examples 2 to 16 and Comparative Examples 1 to 3)
The Ct value was determined in the same manner as in Example 1 (Table 5), except that the type of coating material and the stock concentration of the coating material were changed as shown below and in Table 5. In Table 5, the stock concentration refers to the concentration of the coating material solution used to coat the carrier. In Comparative Example 3, activated carbon was used as is, that is, uncoated activated carbon was used.

Details of the coating materials used are as follows:
Polysorbate 20 (manufactured by Nacalai Tesque, product name: Tween 20)
Polysorbate 80 (manufactured by Nacalai Tesque, product name: Tween 80)
Octylphenol ethoxylate (manufactured by Nacalai Tesque, product name: Triton X-100)
Benzalkonium chloride solution (manufactured by Wako Pure Chemical Industries, Ltd., product name: Osban)
Cetyltrimethylammonium bromide (manufactured by Wako Pure Chemical Industries, Ltd., abbreviated as CTAB)
Sodium dodecyl sulfate (Wako Pure Chemical Industries, Ltd., abbreviated as SDS)
Sodium 1-octanesulfonate (Wako Pure Chemical Industries, Ltd.)
Glycine (Wako Pure Chemical Industries, Ltd.)
Skim milk powder (Wako Pure Chemical Industries, product name: Skim Milk)
Bovine serum albumin (manufactured by Wako Pure Chemical Industries, Ltd., abbreviated as BSA)
Polyethylene glycol 6000 (manufactured by Nacalai Tesque, abbreviated name: PEG-6000)
Polyethylene glycol 4000 (manufactured by Nacalai Tesque, abbreviation: PEG-4000)

The Ct value was 36.25 when the carrier was not coated with a coating material (Comparative Example 3). Therefore, when the Ct value is smaller than 36.25, it can be said that the sRNA extraction efficiency was improved when the carrier was coated with the coating material. On the other hand, when the Ct value is larger than 36.25 or is equal to or greater than the measurement limit, it can be said that the sRNA extraction efficiency was reduced when the carrier was coated with the coating material. The Ct value being equal to or greater than the measurement limit means that the Ct value was too large to obtain a Ct value, and that almost no sRNA was extracted from the organism.
When E. coli was not added, the Ct value was above the measurement limit, and therefore samples for which Ct values were obtained in the table were able to rapidly determine the presence or absence of organisms.

From the results of Examples 1 to 16, when the carrier was coated with a specific coating material, for example, a nonionic surfactant, a cationic surfactant, an anionic surfactant, or a protein, the extraction efficiency of sRNA was improved compared to when the carrier was not coated with a coating material. On the other hand, from the results of Comparative Examples 1 and 2, when the carrier was coated with a hydrophilic polymer, the extraction efficiency of sRNA was lower compared to when the carrier was not coated with a coating material or was coated with the specific coating material.
Furthermore, since no change in Ct value was observed depending on the stock concentration of the coating material, it was found that the activated carbon was sufficiently coated in all of the examples.
In addition, in Examples 1 to 16, since a Ct value distinguishable from noise was obtained, it was possible to determine that each sample contained E. coli.

(Example 17)
-Coating of carrier with nonionic surfactant (Tween 20)-
100 mg of coconut shell activated carbon (10-32 mesh; manufactured by Nacalai Tesque) was weighed out into a 1.5 mL tube. 1 mL of sterilized water was added to the weighed coconut shell activated carbon, and then 10 μL was dispensed into separate tubes to obtain a coconut shell activated carbon suspension. 100 μL of a 2% solution of Tween 20 (stock concentration of Tween 20 is 2%) was added to the dispensed 10 μL of coconut shell activated carbon suspension, and the suspension was allowed to stand at 4° C. for 10 minutes. The coconut shell activated carbon that had been allowed to stand was then centrifuged at 14,000 rpm (revolutions per minute) for 2 minutes, the supernatant was removed, 300 μL of sterilized water was added to wash the suspension, and the suspension was centrifuged at 14,000 rpm for 2 minutes, and the supernatant was removed to obtain a water-washed activated carbon coated with Tween 20.

-Biology-
Escherichia coli W3110 (hereinafter simply referred to as "E. coli W3110"), an organism having a cell wall, was used.
E. coli W3110 was applied to an LB plate and cultured, and the colonies were suspended in 2 mL of LB medium and cultured at 37 ° C. and 180 rpm for 24 hours with shaking. Then, 40 μL of the culture solution was collected and inoculated into 4 mL of LB medium, and cultured at 37 ° C. and 180 rpm for 5 hours with shaking. Then, the culture solution was centrifuged at 3000 g (centrifugal acceleration) for 3 minutes, the supernatant was aspirated, and sterile water was added to obtain an E. coli suspension. After suspension, OD600 (optical density (OD) of the sample measured at a wavelength of 600 nm) was measured, and the concentration of the E. coli suspension was adjusted with sterile water so that OD600 = 1.0.

- Adsorption of E. coli W3110 and extraction of sRNA -
To the water-washed activated carbon coated with the nonionic surfactant (Tween 20), 30 μL of a suspension of E. coli W3110, the concentration of which was adjusted to OD600 = 1.0, was added, and the mixture was allowed to stand for 10 minutes at 4°C, thereby allowing E. coli W3110 to be adsorbed onto the coated activated carbon. Next, the solution containing the activated carbon to which E. coli W3110 had been adsorbed was centrifuged at 500 rpm for 3 minutes to remove the supernatant, and then 30 μL of sterilized water was added.
The suspension containing the activated carbon to which the E. coli W3110 had been adsorbed was heat-treated at 95° C. for 5 minutes to extract a sample containing sRNA from the E. coli W3110.

-Quantification of sRNA-
An attempt was made to quantify sRNA from E. coli W3110, focusing on EC-5p-36, a type of sRNA contained in E. coli W3110.
The samples were quantified for EC-5p-36 by real-time PCR. Specifically, after spinning down, 1 μL of the supernatant was collected and real-time PCR was carried out according to the above-mentioned <Method for quantification of sRNA by real-time PCR method>. The primers used were Taqman® Assay primers having a nucleotide sequence corresponding to EC-5p-36. The results of Ct values obtained using a StepOnePlus® Real-Time PCR System (manufactured by Applied Biosystems) are shown in Table 6.

(Examples 18 to 24 and Comparative Examples 4 to 5)
In Examples 18 to 24 and Comparative Examples 4 and 5, the Ct values were determined in the same manner as in Example 17 (Table 6), except that the type of coating material was changed as shown in Table 6. The details of the coating materials used were the same as those described above.

When the carrier not coated with the coating material was used, and the organism adsorbed on the carrier was heat-treated at 95°C for 5 minutes, the Ct value was 30.8 (Comparative Example 5). Therefore, even if the organism adsorbed on the carrier was heated, when the Ct value is smaller than 30.8, it can be said that the sRNA extraction efficiency was improved when the carrier was coated with the coating material. On the other hand, when the Ct value is larger than 30.8, it can be said that the sRNA extraction efficiency was reduced when the carrier was coated with the coating material.
When E. coli was not added, the Ct value was above the measurement limit, and therefore the samples for which the Ct value in the table was obtained are samples for which the presence of the organism could be determined.

From the results of Examples 17 to 24, when the carrier was coated with a specific coating material, for example, a nonionic surfactant, an anionic surfactant, a protein, or an amino acid, the extraction efficiency of sRNA was improved compared to when the carrier was not coated with a coating material. On the other hand, from the results of Comparative Example 4, when the carrier was coated with a hydrophilic polymer, the extraction efficiency of sRNA was lower compared to when the carrier was not coated with a coating material or was coated with the specific coating material.
In addition, the Ct value of a sample in which sRNA was extracted by immersing the activated carbon on which E. coli W3110 was adsorbed in an aqueous solvent was 36.25 (Comparative Example 3). On the other hand, the Ct value of a sample in which sRNA was extracted by heat-treating a suspension containing the activated carbon on which E. coli W3110 was adsorbed at 95° C. for 5 minutes was 30.8 (Comparative Example 5). This shows that the efficiency of sRNA extraction is improved by heat treatment rather than immersion in an aqueous solvent.
Comparison of the results of Examples 1 to 16 and the results of Examples 17 to 24 also revealed that heat treatment improved the sRNA extraction efficiency more than immersion in an aqueous solvent, and furthermore, regardless of whether immersion in an aqueous solvent or heat treatment was used, coating with a specific coating material improved the sRNA extraction efficiency.
In Examples 17 to 24, since Ct values distinguishable from noise were obtained, it was possible to determine that each sample contained E. coli.

From the above, Examples 1 to 24 were able to provide a method for efficiently extracting sRNA from an organism, or a method for quickly determining the state of existence of an organism.

(Reference Example 1) Detection of bacteria belonging to the Enterobacteriaceae family by sRNA13, 14, 15 and 34 Each of Escherichia coli, Citrobacter freundii and Salmonella gallinarum, which are bacteria belonging to the Enterobacteriaceae family, was inoculated on an LB plate and cultured, and the resulting colonies were suspended in 2 mL of LB medium and shake cultured at 37 ° C. and 180 rpm for 24 hours. Then, 40 μL of the culture solution was collected and inoculated into 4 mL of LB medium, and shake cultured at 37 ° C. and 180 rpm for 24 hours. Then, the culture solution was centrifuged at 3000 G for 5 minutes, the supernatant was removed by aspiration, and sterile water was added. At this time, OD600 was measured and adjusted to OD = 1 with sterile water to prepare a bacterial suspension. Three 500 μL samples were prepared from the prepared bacterial suspension and allowed to stand at 5° C., 25° C., or 37° C. for 1 hour.

Next, reverse transcription and real-time PCR were performed on the sRNA (sRNA13, sRNA14, sRNA15, and sRNA34) in the bacterial suspension after standing. Real-time PCR was performed using 1 μL samples of each bacterial suspension after standing according to the above-mentioned <Method for quantifying sRNA using real-time PCR method>. The results of the Ct values obtained using a real-time PCR system (Applied Biosystems) are shown in Table 7. In all cases, the Ct value was 30 or less, and the presence of the Enterobacteriaceae bacteria under investigation was detected. Therefore, it was demonstrated that it is possible to detect Enterobacteriaceae microorganisms using Enterobacteriaceae tRNA and other sRNA.

(Reference Example 2) Study of the influence of additives on sRNA extraction from E. coli E. coli W3110 was applied to an LB plate and cultured, and the resulting colonies were suspended in 2 mL of LB medium and shake cultured at 37 ° C. and 180 rpm for 24 hours. 40 μL of the resulting culture solution was collected and inoculated into 4 mL of LB medium, and shake cultured at 37 ° C. and 180 rpm for 5 hours. Thereafter, the culture solution was centrifuged at 3000 G for 3 minutes, the supernatant was aspirated, and then sterile water was added. OD600 was measured, and the solution was diluted with sterile water so that the OD600 value was 1.0, to obtain an OD1.0 E. coli W3110 suspension.

10 μL of OD1.0 E. coli W3110 suspension was collected, 10 μL of Triton X-100 was added to a final concentration of 1% by mass, and the suspension was diluted with sterile water to an OD600 value of 0.5, and then left at 37°C for 1 hour. EC-5p-36 was quantified by real-time PCR using the liquid sample after being left for 1 hour. Specifically, 1 μL of the liquid sample was collected, and real-time PCR was performed according to the above <Method of quantification of sRNA by real-time PCR method> to obtain the Ct value. Separately, the Ct value was obtained by real-time PCR for a control (Reference Example 13 without additives) prepared in the same manner as above except that Triton X-100 was not added. The Ct value of Reference Example 2 was subtracted from the Ct value of Reference Example 13, and the obtained value (i.e., the decrease in Ct value compared to the control without additives) was taken as the "score". A positive score indicates that the Ct value has decreased, that is, that the amount of sRNA extracted has increased. Conversely, a negative score indicates an increase in the Ct value, i.e., a decrease in the amount of sRNA extracted. In addition, for reference examples in which the score is described as "below the detection limit," no amplification of sRNA was observed and no Ct value was obtained (i.e., the amount of sRNA extracted decreased to below the detection limit). The results for each additive are shown in Table 8.

(Reference examples 3 to 12)
The scores were measured in the same manner as in Reference Example 2, except that the additives shown in Table 8 were used and the final concentrations were as shown in Table 8 so that the concentration of the E. coli W3110 suspension was the same as in Reference Example 2. For example, in the case of Synperiinc F108 in Reference Example 3, 10 μL of a 2% by mass aqueous solution of Synperiinc F108 was added to 10 μL of the OD1.0 E. coli W3110 suspension, so that the OD600 value was 0.5 and the final concentration of Synperiinc F108 (concentration in the obtained liquid sample) was 1% by mass. The results for Reference Examples 3 to 12 are also shown in Table 8.

As can be seen from the results shown in Table 8, when nonionic surfactants, antimicrobial peptides, monovalent or divalent linear, branched or alicyclic C2-C8 alcohols, acetone, and reducing agents were used within the content ranges specified in this disclosure, the inclusion of these additives was confirmed to have the effect of improving the extraction efficiency of sRNA. On the other hand, when anionic surfactants, cationic surfactants, and oxidizing agents were used as additives, it was confirmed that the extraction efficiency of sRNA was inversely reduced.

All publications, patent applications, and technical standards mentioned in this specification are incorporated by reference into this specification to the same extent as if each individual publication, patent application, and technical standard was specifically and individually indicated to be incorporated by reference.

Claims (22)

A method for producing a cell wall-containing organism, comprising: bringing an organism and a carrier coated with a coating material selected from the group consisting of a nonionic surfactant, a cationic surfactant, an anionic surfactant, an amino acid, an oligopeptide, and a protein, into a first liquid, thereby adsorbing the organism onto the coated carrier; and immersing the coated carrier having the organism adsorbed thereon into a second liquid, which may be the same as or different from the first liquid, thereby extracting sRNA derived from the organism into the second liquid.
wherein the second liquid is an aqueous solvent;
The coating material adsorbs the organism but reduces adsorption of sRNA;
Methods for extracting sRNA from organisms. The extraction method according to claim 1, wherein the soaking is carried out under heating. A method for adsorbing an organism having a cell wall onto a carrier coated with a coating material selected from the group consisting of a nonionic surfactant, a cationic surfactant, an anionic surfactant, an amino acid, an oligopeptide, and a protein, by causing the organism to coexist in a first liquid with the carrier;
immersing the coated carrier with the adsorbed organism in a second liquid, which may be the same as or different from the first liquid, thereby extracting sRNA derived from the organism into the second liquid;
Detecting the state of existence of the extracted sRNA; and determining the state of existence of the organism based on the state of existence of the obtained sRNA.
wherein the second liquid is an aqueous solvent;
The coating material adsorbs the organism but reduces adsorption of sRNA;
A method for determining the state of existence of an organism. The method of claim 3 , wherein the sRNA is one or more types of sRNA. The method according to claim 3 or 4 , wherein the immersion is carried out under heating. The method according to claim 3 , wherein the aqueous solvent contains a reagent for nucleic acid amplification. The method according to any one of claims 3 to 6 , wherein the immersion is carried out at a temperature within a range of 0°C to 50°C. The method according to any one of claims 3 to 7 , wherein the carrier is at least one selected from the group consisting of activated carbon, silica, zeolite, porous ceramics, carbon fiber, sand, and glass beads. The method according to any one of claims 3 to 8 , wherein the nonionic surfactant is at least one selected from the group consisting of polysorbate 20, polysorbate 80, and octylphenol ethoxylate. The method according to any one of claims 3 to 8 , wherein the cationic surfactant is at least one selected from the group consisting of benzalkonium chloride and cetyltrimethylammonium bromide. The method according to any one of claims 3 to 8 , wherein the anionic surfactant is at least one selected from the group consisting of sodium dodecyl sulfate and sodium 1-octanesulfonate. The method according to any one of claims 3 to 8 , wherein the amino acid is glycine. The method according to any one of claims 3 to 8 , wherein the protein is at least one selected from the group consisting of skim milk powder and bovine serum albumin. The method according to any one of claims 3 to 13 , wherein the sRNA is present in the organism. The method according to any one of claims 3 to 14 , wherein determining the existence state of the organism comprises determining the overall existence state of two or more organisms. The method according to any one of claims 3 to 15 , wherein detecting the state of existence of the sRNA comprises determining the amount of the sRNA present. The method according to any one of claims 3 to 16 , wherein the organism comprises at least one organism having a cell wall selected from the group consisting of Escherichia coli, Citrobacter freundii, and Salmonella gallinarum. The method according to any one of claims 3 to 17, wherein the organism comprises a plant. The method for determining whether or not a pathogen is present in the organism is described in any one of claims 3 to 18 , wherein the organism comprises a verotoxin-producing bacterium. The method according to any one of claims 3 to 19 , wherein the number of bases of each of the sRNAs is within the range of 5 to 500. The method according to any one of claims 3 to 20, wherein the sRNA comprises at least one selected from the group consisting of EC-5p-36 having a nucleotide sequence represented by SEQ ID NO: 1, EC-3p-40 having a nucleotide sequence represented by SEQ ID NO: 2, EC-5p-79 having a nucleotide sequence represented by SEQ ID NO: 3, EC-3p-393 having a nucleotide sequence represented by SEQ ID NO: 4, fox_milRNA_5 having a nucleotide sequence represented by SEQ ID NO: 5, miR156 having a nucleotide sequence represented by SEQ ID NO: 6, and miR716b having a nucleotide sequence represented by SEQ ID NO: 7. The method according to any one of claims 3 to 21, wherein the detection of the presence of the sRNA is carried out by PCR.