JP7660351B2 - Method for determining the state of existence of organisms having cell walls, and method for identifying organisms having cell walls - Google Patents

Description

The present disclosure relates to a method for determining the state of existence of an organism having a cell wall, and a method for identifying an organism having a cell wall.

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 bacteria in the environment are harmless, but some bacteria can cause food poisoning, spoilage, or deterioration of products, and become a major hindrance to people's lives.

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

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

Other methods for detecting microorganisms other than the above include methods for detecting microorganisms based on genes contained in the microorganisms (genetic methods). Genetic methods are methods for identifying the type of microorganism present by examining the presence or absence of a nucleic acid sequence that constitutes part of a gene contained in the microorganism to be detected. Examples of nucleic acid molecules containing a nucleic acid sequence used for identification include DNA and ribosomal RNA called 16S rRNA. For example, in JP 2013-93 A, Mycobacterium avium and Mycobacterium intracellulare are detected based on the nucleotide sequence of 16S rRNA.

DNA is a nucleic acid possessed by almost all living organisms, and 16S rRNA is a nucleic acid possessed by almost all living organisms. These nucleic acids exist in large quantities within living organisms. For this reason, DNA is suitable as a detection target, and has been used in the past in research on the detection of microorganisms based on gene sequences. Similarly, 16S rRNA is also suitable as a detection target, and has been used in the past in research on the detection of microorganisms based on gene sequences. In the DNA sequence, there are parts whose sequences are conserved among biological species, and parts whose sequences differ for each biological species. Based on the parts whose sequences differ for each biological species, the biological species can be identified. Similarly, in the 16s rRNA sequence, there are parts whose sequences are conserved among biological species, and parts whose sequences differ for each biological species. Based on the parts whose sequences differ for each biological species, the biological species can be identified.

An object of one embodiment of the present disclosure is to provide a method for easily determining the state of existence of an organism in a measurement sample, and a method for easily identifying an organism contained in a measurement sample.

<1> Preparing a liquid sample by immersing a measurement sample in an aqueous solvent, or preparing a measurement sample that is an aqueous solvent as a liquid sample;
Detecting the presence of one or more types of sRNA in the liquid sample;
determining the state of existence of an organism having a cell wall based on the state of existence of the one or more types of sRNA;
Including,
The one or more types of sRNA include at least one type of sRNA represented by SEQ ID NOs: 15 to 54;
A method for determining the state of existence of an organism having a cell wall.
<2> The method according to <1>, wherein the one or more types of sRNA include at least one type of sRNA represented by SEQ ID NOs: 19 to 21 and 40.
<3> The method according to <1> or <2>, wherein the one or more types of sRNAs exhibit a specific expression state in an organism having a cell wall.
<4> The method according to any one of <1> to <3>, wherein determining the existence state of the organism having a cell wall includes determining the overall existence state of two or more types of organisms having a cell wall.
<5> The method according to any one of <1> to <4>, wherein detecting the state of existence of the sRNA includes detecting the amount of the sRNA present.
<6> The method according to any one of <1> to <5>, wherein the organism having a cell wall includes at least one selected from the group consisting of Escherichia coli, Citrobacter freundii, and Salmonella gallinarum.
<7> The method according to any one of <1> to <5>, wherein the organism having a cell wall includes a verotoxin-producing bacterium.
<8> The method according to any one of <1> to <7>, wherein the measurement sample is obtained by collecting airborne matter, and determining the presence state of organisms having cell walls includes determining the presence state of organisms having cell walls that are present in the air.
<9> The method according to any one of <1> to <8>, wherein the immersion is performed at a temperature within a range of 0° C. to 50° C.
<10> The method according to any one of <1> to <9>, wherein the number of bases of each of the one or more types of sRNA is within a range of 5 to 500.
<11> The method according to any one of <1> to <10>, wherein the one or more types of sRNA further include at least one type 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: 10, EC-3p-393 having a nucleotide sequence represented by SEQ ID NO: 11, fox_milRNA_5 having a nucleotide sequence represented by SEQ ID NO: 9, miR156 having a nucleotide sequence represented by SEQ ID NO: 4, and miR716b having a nucleotide sequence represented by SEQ ID NO: 5.
<12> The method according to any one of <1> to <11>, wherein the aqueous solvent contains a reagent for nucleic acid amplification.
<13> The method according to any one of <1> to <12>, wherein the detection of the presence state of the one or more types of sRNA is carried out by isothermal gene amplification.
<14> The method according to <13>, wherein the isothermal gene amplification is carried out at a temperature within a range of 10°C to 40°C.
<15> The method according to any one of <1> to <12>, wherein the detection of the presence of the one or more types of sRNA is carried out by PCR.
<16> Preparing a liquid sample by immersing a measurement sample in an aqueous solvent, or preparing a measurement sample that is an aqueous solvent as a liquid sample;
Detecting the presence of one or more types of sRNA in the liquid sample;
Identifying an organism having a cell wall present in the measurement sample based on the state of presence of the one or more types of sRNA;
Including,
The one or more types of sRNA include at least one type of sRNA represented by SEQ ID NOs: 15 to 54;
A method for identifying organisms that have cell walls.
<17> The method of identifying according to <16>, wherein the one or more types of sRNA include at least one type of sRNA represented by SEQ ID NOs: 19 to 21 and 40.
<18> The method according to <16> or <17>, wherein the one or more types of sRNAs exhibit a specific expression state depending on an organism having a cell wall.
<19> The method according to any one of <16> to <18>, wherein identifying an organism having a cell wall present in the measurement sample includes identifying one or more of two or more types of candidate organisms as being present.
<20> The method of identifying any one of <16> 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 <16> to <20>, wherein the measurement sample is obtained by collecting airborne matter, and identifying the organism having a cell wall includes identifying an organism having a cell wall present in the air.
<22> The identification method according to any one of <16> to <21>, wherein the immersion is performed at a temperature within a range of 0° C. to 50° C.
<23> The method of identifying any one of <16> to <22>, wherein the number of bases of each of the one or more types of sRNA is within a range of 5 to 500.
<24> The method of identification according to any one of <16> to <23>, wherein the one or more types of sRNA further include at least one type 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: 10, EC-3p-393 having a nucleotide sequence represented by SEQ ID NO: 11, fox_milRNA_5 having a nucleotide sequence represented by SEQ ID NO: 9, miR156 having a nucleotide sequence represented by SEQ ID NO: 4, and miR716b having a nucleotide sequence represented by SEQ ID NO: 5.
<25> The method of identifying any one of <16> to <24>, wherein the aqueous solvent contains a reagent for nucleic acid amplification.
<26> The method according to any one of <16> to <25>, wherein the detection of the presence state of the one or more types of sRNA is carried out by isothermal gene amplification.
<27> The method according to <26>, wherein the isothermal gene amplification is carried out at a temperature within a range of 10°C to 40°C.
<28> The method according to any one of <16> to <25>, wherein the detection of the presence state of the one or more types of sRNA is carried out by PCR.

The present disclosure provides a method for easily determining the presence of organisms having cell walls in a measurement sample, and a method for easily identifying organisms having cell walls contained in a measurement sample.

1 shows the results of detection of E. coli-derived sRNA by isothermal gene amplification in Reference Example 5. FIG. 13 shows the results of electrophoresis of the nucleic acid extract in Reference Example 10.

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 a stepwise manner, 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 a stepwise manner. 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 the values shown in the Examples and Reference 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.
In the present disclosure, when multiple elements are listed using "or", unless otherwise expressly stated, it does not exclude the selection of a combination of the multiple elements unless a technical contradiction arises.
In the present disclosure, even if an element is described in the singular form, it does not exclude the presence of a plurality unless there is a technical contradiction, unless otherwise expressly stated.
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.

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.

Furthermore, in the conventional genetic method, the operation of extracting DNA or 16s rRNA from microorganisms by denaturing the cell membrane is time-consuming and not simple. Specifically, DNA or 16s rRNA 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 DNA and 16S rRNA, it is 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. Thus, in the conventional genetic method, the operation of treating with a denaturant and the operation of removing or neutralizing the denaturant are time-consuming, so the operation is more complicated and not simpler than the culture method and the immunochromatography method. There is also a method of extracting nucleic acid from cells by heating. As an example of this method, a solution containing bacteria is heated at 100°C for 10 minutes and used for nucleic acid amplification. However, a dedicated device is required to use heat treatment, and safety during heating must be taken into consideration.

Considering the above situation, the inventors of the present application have conducted extensive 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, immersing the cells in a hypotonic solution may cause the cell membrane to rupture due to osmotic pressure, and DNA and 16s RNA may be extracted 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.

The method for determining the state of existence of an organism having a cell wall according to the present disclosure includes the steps of:
A measurement sample is immersed in an aqueous solvent to prepare a liquid sample, or a measurement sample which is an aqueous solvent is prepared as a liquid sample;
Detecting the presence of one or more types of sRNA in the liquid sample;
determining the state of existence of an organism having a cell wall based on the state of existence of the one or more types of sRNA;
Including,
The one or more types of sRNA include at least one type of sRNA represented by SEQ ID NOs: 15 to 54;
The present invention relates to a method for determining the state of existence of an organism having a cell wall (hereinafter, also simply referred to as the "determination method according to the present disclosure"). In addition, the present invention relates to a method for identifying an organism having a cell wall,
A measurement sample is immersed in an aqueous solvent to prepare a liquid sample, or a measurement sample which is an aqueous solvent is prepared as a liquid sample;
Detecting the presence of one or more types of sRNA in the liquid sample;
Identifying an organism having a cell wall present in the measurement sample based on the state of presence of the one or more types of sRNA;
Including,
The one or more types of sRNA include at least one type of sRNA represented by SEQ ID NOs: 15 to 54;
The present invention relates to a method for identifying organisms having cell walls (hereinafter, also simply referred to as the "identification method according to the present disclosure").

The determination method according to the present disclosure allows for easy determination of the presence of a cell wall-bearing organism in a measurement sample. In addition, the identification method according to the present disclosure allows for easy identification of the type of cell wall-bearing organism contained in a measurement sample. The determination and identification methods according to the present disclosure do not require the use of strong denaturants such as phenol, guanidine salts, ionic surfactants, alcohols other than short-chain alcohols (monovalent or divalent straight-chain, branched-chain or alicyclic C2-C8 alcohols) described below, or sodium hydroxide, allowing for easy determination or identification.

The determination method according to the present disclosure and the identification method according to the present disclosure can be collectively expressed as the method described below:
A measurement sample is immersed in an aqueous solvent to prepare a liquid sample, or a measurement sample which is an aqueous solvent is prepared as a liquid sample;
Detecting the presence of one or more types of sRNA in the liquid sample;
Obtaining the presence or absence of organisms having cell walls in the measurement sample or identifying the organisms present based on the presence or absence of the one or more types of sRNA;
wherein the one or more types of sRNA include at least one type of sRNA represented by SEQ ID NOs: 15 to 54 (hereinafter also referred to as method A according to the present disclosure).
In addition, the more specific embodiments described in the section "Means for solving the problems" can also be applied to the above method A.

Organisms contain short RNA fragments called small RNA. Small RNA (hereinafter also referred to as "sRNA") plays an important role in controlling life processes such as development, differentiation, transposon silencing, and virus defense. Since sRNA has an important function in cells, sRNA expressed within the same species has nucleotide sequence conservation, that is, the expression profile of various sRNAs is common among multiple cells of the same species. On the other hand, between different species, the expression profile of sRNA is differential according to the species. Therefore, like DNA and 16S rRNA, information on sRNA present in a measurement sample can be used to distinguish species. It was discovered for the first time in this disclosure that 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.

Although the existence of sRNA has been known for some time, it was thought that, like DNA and 16S rRNA, 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 in 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 the measurement sample. Therefore, according to the present disclosure, a specific organism of interest can be detected easily and without much effort, and the type of organism present in the measurement sample can be identified.

Furthermore, because sRNA has a short chain length, it is less susceptible to attack by RNase compared to 16S rRNA, allowing for more stable analysis. In addition, the number of copies of sRNA within cells can range from several thousand to tens of thousands, making it possible to obtain information about the state of existence of sRNA with high sensitivity.

The following describes the determination method according to the present disclosure and the identification method according to the present disclosure together. Unless otherwise specified, the matters described below apply to both the determination method according to the present disclosure and the identification method according to the present disclosure, and also apply to method A according to the present disclosure.

<Organisms with cell walls>
The organism having a cell wall in the determination method and the identification method according to the present disclosure is not particularly limited, and may be any organism having a cell wall. Generally, cells other than animal cells, such as plant cells and microbial cells, have cell walls. The organism having a cell wall may be a fungus such as yeast, slime mold, or mold, or a bacterium such as Escherichia coli. The object to be determined or identified does not have to be the whole organism, but may be a part of the organism. sRNA is also present in the part of the organism, and even detection of a part of the organism gives information about the existence of the organism. The part of the organism may be a part of a multicellular organism, such as pollen of a plant. However, the part of the organism is preferably a part that maintains the shape of the cell. If the part of the organism does not maintain the shape of the cell, intracellular components may have already flowed out into the surrounding medium before immersion in an aqueous solvent or before preparing a measurement sample that is an aqueous solvent. As described above, the determination method according to the present disclosure describes determining the existence state of an organism having a cell wall, and the identification method according to the present disclosure describes identifying an organism having a cell wall, but the organism having a cell wall here represents a concept that includes a part of the organism. Therefore, it is sufficient that the determined existence state or the identification provides information about a biological species or a group of biological species. In fact, for example, in the case of detecting herbaceous plants, the measurement sample will generally contain not the whole organism but a part of it, but the determination method or identification method according to the present disclosure can provide a determination result or identification result regarding the existence of a biological species or a group of biological species. Therefore, the organism in the determination method and identification method according to the present disclosure may be a plant, and in this case, the component contained in the measurement 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 the cell wall may be a bacterium belonging to the family Enterobacteriaceae. Examples of such bacteria include bacteria of the genus Citrobacter, such as Citrobacter freundii; bacteria of the genus Edwardsiella, such as Edwardsiella tarda; bacteria of the genus Enterobacter, such as Enterobacter aerogenes, E. cloacae, E. gergoviae, and E. sakazakii; bacteria of the genus Escherichia, such as Escherichia coli and E. albertii; bacteria of the genus Hafnia, such as Hafnia alvei; bacteria of the genus Klebsiella, such as Klebsiella oxytoca and K. pneumoniae; bacteria of the genus Kluyvera, such as Kluyvera ascorbata and K. cryocrescens; bacteria of the genus Morganella, such as Morganella morganii; bacteria of the genus Proteus, such as Proteus mirabilis and P. vulgaris; bacteria of the genus Providencia alcalifaciens, P. rettgeri, and P. Examples of bacteria that can be used include bacteria of the genus Providencia such as Salmonella enterica, bacteria of the genus Serratia such as Serratia liquefaciens and S. marcescens, bacteria of the genus Shigella such as Shigella boydii, S. dysenteriae, S. flexneri, and S. sonnei, and bacteria of the genus Yersinia such as Yersinia enterocolitica, Y. pestis, and Y. pseudotuberculosis. 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, and the plant may be a Japanese black pine. In another embodiment, the organism having a cell wall includes a verotoxin-producing bacterium.

The organisms having cell walls contained in the aqueous solvent preferably include organisms having cell walls whose cell walls and cell membranes are not destroyed, or organisms having no cell walls whose cell membranes are not destroyed. In other words, it is preferable that the organisms having cell walls have not been subjected to cell disruption treatment, membrane destruction treatment, etc. This is because if the cell wall or cell membrane is destroyed, sRNA has already leaked out of the organism, and there is a risk that sRNA cannot be extracted. In addition, in the determination method and identification method disclosed herein, it is preferable that the process up to the detection of the state of the presence of sRNA does not include treatments that destroy the cell walls and/or cell membranes of organisms having cell walls (cell disruption treatment, membrane destruction treatment, etc.).

In the determination method and identification method disclosed herein, sRNA that has leaked from cells having a cell wall into an aqueous solvent is used for detection, so treatment may be carried out in advance to remove substances that may hinder such leakage.

<Measurement sample>
The measurement sample in the determination method according to the present disclosure is a sample used to determine the state of existence of an organism having a cell wall to be detected, and the measurement sample in the identification method according to the present disclosure is a sample used to identify an organism having a cell wall. When an organism having a cell wall is present in an object (hereinafter also referred to as an analyte) to be analyzed for the state of existence of an organism having a cell wall, there is no particular limitation on the measurement sample as long as the measurement sample is prepared to contain the organism, and the measurement sample may be the analyte itself or a sample prepared from the analyte by any treatment. The analyte 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 body of unknown biological species, etc. From the viewpoint of making the operation easier, it is preferable that the measurement sample is the analyte itself.

For example, if one wishes to analyze organisms with cell walls present in a liquid as the subject of analysis, the liquid itself may be used as the measurement sample, or the liquid may be treated by dilution or the like and used as the measurement sample. Also, if one wishes to analyze organisms present on the surface of an object as the subject of analysis (e.g., the surface of a workbench), the surface may be wiped with a wipe, cotton swab, etc., and the wipe, cotton swab, etc., or a part of it, may be used as the measurement sample.

When it is desired to analyze organisms having cell walls present inside an object to be analyzed (e.g., inside food), the whole or part of the object may be used as the measurement sample, or the object may be immersed in a liquid and the liquid obtained may be used as the measurement sample. When organisms having cell walls are present in the object, it is believed that the organisms will transfer part or all of their total mass into the liquid upon immersion. When it is desired to analyze organisms present in air using air as the object to be analyzed, the measurement sample may be a sample obtained by collecting airborne matter present in the air. The collection may be performed by using a filter, centrifugation (e.g., cyclone separation), or simply leaving the container open (e.g., leaving a petri dish or the like with the lid open), etc.

Thus, the measurement sample can be, for example, a liquid to be analyzed, a diluted version of the liquid, a wipe or cotton swab used to wipe a surface to be analyzed, a collected material on an air filter, a liquid obtained by immersing the collected material in a liquid, a material adhering to the inside of a container open to the surrounding atmosphere, etc. Alternatively, the organism itself can be used as the measurement sample for the purpose of identifying the type of organism with a cell wall that has already been obtained.

<Aqueous Solvent>
The aqueous solvent in the determination method and the identification 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 (hereinafter also referred to as "coexisting components") in addition to water, so 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 contains only 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 of components that do 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. 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-C8 alcohol in the aqueous solvent is not particularly limited, and may be 0% by mass (non-containing). 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.

In order to prevent the denaturation of cell membranes under strongly acidic or strongly alkaline conditions, the pH of the aqueous solvent is preferably within the range of pH 5 to pH 9, more preferably within the range of pH 6 to pH 8, and even more preferably within the range of pH 6.5 to pH 7.5. The aqueous solvent may contain coexisting components other than the solvent as described above. The total content of coexisting components other than the solvent 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 other than the solvent 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 them 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 preparing the liquid sample by immersion, and identification of organisms having cell walls 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 Tween series surfactants (e.g. Tween-20), NP-40, and Triton series surfactants (e.g. 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 a liquid sample containing sRNA leaked from cells having cell walls, without carrying out a reagent addition operation or a temperature cycle operation.

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.

<Immersion>
In the determination method according to the present disclosure and the identification method according to the present disclosure, the immersion may be performed at room temperature or with heating or cooling. In order to minimize denaturation of the cell membrane, the immersion 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 immersion may be performed at room temperature.
The immersion time is not particularly limited as long as the sRNA leaks out of the cell in an amount sufficient for detection. The immersion time is, for example, 10 seconds to 30 hours, and may be 10 seconds to 10 hours, 1 minute to 5 hours, or 5 minutes to 1 hour. The immersion time may alternatively be 0.5 hours to 6 hours, 0.7 hours to 4.5 hours, or 0.8 hours to 2 hours. The method of immersion is not particularly limited, and any treatment that brings the organism into contact with an aqueous solvent may be used if the measurement sample contains an organism having a cell wall. When the measurement sample is a solid sample (e.g., a cotton swab, a wipe, a filter, the analyte itself or a part thereof, or a bacterial body), the immersion can be performed, for example, by immersing the solid sample as the measurement sample in an aqueous solvent stored in a container.
As described above, the organism having a cell wall itself can be used as the measurement sample, so the organism having a cell wall may be immersed alone in the aqueous solvent, or the organism having a cell wall attached to another object may be immersed together with the object in the aqueous solvent.
When an organism having a cell wall is present in the measurement sample, the immersion causes leakage of sRNA outside the cells, and a liquid sample containing sRNA in the aqueous solvent is obtained.

<Measurement sample as aqueous solvent>
If the analyte is in the form of an aqueous solvent from the beginning, the analyte itself can be used as a measurement sample and as a liquid sample as it is. For example, tap water can be used as a liquid sample as it is to determine the presence of organisms having cell walls in tap water, or to identify organisms having cell walls contained in tap water. Since the analyte may contain organisms having cell walls, the aqueous solvent as the measurement sample may contain organisms having cell walls.
When the analyte is a liquid but needs to be pretreated due to a large amount of impurities, a measurement sample that is an aqueous solvent may be prepared by removing the impurities by filtration, centrifugation, dialysis, etc., and this measurement sample may be used as a liquid sample. For example, when testing organisms having cell walls in muddy water, the mud in the muddy water is removed by filtration, etc., and the filtrate (a measurement sample that may contain organisms having cell walls) can be used as a liquid sample to determine the presence of organisms having cell walls or identify organisms having cell walls. As described above, the aqueous solvent may contain coexisting components other than water, so that organisms having cell walls that may be contained in the measurement sample may be contained as coexisting components in the aqueous solvent.
The measurement sample, which is an aqueous solvent, can also be prepared by immersing the analyte in an aqueous solvent. Specifically, an organism having a cell wall is transferred from the analyte to the aqueous solvent to obtain a measurement sample, which is an aqueous solvent. Thus, the immersion may be for the purpose of preparing a measurement sample, which is an aqueous solvent, by transferring an organism having a cell wall to the aqueous solvent, instead of extracting sRNA. The immersion conditions in this case are the same as those described above.
In a preferred embodiment, the aqueous solvent does not contain any other coexisting components than the above-mentioned cell wall-bearing organisms. If the cell wall-bearing organisms that may be contained in the measurement sample are allowed to contact the aqueous solvent for a certain period of time (for example, the period exemplified as the above-mentioned immersion time), sRNA will leak from the organism (if the measurement sample actually contains an organism with a cell wall), and a liquid sample containing sRNA similar to that obtained by the above-mentioned immersion can be obtained. The embodiment using the measurement sample that is an aqueous solvent can be used, for example, to determine the presence of organisms with cell walls in water (for example, tap water, sewage, etc.) as the analysis target, or to identify organisms with cell walls contained in water (for example, tap water, sewage, etc.).
As long as the operation is that an organism having a cell wall comes into contact with an aqueous solvent, the operation is included in the above-mentioned immersion of a measurement sample in an aqueous solvent to prepare a liquid sample, or preparation of a measurement sample that is an aqueous solvent as a liquid sample. When the measurement sample is immersed in an aqueous solvent to prepare a liquid sample, the operation may be an operation in which the aqueous solvent in which the measurement sample is immersed is left to stand or stirred, etc., and artificially aged for the extraction of sRNA. When the measurement sample is a liquid sample, it may also be left to stand or stirred, etc., and artificially aged for the extraction of sRNA.

The leakage of sRNA from cells having cell walls, as explained in the above sections "immersion" and "measurement sample being an aqueous solvent", is surprisingly not a phenomenon that occurs with nucleic acids in general, but a phenomenon that occurs specifically with sRNA. For this reason, even if cells having cell walls are immersed in an aqueous solvent in an attempt to cause 16S rRNA, which has traditionally been used to distinguish biological species, to leak into an aqueous solvent, 16S rRNA does not leak at all from cells having cell walls, or, even if it does leak, it leaks only in an amount that is insufficient for use in detecting biological species. 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 cells having cell walls 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 cells having cell walls unless an extraction operation involving denaturation of the cell membrane is performed.

<sRNA>
In the determination method and the identification 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.

Research is ongoing on sRNAs present in organisms with cell walls, and sequence information on 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. Therefore, 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 one or more organisms in which a particular sRNA is expressed, and also to search for one or more sRNAs expressed in a particular organism. In the determination method according to the present disclosure, it is preferable that the one or more sRNAs exhibit a specific expression state in a particular organism of interest. In this way, the state of existence of the organism can be better determined from the state of existence of the sRNA.

Here, the term "specific expression state" does not necessarily refer to an sRNA that is expressed only in an organism having the specific cell wall, or that is not expressed only in an organism having the specific cell wall, but also refers to a concept that includes an sRNA that is not completely specific only to an organism having the specific cell wall, such as an sRNA that is expressed only in a group of organisms having a specific cell wall including the organism having the specific cell wall, or that is not expressed only in a group of organisms having a specific cell wall including the organism having the specific cell wall.
In the identification method according to the present disclosure, it is also preferable that the one or more types of sRNA to be detected for their presence state exhibit a specific expression state depending on the organism having a cell wall.

As described above, in the determination method and identification method of the present disclosure, sRNA can be leaked into the aqueous solvent outside the cell having a cell wall without denaturing the cell membrane with strong alkali treatment, guanidine treatment, phenol, alcohols other than the above-mentioned short-chain alcohols (monovalent or divalent linear, branched or alicyclic C2-C8 alcohols), ionic surfactants, etc. This eliminates the need to use a denaturant and the time required for treatment with a denaturant, and the aqueous solvent into which the sRNA has leaked can be used for subsequent treatment (e.g., nucleic acid amplification treatment for sRNA detection, etc.) as is.

<State of Existence>
In the determination method and the identification method according to the present disclosure, the term "existence state" may simply refer to the presence or absence of a substance, or may refer to the amount of a substance present (including the case where the amount of a substance is 0, i.e., the case where the substance does not exist). In other words, "detecting the existence state" may be obtaining binary information of whether or not a substance exists, but may also obtain information on the amount of a substance present in addition to whether or not a substance exists. The information on the amount of a substance present also contains information on the presence or absence of a substance. Similarly, "determining the existence state" may be performing a binary determination of whether or not a substance exists, but may also perform a determination of the amount of a substance present in addition to whether or not a substance exists. Here, the amount of a substance present is not limited to an absolute amount of a substance present, and may be a relative amount of a substance present relative to a comparison target such as a negative control or a positive control.

<Detection of Presence State>
In the determination method and the identification method of 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 one or more types of 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, such as the LAMP method or the RCA method. The RCA method is particularly exemplified by the SATIC (termed signal amplification by ternary initiation complexes) method. Isothermal gene amplification is preferable in that it does not require the preparation of an equipment for amplification (equipment that changes the temperature according to the temperature cycle). Isothermal gene amplification can be performed at a temperature within the range of, for example, 10°C to 40°C, so it can be performed at room temperature. In the present disclosure, room temperature may be a temperature within the range of 20°C to 40°C, except as otherwise specified in the Examples and Reference Examples.
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 aqueous solvent after immersion is complete, or may be pre-contained in the aqueous solvent before immersion. If the sRNA detection reagent is pre-contained in the aqueous solvent before immersion, this is preferable in that no addition operation is required after immersion is complete. In addition, when a measurement sample that is an aqueous solvent is used as a liquid sample, the sRNA detection reagent may be added to the liquid sample, or, if there is a process for preparing a measurement sample that is an aqueous solvent, the sRNA detection reagent may be added during this preparation process.

The amplified nucleic acid can be detected by using existing methods for detecting amplified nucleic acid, 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 of the 3'-terminal region of the sRNA.

The detection of the presence of the sRNA may be performed simultaneously with the extraction of sRNA from cells having a cell wall. For example, a sample that may contain an organism having a cell wall may be directly immersed or added to a reaction solution for an isothermal gene amplification method such as PCR or SATIC, so that leakage and detection of 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 liquid sample.

<Determination of the state of existence of organisms having cell walls>
The detection of sRNA specific to an organism having a cell wall in a measurement sample indicates the presence of the organism in the measurement sample. Information on the amount of sRNA specific to an organism having a cell wall in a measurement sample can also provide information on the amount of the organism in the measurement sample. It is the first time that the present disclosure has found that the organism can be identified based on the state of sRNA.

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.

In the determination method according to the present disclosure, the term "organism having a cell wall" refers to an organism having a cell wall to be detected, and may be any organism having a cell wall to be detected. The state of existence of the organism can be determined by detecting the state of existence of sRNA that is specifically expressed in the organism compared to other organisms. Detection of sRNA that is specifically 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 an expression state specific to the organism as the sRNA for detecting the state of existence, depending on the type of organism whose state of existence is to be detected in the measurement sample. The specific expression state of the sRNA may be specifically expressed in the organism whose state of existence is to be detected, or may be specifically non-expressed in the organism whose state of existence is to be detected. However, it is preferable that at least one of the one or more types of sRNA whose state of existence is to be detected is an sRNA that is specifically expressed in the organism whose state of existence is to be detected.

Note that one type of sRNA is not necessarily completely specific 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 specifically non-expressed in the organism can also be used. The detection or non-detection of sRNA that is specifically non-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 specifically expressed in the organism, it is possible to further narrow down candidates for organisms that show the expression profile of those sRNAs.

As the type or types of sRNAs whose presence state is detected in the determination method according to the present disclosure, it is preferable to select sRNAs whose expression profile allows a specific organism of interest to be better distinguished from other organisms.

However, it is not essential in the determination method according to the present disclosure to limit the candidates for organisms having a cell wall to one type, and it is sufficient to simply identify a group of candidates consisting of multiple types of organisms. For this reason, the detection method according to the present disclosure may determine the presence state of multiple types of organisms, in which case the detection method does not determine the presence state of each of the multiple types of organisms individually, but determines the presence state of the multiple types of organisms as a whole. In other words, even if the detection method cannot determine which of the multiple types of organisms is actually present, it is sufficient if it can determine that at least one of the multiple types of organisms that make up 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 present.

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 tests may be performed based on this determination result, or the analysis subject from which the measurement sample was obtained may be purified 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 existence of one or more types of sRNA, the amount of sRNA present can also be measured, making it possible to determine the amount of the organism 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, or using qPCR.

<Identification of organisms with cell walls present in the measurement sample>
From the state of existence of one or more types of sRNA obtained from a liquid sample, one or more types of organisms having an sRNA expression profile that matches the state of existence of the sRNA can be identified. The one or more types of sRNA are preferably sRNAs that show a specific expression state depending on the organism. Detecting the state of existence of one or more types of sRNA in a liquid sample may include comprehensively detecting the sRNA present in the liquid sample, but may also include measuring the state of existence of one or more types of sRNA selected in advance in consideration of the type of organism that may be present in the measurement sample.

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

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

In other words, in the identification method according to the present disclosure, identifying the organisms present in the measurement 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. In the identification method according to the present disclosure, when identifying the organisms present in the measurement 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 tests may be performed based on this identification result, or the analysis target from which the measurement sample was obtained may be purified (appropriate purification according to the type of candidate microorganism) 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 existence of one or more types of sRNA, the amount of sRNA present can be measured to identify the amount of the organisms present. This is because the amount of sRNA present in a liquid sample is thought to reflect the amount of organisms expressing the sRNA (in the simplest case, it is 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 of one or more types of sRNA in a liquid sample in the identification method of the present disclosure will be described. According to a Nucleotide BLAST search, the sRNA EC-5p-36 (SEQ ID NO: 1; 5'-UGUGGGGCACUCGAAGAUACGGAU-3', see Curr Microbiol. 2013 Nov; 67(5):609-13) is commonly expressed in Escherichia, Shigella, Salmonella, and Citrobacter bacteria. Therefore, when EC-5p-36 is detected as being present in a liquid sample, the organism present can be identified as one or more of Escherichia, Shigella, Salmonella, and Citrobacter bacteria. In addition, according to a Nucleotide BLAST search, EC-3p-40, which is an sRNA, is commonly expressed in bacteria of the genus Shigella, Salmonella, Escherichia, and Citrobacter, as well as in bacteria of the genus Klebsil. Therefore, when EC-3p-40 (SEQ ID NO: 2; 5'-GUUGUGAGGUUAAGCGACU-3') is detected to be present in a liquid sample, the organisms present can be identified as one or more of bacteria of the genus Escherichia, Shigella, Salmonella, Citrobacter, and Klebsil. In addition, these results may be combined to perform the identification. For example, EC-5p-36 is not expressed in Klebsilla bacteria, but EC-3p-40 is expressed in Klebsilla bacteria, so if EC-3p-40 is detected to be present in a liquid sample but EC-5p-36 is not, the organism can be identified as a Klebsilla bacterium.

Organisms may also be identified based on the presence of sRNA with a specific function. For example, 24B_1 (SEQ ID NO: 3; 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, when 24B_1 is detected to be present in a liquid sample, 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 types 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: 10; 5'-UUUGCUCUUUAAAAAAUC-3') and EC-3p-393 (SEQ ID NO: 11; 5'-CUCGAAGAUACGGAUUCUUAAC-3') are expressed in Escherichia coli, Citrobacter freundii, and Salmonella gallinarum. From the above, examples of the correspondence between sRNA and biological species include 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, a combination of fox_milRNA_5 having a nucleotide sequence represented by SEQ ID NO:9 with Fusarium oxysporum, a combination of miR156 having a nucleotide sequence represented by SEQ ID NO:4 with Pinus thunbergii, and a combination of miR716b having a nucleotide sequence represented by SEQ ID NO:5 with 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, sRNA19 to sRNA38 are tRNA sequences.

In addition, for sRNA15, 16, 19-21, 23-25, 27-30, 32, and 33, 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 used in the determination method and identification method of the present disclosure. In the determination method and identification method of the present disclosure, the one or more types of sRNAs for detecting the presence state include at least one type of sRNAs represented by SEQ ID NOs: 15 to 54 (i.e., at least one type of sRNA15 to sRNA54). It is preferable that the one or more types of sRNAs for detecting the presence state include at least one type of sRNAs represented by SEQ ID NOs: 19 to 21 and 40 (i.e., at least one type of sRNA19 to sRNA21 and sRNA40).

The one or more types of sRNA for detecting the existence state may include at least one type of sRNA represented by SEQ ID NOs: 15 to 54 (i.e., at least one type of sRNA15 to sRNA54) as well as other sRNAs. By including other sRNAs, it is possible to further increase the accuracy of the determination or identification and/or to determine the existence state of organisms having cell walls or identify organisms having cell walls for a wider range of biological species.

For this reason, for example, one or more types of sRNA for detecting the presence state may further include at least one type of sRNA of SEQ ID NOs: 1 to 5 and 9 to 11 in addition to at least one type of sRNA represented by SEQ ID NOs: 15 to 54 (preferably at least one type of sRNA represented by SEQ ID NOs: 19 to 21 and 40).

For sRNAs other than those mentioned above, the correspondence between sRNAs and organisms required for determination or identification can be easily obtained from academic papers, the above-mentioned databases, etc.

In consideration of the above-described embodiments, the present disclosure also provides the following embodiments. That is, the method for detecting a target organism according to the present disclosure includes:
Immersing a target organism contained in a measurement sample in an aqueous solvent to prepare a liquid sample in which one or more types of sRNA specific to the target organism are leaked into the aqueous solvent;
detecting the one or more sRNAs in the liquid sample;
The one or more types of sRNA include at least one type of sRNA represented by SEQ ID NOs: 15 to 54. The method of immersing the target organism in an aqueous solvent is not particularly limited as long as the target organism contacts the aqueous solvent, and can be performed in the same manner as the immersion of the measurement sample in the determination method according to the present disclosure and the identification method according to the present disclosure or the preparation of the measurement sample which is an aqueous solvent. In addition, the one or more types of sRNA specific to the target organism may be sRNAs that are not specifically expressed in the target organism as long as the one or more types of sRNA include at least one type of sRNA that is specifically expressed in the target organism.

As described above, in the determination method and the identification method of the present disclosure, sRNA can be easily leaked from cells having a cell wall into an aqueous solvent without undergoing a membrane denaturation operation using a strong denaturant. Then, by detecting the state of existence of the leaked sRNA (for example, by detecting sRNA by nucleic acid amplification), the state of existence of a specific organism of interest can be determined with less effort than the method of denaturing the membrane and extracting 16S rRNA, and the organism present in the measurement sample can be identified. In particular, when an isothermal gene amplification method (for example, RCA method) in which the reaction proceeds at room temperature is used as the nucleic acid amplification method, the state of existence of the organism can be easily determined or the organism contained in the measurement sample can be identified without using a special device (for example, a device for performing temperature cycles) from start to finish.

The determination method and identification method according to the present disclosure, unlike the culture method, do not require culturing organisms that may be contained in the measurement sample. Therefore, while it is possible to perform determination or identification in a short time, it is also possible to achieve high sensitivity by using nucleic acid amplification or the like. Furthermore, the determination method and identification method according to the present disclosure can be applied to bacteria that are difficult to culture. Furthermore, unlike the culture method, the determination method and identification method according to the present disclosure require a small amount of sample, and materials used in processing can be easily disposed of. The determination method and identification method according to the present disclosure maintain the advantages of the genetic method, while still allowing the determination of the state of the organism and the identification of the organism contained in the measurement sample to be performed with simpler operations than conventional genetic methods.

The determination method and the identification method disclosed herein can be used, for example, for simple microbial testing in locations 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 food poisoning accidents, 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 and reference examples are based on mass unless otherwise specified. In the following examples and reference examples, "room temperature" was approximately 30°C.

<Method for quantification of sRNA by real-time PCR method>
In Reference Examples 1 to 3, 6 to 8, Example 9 and Reference Example 11, the sRNA to be subjected to the determination of its presence state was quantified by the method described below.
The sRNA in the reaction solution in each of Reference Examples 1 to 3, Reference Examples 6 to 8, Example 9, and Reference Example 11 was quantified by real-time PCR. A quantitative reagent (Taqman (registered trademark) microRNA Assays, Applied Biosystems) and a 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.

Reference Example 1 Simplified Extraction of sRNA (EC-5p-36) Contained in Escherichia coli Simplified extraction from Escherichia coli W3110 (hereinafter simply referred to as "E. coli W3110") of EC-5p-36, which is a type of sRNA contained in E. coli W3110, was attempted.
A colony of E. coli W3110 was suspended in 100 μL of pure water to prepare a 90% by mass E. coli W3110 cell suspension. Based on this 90% by mass E. coli W3110 cell suspension, the following three types of samples were prepared.

Sample 1) An RNase inhibitor (manufactured by Toyobo Co., Ltd.) was added to a 90% by mass suspension of Escherichia coli W3110 cells to a final concentration of 0.4 U/μL to prepare 10 μL of a reaction solution. The reaction solution was left to stand at room temperature for 4 hours to prepare Sample 1.
Sample 2) A 90% by mass suspension of Escherichia coli W3110 cells was immediately purified using a miRNeasy kit (a cell lysis reagent that contains phenol and causes cell membrane denaturation; manufactured by QIAGEN), and total sRNA was extracted to prepare Sample 2.

In addition, for comparison, 10 μL of pure water was used as sample 0.

EC-5p-36 was quantified by real-time PCR for samples 0 to 2. The amount of sRNA in samples 0 to 2 obtained by quantification was divided by the amount of sRNA in sample 2, and the obtained value was taken as the relative extraction rate (Table 5).

As can be seen from the results shown in Table 5, sRNA can be easily extracted from bacterial cells and measured. sRNA could be extracted using pure water alone.

(Reference Example 2) Simple extraction of sRNA (miR156) contained in black pine pollen Simple extraction from black pine pollen of miR156 (sequence number 4; 5'-CAGAAGAUAGAGAGCACAUC-3'; see http://www.mirbase.org/; pta-miR156a), a type of sRNA contained in black pine pollen, was attempted.
10 mg of black pine pollen (Biosta) was suspended in 1 mL of pure water and allowed to stand at room temperature for 1 hour. Then, miR156 was quantified by real-time PCR. RNase-free water was used as a negative control, and water containing 1 nM synthetic miR156 (SEQ ID NO: 4; Eurofins Genomics) was used as a positive control to compare whether sRNA (miR156) could be detected. As a result, it was confirmed that sRNA could be extracted from black pine pollen simply by suspending it in pure water.

(Reference Example 3) Simple extraction of sRNA (miR716b) contained in yeast Simple extraction of sRNA from Saccharomyces cerevisiae (hereinafter simply referred to as baker's yeast) was attempted using miR716b (SEQ ID NO: 5; 5'-GAGAUCUUGGUGGUAGUAGCAAAUA-3'; see Sci. Rep., 2015, 5, 7763), a type of sRNA contained in baker's yeast.
Baker's yeast was cultured on an LB plate, 10 mg of baker's yeast was scraped off, suspended in 1 mL of pure water, and allowed to stand at room temperature for 1 hour. After standing, an attempt was made to extract miR716b by real-time PCR. RNase-free water was used as a negative control, and 1 nM of synthetic miR716b (SEQ ID NO: 5; Eurofins Genomics) was used as a positive control to compare whether sRNA could be detected. As a result, it was confirmed that sRNA derived from baker's yeast could be extracted simply by suspending it in pure water, and it was also confirmed that the amount of extracted sRNA could be quantitatively detected.

(Reference Example 4) Detection of Escherichia coli-derived sRNA (EC-5p-36) by isothermal gene amplification method EC-5p-36, a type of sRNA contained in Escherichia coli, was targeted. Detection was attempted using an isothermal gene amplification method (SATIC method; Anal Chem. 2016 Jul 19; 88(14):7137-44)) of sRNA simply extracted from E. coli with an aqueous solvent.

<Synthesis of cyclized T1B-EC-5p-36>
First, a primer, which is a synthetic single-stranded DNA having the following nucleotide sequence, was ordered and obtained from Eurofin genomics.
Primer sequence (SEQ ID NO: 6): 5'-CCCCAAAAAAATCCGTATCTTCGAGTGCCCACAAAAAAGAAGCTGTTGTATTGTTGTCGAAGAAGAAAAGT-3'(5' phosphorylated, HPLC purified)

Next, the synthetic single-stranded DNA was circularized using CircLigase II ssDNA Ligation kit (manufactured by Lucigen) according to the manual. That is, 15 μL of RNase-free water (manufactured by Qiagen), 1 μL of the synthetic single-stranded DNA at 10 pmol/μL, 2 μL of CircLigase II 10×Reaction Buffer, 1 μL of 50 mM MnCl ₂ , and 1 μL of CircLigase II ssDNA Ligase were added to a PCR tube. After mixing, the PCR tube was treated on a thermal cycler at 60° C. for 1 hour and at 80° C. for 10 minutes to obtain 500 nM of circularized DNA (hereinafter referred to as “circularized T1B-EC-5p-36”). This was diluted 5-fold with RNase-free water to give 100 nM circularized T1B-EC-5p-36.

<Synthesis of cyclized T2>
First, a primer, which is a synthetic single-stranded DNA having the following nucleotide sequence, was ordered and obtained from Eurofin genomics.
Primer sequence (SEQ ID NO: 7): 5'-CCCAACCCTACCCACCCTCAAGAAAAAAAAAGTGATAATTGTTGTCGAAGAAGAAAAAAAAATT-3'(5' phosphorylated, HPLC purified)

Next, using CircLigase II ssDNA Ligation kit (Lucigen), the same procedure as for the synthesis of circularized T1B-EC-5p-36 was carried out, except that the primer (T2) of sequence number 7 was used instead of the primer (T1B-EC-5p-36) of sequence number 6, to obtain 500 nM circularized DNA (hereinafter referred to as "circularized T2"). This was diluted 1.25-fold with RNase-free water to obtain 400 nM circularized T2.

<Synthesis of primer P2>
Primers having the following nucleotide sequences were ordered from Eurofin genomics, and synthetic primers were obtained.
Primer sequence (SEQ ID NO: 8): 5'-GAAGCTGTTGTTATCACT-3' (unmodified, HPLC purified)

The primer was diluted with RNase-free water to prepare 480 nM primer P2.

Detection of E. coli-derived sRNA by isothermal gene amplification method
Detection of E. coli-derived sRNA was attempted using φ29 DNA polymerase (a kit manufactured by New England Biolabs). That is, per sample, 5.8 μL of RNase-free water, 2 μL of 480 μM primer P2, 2 μL of 100 nM circularized T1B-EC-5p-36, 2 μL of 400 nM circularized T2, 2 μL of 10 mM dNTP Mix, 2 μL of 10 × φ29 DNA polymerase Reaction buffer, 0.2 μL of BSA included in the kit, and 2 μL of φ29 DNA polymerase were mixed in a PCR tube to prepare a premix. To this premix, 2 μL of a suspension of colonies cultured in LB medium of E. coli W3110 in RNase-free water was added to prepare a sample. In addition, 2 μL of 10 nM synthetic EC-5p-36 (SEQ ID NO: 1; Eurofins Genomics) was added to the premix to prepare a positive control. Furthermore, 2 μL of RNase-free water was added to the premix to prepare a negative control.

The sample, positive control, and negative control were incubated at 37°C for 16 hours. Then, 2 μL of a 100-fold diluted solution of SYBR Gold Nucleic Acid Gel Stain (Invitrogen), a reagent whose fluorescence intensity increases when bound to nucleic acids, was added to the incubated solution and mixed. The reaction solution thus obtained was irradiated with black light (365 nm) to observe the fluorescence color development. As a result, strong yellow-green fluorescence was observed in the sample and positive control, but only weak yellow fluorescence derived from SYBR Gold Nucleic Acid Gel Stain was observed in the negative control. Therefore, it was shown that in the sample, sRNA extracted from E. coli W3110 with an aqueous solvent was amplified by isothermal gene amplification, and the amplified product was detected by fluorescence. A comparison of the fluorescence between the negative control and the sample is shown in Figure 1. In Figure 1, + indicates the sample and - indicates the negative control.

(Reference Example 5) Extraction of sRNA from Fusarium oxysporum Fusarium oxysporum IFO5942 was cultured on an LB plate for 3 days. Then, 1 mg of the cells was scraped off and suspended in 1 mL of water containing 1% by mass of RNase inhibitor (manufactured by Toyobo Co., Ltd.). Immediately after suspension or after standing at 25 ° C for 16 hours, the suspension was filtered with a 100K Amicon Ultra 0.5 filter (manufactured by Merck Millipore Co., Ltd.), and 10 μL of the filtrate was diluted with 90 μL of sterile water. After standing at 25 ° C for 16 hours, 10 μL of 1000-fold diluted SYBR Gold Nucleic Acid Gel Stain (manufactured by Thermo scientific Co., Ltd.) was added. The fluorescence of the suspension (excitation wavelength: 495 nm, emission wavelength: 540 nm) was measured using a fluorescence plate reader (Spectramax 3i, Molecular probes). The fluorescence intensity was 3.4×10 ⁶ RFU immediately after suspension, but increased to 4.2×10 ⁶ RFU after 2 hours. This confirmed that sRNA was released from Fusarium by suspending Fusarium in water, and that quantitative measurement of the release was possible.

(Reference Example 6) Detection of E. coli-derived sRNA (EC-5p-36) and Fusarium oxysporum-derived sRNA (fox_milRNA_5) Fusarium oxysporum IFO5942 was cultured on an LB plate at 25°C for 3 days. Next, the cells were scraped off, suspended in 1 mL of pure water, and allowed to stand at room temperature for 1 hour. The Fusarium oxysporum cell suspension after standing was diluted with pure water so that the OD600 value was 0.01, to give an OD0.01 Fusarium cell suspension.

E. coli W3110 was cultured on an LB plate at 37° C. for 1 day. Then, a colony was scraped off, suspended in 100 μL of pure water, and allowed to stand at room temperature for 1 hour. The E. coli W3110 cell suspension after standing was diluted with pure water to an OD600 value of 0.1 to obtain an OD0.1 E. coli W3110 cell suspension.
Using the thus obtained bacterial cell suspension, EC-5p-36 and fox_milRNA_5 were quantified by real-time PCR. EC-5p-36 sRNA (SEQ ID NO: 1) is an sRNA that is expressed in E. coli W3110 but not in Fusarium oxysporum IFO5942. On the other hand, fox_milRNA_5 sRNA (SEQ ID NO: 9; 5'-UCCGGUAUGGUGUAGUGGC-3', see PLoS One. 2014 Aug 20; 9(8): e104956.) is an sRNA that is not expressed in E. coli W3110 but is expressed in Fusarium oxysporum IFO5942.

Real-time PCR was performed using 1 μL samples of the Fusarium cell suspension at OD 0.01 and the E. coli W3110 cell suspension at OD 0.1 according to the above-mentioned <Method for quantifying sRNA using real-time PCR method>. When the detection target was fox_milRNA_5 and when the detection target was EC-5p-36, Taqman® Assay primers having a nucleotide sequence corresponding to the detection target were used. The results of Ct values obtained using the StepOnePlus® Real-time PCR System (Applied Biosystems) are shown in Table 8.

The results in Table 8 show that fox_milRNA_5 can be specifically detected from Fusarium, and EC-5p-36 can be specifically detected from E. coli W3110. It was also shown that quantitative detection of sRNA is possible using real-time PCR.

(Reference Example 7) Study of temperature conditions during extraction treatment Escherichia coli DH5α was inoculated on an LB plate and cultured, and the resulting 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 4 hours with shaking. Then, the culture solution was centrifuged at 3000G for 5 minutes, the supernatant was removed by suction, and sterile water was added. At this time, OD600 was measured and adjusted to OD=0.1 with sterile water to prepare an E. coli suspension.

Three 500 μL samples were prepared from the prepared E. coli suspension and left to stand for 1 hour at 5°C, 25°C, or 37°C. The E. coli suspension was then filtered through a 0.22 μm filter.

Reverse transcription and real-time PCR were performed on EC-5p-36 and 16S rRNA in the filtrate. 1 μL of the filtrate was collected as an RNA sample, and real-time PCR was performed for EC-5p-36 according to the above-mentioned <Method for quantification of sRNA by real-time PCR method>, and for 16S rRNA, real-time PCR was performed according to the <Method for quantification of 16S rRNA by real-time PCR method> described below. The results of the Ct values obtained by the real-time PCR method are shown in Table 9. It was found that 16S rRNA was only extracted at low concentrations. On the other hand, EC-5p-36 was extracted in sufficient amounts to determine its presence at all temperatures of 10°C, 25°C, and 37°C. In addition, since the Ct value decreased as the temperature during extraction (immersion) increased, it can be observed that the higher the temperature, the easier it is to extract. In this way, it can be seen that EC-5p-36 (23 bases), which has a relatively small number of bases, has a low Ct value and can be stably detected. In contrast, 16S rRNA, which has a relatively large number of bases (approximately 1,500 bases), has a high Ct value and is difficult to detect.

The quantitative determination method of 16S rRNA by real-time PCR is described below.
<16S rRNA quantification method using real-time PCR method>
The 16S rRNA to be determined as the presence state was quantified by the method described below. 1 μL of RNA sample was used, and the liquid volume was adjusted to 10 μL. The reverse transcription reaction was performed in 10 μL of reaction solution, which contained 100 nM primer (SEQ ID NO: 12: CCGGGAACGTATTCACC), 0.4 mass% Moloney Murine Leukemia Virus Reverse Transcriptase (manufactured by Promega), 20 volume% 5X Reaction Buffer, 0.4 mM each of dNTP, 0.1 mass% RNase inhibitor (manufactured by Toyobo Co., Ltd.), and 10 volume% of the extract (RNA sample). The temperature profile of the reverse transcription reaction included the steps of (1) 16°C for 30 min, followed by (2) 42°C for 20 min, and (3) 85°C for 5 min.

Next, real-time PCR was carried out using the obtained reverse transcription reaction solution. 2 μL of the reverse transcription reaction solution was used, and the liquid volume was adjusted to 20 μL. The PCR reaction solution contained 300 nM primer 1 (SEQ ID NO: 13: AGAGTTTGATCATGGCTCAG), 300 nM primer 2 (SEQ ID NO: 14: CCGGGAACGTATTCACC), 200 μM each of dNTPs (CleanAmp™ Hot Start dNTP Mix, obtained from Sigma-Aldrich, USA) (Note: previously filtered and purified through an Amicon Ultra 50 K centrifugal filter from Merck Millipore), 50 mM KCl, 2.25 mM MgCl ₂ , 10 mM Tris-HCl (pH 8.3), 1× EvaGreen (Biotium The reverse transcription reaction solution used was a solution containing 0.05 units/μL of eukaryote-made thermostable DNA polymerase (see J Clin Microbiol. 2011 49(9) 3316-3320), and 10% by volume of the above reverse transcription reaction solution. The temperature profile of this real-time PCR reaction included a denaturation step at 95°C for 10 minutes, followed by 40 cycles of 94°C for 10 seconds, 65°C for 20 seconds, 72°C for 30 seconds, and 85°C for 10 seconds. Using a StepOnePlus (registered trademark) real-time PCR system (Applied Biosystems), Ct values were calculated by automatic calculation using the StepOnePlus software under the conditions of reagents = "SYBR Green reagents" and ramp speed = "Standard" (all other settings were default).

(Reference Example 8) Detection of bacteria belonging to the Enterobacteriaceae family Escherichia coli, Citrobacter freundii, and Salmonella gallinarum, which are bacteria belonging to the Enterobacteriaceae family, were inoculated and cultured on an LB plate, 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 aspirated, 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 left to stand for 1 hour at 5°C, 25°C, or 37°C. The bacterial suspension was then filtered through a 0.22 μm filter.

Reverse transcription and real-time PCR were performed on the sRNAs (EC-5p-36, EC-3p-40, and EC-5p-79) in the filtrate. Real-time PCR was performed using 1 μL samples of each filtrate 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 10. In all cases, the Ct value was 30 or less, and the presence of the Enterobacteriaceae bacteria being investigated was detected.

(Example 9) Detection of bacteria belonging to the family Enterobacteriaceae using sRNA19, 20, 21, and 40 The procedure up to preparation of a bacterial suspension with OD = 1 was carried out in the same manner as in Reference Example 8. 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 (sRNA19, sRNA20, sRNA21, and sRNA40) 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 11. 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 10) Electrophoresis of nucleic acid extract Escherichia coli W3110 was inoculated on an LB plate and cultured, and the resulting colonies were suspended in 2 mL of LB medium and cultured at 37 ° C. and 180 rpm for 24 hours with shaking. Then, 100 μL of the culture solution was collected and inoculated into 10 mL of LB medium, and cultured at 37 ° C. and 180 rpm for 4 hours with shaking. Then, the culture solution was centrifuged at 3000 G for 5 minutes, the supernatant was removed by suction, and sterile water was added. At this time, OD600 was measured and adjusted to OD = 1 with sterile water to prepare a bacterial suspension.

500 μL of the bacterial suspension was left to stand at 37°C for 1 hour. The suspension was then centrifuged at 3000G for 5 minutes, and the supernatant was filtered through a 0.22 μm filter. 10 μL of the filtrate was mixed with 2 μL of 6x Loading buffer (manufactured by Takara Bio Inc.), added to a 2% by mass agarose gel (LO3, manufactured by Takara Bio Inc.), and electrophoresed in TAE (Tris-acetate with EDTA) buffer. The results are shown in Figure 2. Gene ladder wide 1 (manufactured by Nippon Gene Co., Ltd.) was used as the ladder (lane 2 on the right). 0.01% by mass SYBR Green II (manufactured by Takara Bio Inc.) was used as the fluorescent substance that stains nucleic acids. Lane 1 on the left is the lane into which the filtrate was loaded.

As a result of electrophoresis, fluorescence was observed in regions of 500 bases or less in length, and particularly in regions of 100 bases or less in length. Fluorescence depends on the concentration of the substance, but if the substance is a low-molecular-weight substance, the number of molecules will be calculated to be large even with the same fluorescence, so it was shown that the substance concentration (number of molecules) of nucleic acids 100 bases or less in length is particularly high.

(Reference Example 11) Detection of airborne bacteria Escherichia coli DH5α was inoculated on an LB plate and cultured, and the resulting colonies were suspended in 2 mL of LB medium and cultured with shaking at 37°C and 180 rpm for 24 hours. Then, 100 μL of the culture solution was collected and inoculated into 10 mL of LB medium and cultured with shaking at 37°C and 180 rpm for 4 hours. Then, the culture solution was centrifuged at 3000G for 5 minutes, the supernatant was removed by suction, and sterile water was added. At this time, OD600 was measured and adjusted to OD=1 with sterile water to prepare an E. coli suspension. The obtained E. coli suspension was placed in a 30 mL glass spray.

An empty petri dish (diameter 9 cm) for airborne bacterial cell collection and a petri dish containing LB solid medium (diameter 9 cm) as a positive control were placed on a table, and E. coli suspension was sprayed once from 30 cm above the surface using a glass sprayer. The petri dish containing LB solid medium was covered and placed in an incubator at 37°C for one day of culture. 500 μL of sterile water was added to the empty petri dish, and the sterile water was spread throughout the petri dish using a cone rod, after which the liquid in the petri dish was collected and allowed to stand at 37°C for one hour. The amount of EC-5p-36 present in the liquid collected from the empty petri dish was measured by real-time PCR according to the above-mentioned <Method for quantifying sRNA using real-time PCR method>, and the Ct value was 28 or less, confirming the presence of EC-5p-36. On the other hand, real-time PCR was performed directly on sterile water as a negative control to measure the amount of EC-5p-36 present, and the Ct value was 33 or more, denying the presence of EC-5p-36. Additionally, colony formation was observed in the positive control LB dish.

The results of Reference Example 11 suggest that the determination method and identification method disclosed herein can also be applied to measurement samples obtained by collecting airborne bacterial cells.

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 (26)

A measurement sample is immersed in an aqueous solvent at a temperature range of 0°C to 50°C to prepare a liquid sample, or a measurement sample which is an aqueous solvent is prepared as a liquid sample;
Detecting the presence of one or more types of sRNA in the liquid sample;
determining the presence of bacteria belonging to the family Enterobacteriaceae having a cell wall based on the presence of the one or more types of sRNA;
Including,
Does not involve disrupting cell walls and/or cell membranes;
The aqueous solvent does not contain a denaturing component that causes denaturation of a cell membrane, or contains a denaturing component in an amount that does not cause denaturation of a cell membrane,
The one or more types of sRNA include at least one type of sRNA represented by SEQ ID NOs: 15 to 54;
A method for determining the presence or absence of Enterobacteriaceae bacteria having a cell wall. The method of claim 1, wherein the one or more types of sRNA include at least one type of sRNA represented by SEQ ID NOs: 19 to 21 and 40. The method of claim 1 or 2, wherein the one or more types of sRNA exhibit a specific expression state in the Enterobacteriaceae bacteria. The method according to any one of claims 1 to 3, wherein determining the presence state of bacteria of the Enterobacteriaceae family includes determining the overall presence state of two or more types of bacteria of the Enterobacteriaceae family having a cell wall. The method according to any one of claims 1 to 4, wherein detecting the state of existence of the sRNA includes detecting the amount of the sRNA present. The method according to any one of claims 1 to 5, wherein the bacteria of the Enterobacteriaceae family include at least one species selected from the group consisting of Escherichia coli, Citrobacter freundii, and Salmonella gallinarum. The determination method according to any one of claims 1 to 5, wherein the bacteria of the Enterobacteriaceae family include verotoxin-producing bacteria. The method according to any one of claims 1 to 7, wherein the measurement sample is a measurement sample obtained by collecting airborne matter, and determining the presence state of bacteria of the Enterobacteriaceae family includes determining the presence state of bacteria of the Enterobacteriaceae family having a cell wall that is present in the air. The method according to any one of claims 1 to 8 , wherein the number of bases of each of the one or more types of sRNA is within the range of 5 to 500. The method according to any one of claims 1 to 9, wherein the one or more types of sRNA further comprises at least one type 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: 10, EC-3p-393 having a nucleotide sequence represented by SEQ ID NO: 11, fox_milRNA_5 having a nucleotide sequence represented by SEQ ID NO: 9 , miR156 having a nucleotide sequence represented by SEQ ID NO: 4, and miR716b having a nucleotide sequence represented by SEQ ID NO: 5. The method according to any one of claims 1 to 10 , wherein the aqueous solvent contains a reagent for nucleic acid amplification. The method according to any one of claims 1 to 11 , wherein the detection of the presence state of the one or more types of sRNA is carried out by isothermal gene amplification. The method according to claim 12 , wherein the isothermal gene amplification is carried out at a temperature within a range of 10°C to 40°C. The method according to any one of claims 1 to 11 , wherein the detection of the presence of the one or more types of sRNA is carried out by PCR. A measurement sample is immersed in an aqueous solvent at a temperature range of 0°C to 50°C to prepare a liquid sample, or a measurement sample which is an aqueous solvent is prepared as a liquid sample;
Detecting the presence of one or more types of sRNA in the liquid sample;
Identifying bacteria belonging to the Enterobacteriaceae family that have a cell wall and are present in the measurement sample based on the presence state of the one or more types of sRNA;
Including,
Does not involve disrupting cell walls and/or cell membranes;
The aqueous solvent does not contain a denaturing component that causes denaturation of a cell membrane, or contains a denaturing component in an amount that does not cause denaturation of a cell membrane,
The one or more types of sRNA include at least one type of sRNA represented by SEQ ID NOs: 15 to 54;
A method for identifying bacteria of the family Enterobacteriaceae that have a cell wall. The method of claim 15 , wherein the one or more types of sRNA include at least one type of sRNA represented by SEQ ID NOs: 19 to 21 and 40. The method of claim 15 or 16 , wherein the one or more types of sRNAs exhibit specific expression states depending on the bacteria of the Enterobacteriaceae family having a cell wall. The method of any one of claims 15 to 17 , wherein identifying bacteria belonging to the family Enterobacteriaceae having a cell wall present in the measurement sample comprises identifying one or more of two or more types of candidate bacteria as being present. The method of any one of claims 15 to 18 , wherein detecting the state of existence of the sRNA comprises detecting the amount of the sRNA present. The method of any one of claims 15 to 19, wherein the measurement sample is obtained by collecting airborne matter, and identifying the bacteria of the Enterobacteriaceae family includes identifying bacteria of the Enterobacteriaceae family that have a cell wall and are present in the air. The method according to any one of claims 15 to 20 , wherein the number of bases of each of the one or more types of sRNA is within the range of 5 to 500. The method of any one of claims 15 to 21, wherein the one or more types of sRNA further comprises at least one type 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: 10, EC-3p-393 having a nucleotide sequence represented by SEQ ID NO: 11, fox_milRNA_5 having a nucleotide sequence represented by SEQ ID NO: 9, miR156 having a nucleotide sequence represented by SEQ ID NO: 4 , and miR716b having a nucleotide sequence represented by SEQ ID NO: 5 . The method according to any one of claims 15 to 22 , wherein the aqueous solvent contains a reagent for nucleic acid amplification. The method according to any one of claims 15 to 23 , wherein the detection of the state of existence of the one or more types of sRNA is carried out by isothermal gene amplification. The method of claim 24 , wherein the isothermal gene amplification is carried out at a temperature within the temperature range of 10°C to 40°C. The method according to any one of claims 15 to 23 , wherein the detection of the state of existence of the one or more types of sRNA is carried out by PCR.