Composition for use in suppressing lysozyme-induced nucleic acid amplification inhibition, and use thereof

Abstract

To provide a composition capable of suppressing lysozyme-induced nucleic acid amplification inhibition.SOLUTION: The present invention provides a composition for use in suppressing lysozyme-induced nucleic acid amplification inhibition, the composition comprising guanidine or a salt thereof.SELECTED DRAWING: Figure 3

Description

Technical Field

[0001] The present disclosure relates to a composition for use in suppressing the inhibition of nucleic acid amplification by lysozyme and its use.

Background Art

[0002] Microorganisms such as bacteria have cell walls. Therefore, when amplifying nucleic acids in the microorganisms, the cell walls are destroyed to make the nucleic acids in the microorganisms accessible. And in this state, procedures for amplifying nucleic acids in the microorganisms are taken.

[0003] For the destruction of the cell walls of the microorganisms, particularly the cell walls of Gram-positive bacteria, lysozyme is generally used. However, it is known that the lysozyme inhibits nucleic acid amplification reactions in vitro (Non-Patent Document 1).

Prior Art Documents

Non-Patent Documents

[0004]

Non-Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0005] Therefore, for a sample containing the lysozyme, for the purpose of removing the lysozyme, nucleic acids in the sample are isolated before performing a nucleic acid amplification reaction, which requires time and cost until the nucleic acid amplification reaction is performed.

[0006] Therefore, an object of the present disclosure is to provide a composition capable of suppressing the nucleic acid amplification inhibitory action by lysozyme.

Means for Solving the Problems

[0007] To achieve the above object, a composition for use in suppressing nucleic acid amplification inhibition by the lysozyme of the present disclosure contains guanidine or a salt thereof.

[0008] A kit for use in nucleic acid amplification of the present disclosure contains guanidine or a salt thereof, lysozyme, and a nucleic acid amplification reagent.

[0009] The nucleic acid amplification method of the present disclosure includes an amplification step of amplifying a target nucleic acid in the sample in the coexistence of a sample of interest, lysozyme, and guanidine or a salt thereof.

Effects of the Invention

[0010] According to the present disclosure, the nucleic acid amplification inhibitory action by lysozyme can be suppressed.

Brief Description of the Drawings

[0011]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Modes for Carrying Out the Invention

[0012] <Definition> As used herein, "nucleic acid", "polynucleotide", or "oligonucleotide" means a polymer of deoxyribonucleotides (DNA), ribonucleotides (RNA), and / or modified nucleotides. The nucleic acid may be a single-stranded nucleic acid molecule or a double-stranded nucleic acid molecule. The polynucleotide may be composed of natural nucleotides, modified or artificial nucleotides, or both.

[0013] As used herein, "lysozyme" means an enzyme that acts on peptidoglycan in the cell wall of bacteria and hydrolyzes the β-1,4 bond between N-acetylmuramic acid and N-acetylglucosamine. When the lysozyme acts directly on living bacteria, it decomposes the cell wall of the bacteria and causes lysis. Examples of the lysozyme include animal-derived lysozymes such as egg white lysozyme (lysozyme derived from chicken) and human-derived lysozyme; and bacteria-derived lysozyme.

[0014] As used herein, a composition consisting of "unit packaging form per test" means a composition in a form in which the amount to be tested per test is predetermined. As used herein, the composition includes, for example, forms such as inspection reagents, research reagents, and test reagents. Examples of the unit packaging form per test include containers in a form that defines a certain amount, such as microtubes, centrifuge tubes, test tubes, flasks, specimen containers, feces containers, packs, packages, bottles, microplates, ampoules, and cartridges. When the composition is a liquid, examples of the unit packaging form per test include a form in which a certain amount can be defined by the above packaging or the like, or a form in which the dosage per test is indicated on the container, instruction manual, or description.

[0015] As used herein, "subject" means an animal or cells, tissues or organs derived from an animal, and is used in particular to include humans. The animal means a human and a non-human animal. Examples of the non-human animal include mammalian animals such as mice, rats, rabbits, dogs, cats, cows, horses, pigs, monkeys, dolphins, and deer.

[0016] As used herein, "sample" may contain nucleic acids or may potentially contain nucleic acids. The sample may, for example, contain microorganisms or may potentially contain microorganisms. Examples of the sample include biological samples (e.g., biological specimens or samples, etc.), environmental samples, etc. Examples of the biological sample include samples containing body fluids, cells, tissues, organs, etc. Specific examples include feces, saliva, samples derived from the lower respiratory tract (e.g., sputum, tracheal aspirate, etc.), oral rinse, pharyngeal rinse, nasopharyngeal rinse, nasal rinse, nasal discharge, conjunctival rinse, whole blood, serum, plasma, cerebrospinal fluid, urine, sweat, semen, vaginal fluid, puncture fluid, gastric juice, endometrium, vaginal skin, sebum, dental plaque, tongue coating, bile, and other samples. The biological sample is preferably a biological sample containing microorganisms. Examples of the "environmental sample" include substances wiped from devices, equipment, or articles, etc., food, air, soil, dust, sludge, water, and the like. The sample may be liquid or solid. When the sample is solid, in the present disclosure, it is preferable to mix the solid sample with a liquid to prepare a liquid sample. Examples of the liquid include water; physiological saline; buffers such as Hank's buffer, Good's buffer (HEPES buffer, Tris buffer, etc.), Tris buffer, phosphate buffer, glycine buffer, and the like.

[0017] As used herein, "exogenous" means being of a different origin from the origin of the sample. As used herein, "exogenous lysozyme" means that the origin of the sample and the origin of the lysozyme are different, that is, the animal species from which the sample was isolated is different from the animal species from which the lysozyme is derived.

[0018] As used herein, "microorganism" includes, for example, bacteria such as Gram-positive bacteria and Gram-negative bacteria; fungi such as molds and yeasts; archaea; protists; and parasites.

[0019] As used herein, "isolation of nucleic acid" means, for example, separation, purification, and / or concentration of a desired nucleic acid from a degradation product (mixture) obtained in a degradation step. The desired nucleic acid includes, for example, nucleic acids derived from microorganisms. Also, as used herein, "not including the step of isolating nucleic acid" means, for example, not subjecting the degradation product to a treatment for isolating the nucleic acid. More specifically, "not including the step of isolating nucleic acid" means, for example, not subjecting the degradation product to alcohol precipitation (e.g., ethanol precipitation, isopropanol precipitation, etc.), polyethylene glycol precipitation, phenol treatment (e.g., phenol-chloroform treatment, etc.), gel filtration, adsorption by silica or cellulose (e.g., spin column method, magnetic bead method, etc.), electrophoresis, dialysis, chromatography such as affinity, etc.

[0020] As used herein, "kit" generally means a unit in which provided components (e.g., assay reagents, diagnostic reagents, test reagents, labels, instructions, etc.) are provided in two or more compartments. The kit can be suitably used for providing a composition that is preferably not provided as a mixture for reasons such as stability and is mixed and used immediately before use. The kit preferably includes, for example, an instruction manual or description regarding the usage method of the provided components (e.g., assay reagents, diagnostic reagents, test reagents, etc.), or an instruction manual or description in which the treatment of the components is described. As used herein, when the kit is used as a reagent kit, the kit may include an instruction manual or the like in which the usage methods of assay reagents, diagnostic reagents, test reagents, etc. are described.

[0021] In this specification, the "Instruction Manual" or "Specification" describes how to use the present disclosure and provides explanations for physicians or other users. The Instruction Manual describes, for example, instructions regarding the suppression method or amplification method of the present disclosure, or how to use the composition or kit. The Instruction Manual is prepared in accordance with the format specified by the regulatory authorities of the country where the present disclosure is implemented (for example, in the case of Japan, the Ministry of Health, Labour and Welfare; in the case of the United States, the Food and Drug Administration (FDA); in the case of Europe, the European Medicines Agency (EMA), etc.), and may specify that it has been approved by the regulatory authority. The Instruction Manual may be a so-called package insert, and is usually provided in a paper medium, but is not limited thereto, and may be provided in a form such as an electronic medium (for example, a homepage provided on the Internet, an e-mail).

[0022] In this specification, "gene" refers to a factor that defines a genetic trait, and "gene" may refer to "polynucleotide", "oligonucleotide", and "nucleic acid".

[0023] In this specification, "label" means something for identifying a target molecule or substance from other molecules or substances. Examples of the label include fluorescent labels such as fluorescent dyes or fluorescent substances; chemiluminescent labels; enzyme labels such as alkaline phosphatase; radioisotope (RI) labels; and the like.

[0024] The sequence information of the protein described in this specification or the nucleic acid encoding the same (for example, DNA or RNA) is available from Protein Data Bank, UniProt, GenBank, etc. In addition, the nucleic acid sequence of RNA is also available from the base sequence of the corresponding DNA using sequence conversion software or the like as appropriate.

[0025] Hereinafter, the present disclosure will be specifically described with examples. Hereinafter, unless otherwise specified, each disclosure can incorporate the description of other disclosures.

[0026] <Composition for use in suppressing nucleic acid amplification inhibition by lysozyme> In one aspect, the present disclosure provides a composition capable of suppressing the inhibitory effect of lysozyme on nucleic acid amplification. The composition for suppressing the inhibition of nucleic acid amplification by lysozyme of the present disclosure (hereinafter, also referred to as "composition") contains guanidine or a salt thereof.

[0027] As a result of intensive research, the present inventors have found that guanidine or a salt thereof can suppress the inhibitory effect of the nucleic acid amplification reaction by lysozyme, and have established the present disclosure. According to the composition of the present disclosure, since the inhibitory effect of the nucleic acid amplification reaction by the lysozyme can be suppressed, for example, even when the sample contains lysozyme or when lysozyme is added in the treatment of the sample, the nucleic acid amplification can be carried out without performing the removal treatment of the lysozyme, for example, nucleic acid isolation. Therefore, according to the composition of the present disclosure, for example, for a sample containing a microorganism in which the cell wall is lysed by the lysozyme, the amplification of the target nucleic acid in the sample can be carried out more quickly and simply.

[0028] The composition of the present disclosure may contain only guanidine, only a salt of guanidine, or both. Therefore, the composition of the present disclosure may contain one type or a plurality of types of the guanidine or a salt thereof. Hereinafter, unless otherwise specified, the description of guanidine in the present disclosure can be applied to the description of the salt of guanidine.

[0029] The guanidine means a compound represented by HN=C(NH2)2. Examples of the guanidine include a compound registered with the Cas registration number 113-00-8.

[0030] The guanidine, for example, exists as a cation by being protonated. Therefore, the guanidine may form a salt. Examples of the salt of guanidine include guanidine hydrochloride (guanidinium hydrochloride), guanidine thiocyanate, guanidine nitrate, guanidine phosphate, guanidine carbonate, guanidine sulfamate, guanidine hydrobromide, and the like.

[0031] The content of guanidine in the composition may be, for example, 0.5 to 500 mmol, 1 to 250 mmol, 5 to 100 mmol, or 10 to 50 mmol. When the guanidine is guanidine thiocyanate, the content of guanidine in the composition may be, for example, 0.5 to 500 mmol, 0.75 to 250 mmol, 1 to 100 mmol, 2 to 90 mmol, 3 to 80 mmol, 4 to 70 mmol, 5 to 60 mmol, 6 to 50 mmol, 7 to 40 mmol, 8 to 35 mmol, 9 to 30 mmol, or 10 to 30 mmol. When the guanidine is guanidine hydrochloride, the content of guanidine in the composition may be, for example, 0.5 to 500 mmol, 1 to 400 mmol, 5 to 300 mmol, 10 to 300 mmol, 20 to 200 mmol, 30 to 100 mmol, or 30 to 50 mmol.

[0032] The composition of the present disclosure, that is, the guanidine or its salt may be in a form packaged in units to be tested per test.

[0033] When the composition of the present disclosure is a solid reagent, the composition of the present disclosure contains, for example, 0.1 to 100 μmol, 0.5 to 50 μmol, 0.75 to 25 μmol, 1 to 20 μmol, or 2 to 10 μmol of guanidine or its salt as the amount to be tested in one test (the amount to be tested in one test) in the unit, and can more effectively suppress the inhibitory effect of lysozyme on nucleic acid amplification. Therefore, preferably, it contains 1 to 20 μmol, or 2 to 10 μmol. When the guanidine is guanidine thiocyanate, the composition of the present disclosure contains, for example, 0.1 to 100 μmol, 0.5 to 50 μmol, 0.75 to 25 μmol, 1 to 20 μmol, or 2 to 10 μmol of guanidine thiocyanate as the amount to be tested in one test in the unit. When the guanidine is guanidine hydrochloride, the composition of the present disclosure contains, for example, 0.1 to 100 μmol, 0.5 to 50 μmol, 1 to 30 μmol, 2 to 20 μmol, or 5 to 15 μmol of guanidine hydrochloride as the amount to be tested in one test in the unit.

[0034] When the composition of the present disclosure is a liquid reagent, the composition of the present disclosure contains, for example, a liquid containing guanidine or a salt thereof in the unit as a test dose, for example, at a concentration of 0.1 to 100 mol / L (concentration of guanidine or a salt thereof) in an amount of 1 to 100 μL, 2 to 50 μL, 3 to 25 μL or 5 to 15 μL, at a concentration of 0.5 to 50 mol / L in an amount of 1 to 100 μL, 2 to 50 μL, 3 to 25 μL or 5 to 15 μL, at a concentration of 1 to 20 mol / L in an amount of 1 to 100 μL, 2 to 50 μL, 3 to 25 μL or 5 to 15 μL, or at a concentration of 2 to 10 mol / L in an amount of 1 to 100 μL, 2 to 50 μL, 3 to 25 μL or 5 to 15 μL. Since it can more effectively suppress the nucleic acid amplification inhibitory effect by lysozyme, preferably, it contains 1 to 100 μL, 2 to 50 μL, 3 to 25 μL or 5 to 15 μL at a concentration of 1 to 20 mol / L, or 1 to 100 μL, 2 to 50 μL, 3 to 25 μL or 5 to 15 μL at a concentration of 2 to 10 mol / L.

[0035] When the guanidine is guanidine thiocyanate, the composition of the present disclosure contains, for example, a liquid containing guanidine thiocyanate in the unit as a test dose, for example, at a concentration of 0.1 to 100 mol / L (concentration of guanidine thiocyanate) in an amount of 1 to 100 μL, 2 to 50 μL, 3 to 25 μL or 5 to 15 μL, at a concentration of 0.5 to 50 mol / L in an amount of 1 to 100 μL, 2 to 50 μL, 3 to 25 μL or 5 to 15 μL, at a concentration of 1 to 20 mol / L in an amount of 1 to 100 μL, 2 to 50 μL, 3 to 25 μL or 5 to 15 μL, or at a concentration of 2 to 10 mol / L in an amount of 1 to 100 μL, 2 to 50 μL, 3 to 25 μL or 5 to 15 μL.

[0036] When the guanidine is guanidine hydrochloride, the composition of the present disclosure contains, for example, in the unit, a solution containing guanidine hydrochloride, as one test sample amount, at a concentration of, for example, 0.1 to 100 mol / L (concentration of guanidine hydrochloride) in an amount of 1 to 100 μL, 2 to 50 μL, 3 to 25 μL or 5 to 15 μL, at a concentration of 1 to 50 mol / L in an amount of 1 to 100 μL, 2 to 50 μL, 3 to 25 μL or 5 to 15 μL, at a concentration of 2 to 25 mol / L in an amount of 1 to 100 μL, 2 to 50 μL, 3 to 25 μL or 5 to 15 μL, or at a concentration of 5 to 15 mol / L in an amount of 1 to 100 μL, 2 to 50 μL, 3 to 25 μL or 5 to 15 μL.

[0037] The composition of the present disclosure may contain, for example, lysozyme.

[0038] The form of the composition of the present disclosure may be a solid form such as powder or granule, or a liquid form such as slurry (suspension), jelly, solution.

[0039] The composition of the present disclosure can be suitably used, for example, as an inspection reagent, a test reagent or a research reagent for detecting nucleic acid in a sample containing microorganisms.

[0040] <Kit> In another aspect, the present disclosure provides a kit for use in nucleic acid amplification in which the inhibitory effect of lysozyme on nucleic acid amplification is suppressed. The kit for use in nucleic acid amplification of the present disclosure (hereinafter also referred to as "kit") includes guanidine or a salt thereof, lysozyme, and a nucleic acid amplification reagent. According to the kit of the present disclosure, the inhibitory effect of the lysozyme on the nucleic acid amplification reaction can be suppressed. Therefore, the kit of the present disclosure can perform nucleic acid amplification, for example, even when the sample contains lysozyme, without performing a treatment for removing the lysozyme, for example, nucleic acid isolation. Therefore, according to the kit of the present disclosure, for example, for a sample containing a microorganism in which cell wall lysis treatment by the lysozyme is performed, amplification of the target nucleic acid in the sample can be performed more quickly and simply.

[0041] The content of the lysozyme may be, for example, 0.01 to 2500 μg, 0.05 to 1250 μg, 0.1 to 625 μg, 0.5 to 250 μg, or 1 to 100 μg.

[0042] The lysozyme may be in a form packaged in units to be used in the test per test. When the lysozyme is a solid reagent, the lysozyme contains, for example, guanidine or a salt thereof in the unit in an amount (amount for one test) of 0.01 to 100 μg, 0.05 to 50 μg, 0.1 to 25 μg, 0.5 to 10 μg, or 1 to 5 μg for one test. Since it can more effectively decompose the cell wall of the microorganism, it preferably contains 0.1 to 25 μg, 0.5 to 10 μg, or 1 to 5 μg. When the lysozyme is a liquid reagent, the lysozyme contains, for example, a liquid containing guanidine or a salt thereof in the unit in an amount of 1 to 1000 μg / mL at a concentration of 1 to 100 μL, 2.5 to 50 μL, 5 to 40 μL, or 10 to 30 μL, 1 to 100 μL, 2.5 to 50 μL, 5 to 40 μL, or 10 to 30 μL at a concentration of 5 to 500 μg / mL, 1 to 100 μL, 2.5 to 50 μL, 5 to 40 μL, or 10 to 30 μL at a concentration of 10 to 250 μg / mL, or 1 to 100 μL, 2.5 to 50 μL, 5 to 40 μL, or 10 to 30 μL at a concentration of 25 to 100 μg / mL. Since it can more effectively decompose the cell wall of the microorganism, it preferably contains 1 to 100 μL, 2.5 to 50 μL, 5 to 40 μL, or 10 to 30 μL at a concentration of 10 to 250 μg / mL, or 1 to 100 μL, 2.5 to 50 μL, 5 to 40 μL, or 10 to 30 μL at a concentration of 25 to 100 μg / mL.

[0043] The nucleic acid amplification reagent is a reagent capable of amplifying nucleic acid in the sample, and examples thereof include DNA polymerase, primers, substrates, buffers, and the like. The nucleic acid amplification reagent includes one or more types of reagents.

[0044] The DNA polymerase can be selected according to the nucleic acid amplification method. For example, a heat-resistant polymerase can be used as the DNA polymerase. Specific examples include Taq, Tbr, Tfl, Tru, Tth, Tli, Tac, Tne, Tma, Tih, Tfi, Pfu, Pwo, Kod, Bst, GBD, Sac, Sso, Poc, Pab, Mth, Pho, Pfu, ES4, VENT (trademark), DEEPVENT (trademark), and variants thereof. The DNA polymerase may be, for example, a hot start DNA polymerase.

[0045] The primer is, for example, a primer set specific to the target nucleic acid (e.g., a forward primer and a reverse primer), and can be designed according to the base sequence of the nucleic acid to be amplified, that is, the base sequence of the target nucleic acid. The base sequence of the primer can be designed using, for example, a primer design tool (software). The primer can be chemically synthesized using, for example, an automatic DNA synthesizer based on the designed base sequence. The primer may include, for example, one type or a plurality of types, and three or more types may be included according to the target nucleic acid.

[0046] Examples of the substrate include deoxynucleoside triphosphates or a mixture thereof. Specific examples include deoxycytidine triphosphate (dCTP), deoxyguanosine triphosphate (dGTP), deoxyadenosine triphosphate (dATP), deoxythymidine triphosphate (dTTP), deoxyuridine triphosphate (dUTP), derivatives thereof, or a mixture of two or more of these. Examples of the mixture include a mixture of dCTP, dGTP, dATP, and dTTP, a mixture of dCTP, dGTP, dATP, and dUTP, a mixture of dCTP, dGTP, dATP, dTTP, and dUTP, and the like.

[0047] The buffer solution includes, for example, Tris buffer solution, Tris-EDTA (TE) buffer solution, Bis-Tris-Tricine buffer solution, Bis-Tricine buffer solution, phosphate buffer solution, HEPES buffer solution, Mops buffer solution, Tes buffer solution, Taps buffer solution, Pipes buffer solution, Caps buffer solution, MES buffer solution, etc.

[0048] The nucleic acid amplification reagent may contain other components, for example. The other components include, for example, manganese ions (Mn 2+ ), cobalt ions (Co 2+ ), magnesium ions (Mg 2+divalent ions such as; chlorides such as lithium chloride, sodium chloride, potassium chloride, magnesium chloride, manganese chloride, acetates such as lithium acetate, sodium acetate, potassium acetate, magnesium acetate, manganese acetate, sulfates such as potassium sulfate, magnesium sulfate, manganese sulfate, etc.; anionic surfactants such as sodium dodecyl sulfate, sodium cholate, sodium deoxycholate, cationic surfactants such as cetyltrimethylammonium bromide; nonionic surfactants such as octylphenol ethoxylate, polyoxyethylene lauryl ether, polyoxyethylene oleyl ether, polyoxyethylene stearyl ether, polyoxyethylene sorbitan monolaurate, Triton®-X-100, Triton®-X-102, Triton®-X-114, Triton®-X-165, Triton®-X-45, Triton®-X-305, Triton®-X-405, NP40, Brij-35, Brij-58, n-octyl-β-d-glucoside, Tween®20, Tween®40, Tween®60, Tween®65, Tween®80, etc.; zwitterionic surfactants such as 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid, etc.; organic solvents such as methanol, ethanol, isopropanol, acetonitrile, dimethyl sulfoxide, etc.; molecular chaperones such as α-cyclodextrin, β-cyclodextrin, or γ-cyclodextrin and its derivatives, cycloamylose, etc. are mentioned.

[0049] In the kit of the present disclosure, the form of each reagent may be a solid form such as powder or granule, or a liquid form such as slurry (suspension), jelly, solution, etc.

[0050] In the kit of the present disclosure, the nucleic acid amplification reagent may be, for example, such that each component may be separately contained, or a part or all of them may be contained in a mixed or unmixed state. In the kit of the present disclosure, when all the reagents are contained in one container in a mixed or unmixed state, the kit of the present disclosure can also be referred to as, for example, a reagent for nucleic acid amplification.

[0051] The kit of the present disclosure may further include, for example, a container for storing the sample. In this case, since the guanidine or its salt can improve the preservability of the sample and suppress the nucleic acid amplification inhibitory effect by lysozyme when the sample is subjected to a nucleic acid amplification reaction after lysozyme treatment, it is preferably placed in the container. When the guanidine or its salt is placed in the container, the guanidine or its salt is preferably in a liquid state. The container can employ the examples of the aforementioned containers. The container may include, for example, sampling instruments such as a swab, a brush, a cotton swab, a spoon, a scraper, a rod having a tip in the shape of a drill, a spatula, or a fork, and a rod having an opening.

[0052] The kit of the present disclosure may include, for example, an instruction manual or a description.

[0053] The kit of the present disclosure can be suitably used, for example, as an inspection kit, a test kit, or a research kit for detecting nucleic acids in a sample containing microorganisms.

[0054] <Method for suppressing nucleic acid amplification inhibition by lysozyme> In another aspect, the present disclosure discloses a method capable of suppressing the inhibitory effect of lysozyme on nucleic acid amplification. The method for suppressing nucleic acid amplification inhibition by lysozyme of the present disclosure (hereinafter, also referred to as "suppression method") includes a coexistence step of coexisting a target sample, lysozyme, and guanidine or a salt thereof. According to the suppression method of the present disclosure, the inhibitory effect of the nucleic acid amplification reaction by the lysozyme can be suppressed. Therefore, according to the suppression method of the present disclosure, for example, even when the lysozyme is contained, nucleic acid amplification can be carried out without performing the removal treatment of the lysozyme, for example, isolation of nucleic acid derived from the sample. Therefore, according to the suppression method of the present disclosure, for example, for a sample containing a microorganism in which cell wall lysis treatment by the lysozyme is performed, a sample capable of directly amplifying the target nucleic acid in the sample can be prepared.

[0055] The suppression method of the present disclosure can also be, for example, a method (production method) for preparing a lysozyme-containing sample capable of nucleic acid amplification, or a method (production method) for preparing a lysozyme-containing sample with improved nucleic acid amplification efficiency.

[0056] The suppression method of the present disclosure includes a coexistence step of coexisting a target sample, lysozyme, and guanidine or a salt thereof. Thereby, the suppression method of the present disclosure can suppress the inhibitory effect of the nucleic acid amplification reaction by the lysozyme, for example, when the sample after the coexistence is subjected to nucleic acid amplification. The coexistence can be carried out by bringing the target sample, the lysozyme, and the guanidine into contact or mixing. The coexistence is preferably carried out in a liquid system (coexistence system or mixing system) containing water, physiological saline, or the buffer solution. The order of the contact or mixing is not particularly limited and can be in any order. Specific examples of the order include, for example, contact or mixing of the sample and guanidine, followed by contact or mixing of these with the lysozyme, contact or mixing of the sample and lysozyme, followed by contact or mixing of these with the guanidine, and the like. The lysozyme can also be, for example, the lysozyme added to the coexistence system.

[0057] In the coexistence step, the concentration of the lysozyme is, for example, more than 0 mg / mL and 270 μg / mL or less, 4.4 to 220 μg / mL, 8.8 to 176 μg / mL, 17.6 to 88 μg / mL, 44 to 132 μg / mL, 88 to 132 μg / mL. Since the cell wall of the microorganism can be decomposed more effectively, it is preferably 8.8 to 220 μg / mL or 17.6 to 176 μg / mL. The concentration of the lysozyme may be, for example, the total concentration of the lysozyme derived from the sample and the added lysozyme, or the concentration of only the added lysozyme. When the origin of the sample is different from the origin of the lysozyme, the concentration of only the added lysozyme can also be referred to as the concentration of exogenous lysozyme.

[0058] In the present disclosure, the molecular weight of the lysozyme is about 14300. Therefore, in the present disclosure, the concentration of the lysozyme may be converted to a molar concentration using the molecular weight, for example. In this case, in the coexistence step, the lysozyme concentration is, for example, more than 0 nmol / L and 18.87 nmol / L or less, 0.31 to 15.38 nmol / L, 0.61 to 12.3 nmol / L, 3.07 to 9.23 nmol / L, or 6.15 to 9.23 nmol / L.

[0059] In the coexistence step, the concentration of the guanidine is, for example, more than 0 mmol / L and 500 mmol / L or less, 17.6 to 264 mmol / l, 50 to 250 mmol / L, or 60 to 200 mmol / L. Since the inhibitory effect of the lysozyme on nucleic acid amplification can be more effectively suppressed, it is preferably 17.6 to 264 mmol / L, 50 to 250 mmol / L, or 60 to 200 mmol / L. In the coexistence step, by setting the concentration of the guanidine to 264 mmol / L or less, the nucleic acid amplification reaction specific to the target nucleic acid can be promoted more effectively. Also, in the coexistence step, by setting the concentration of the guanidine to 17.6 mmol / L or more, the inhibitory effect of the lysozyme on nucleic acid amplification can be more effectively suppressed. The guanidine may be the concentration of one type of guanidine or its salt, or the total concentration of multiple types of guanidine or its salts.

[0060] When the guanidine is guanidine thiocyanate, the concentration of the guanidine thiocyanate is, for example, more than 0 mmol / L, 500 mmol / L or less, 8.8 to 193.6 mmol / L, 17.6 to 176 mmol / L, 35.2 to 158.4 mmol / L, 52.8 to 140.8 mmol / L because the inhibitory effect of lysozyme on nucleic acid amplification can be more effectively suppressed. By setting the concentration of the guanidine thiocyanate to 158.4 mmol / L or less, the nucleic acid amplification reaction specific to the target nucleic acid can be more promoted. Further, in the coexistence step, by setting the concentration of the guanidine thiocyanate to 17.6 mmol / L or more, the inhibitory effect of lysozyme on nucleic acid amplification can be more effectively suppressed.

[0061] When the guanidine is guanidine hydrochloride, the concentration of the guanidine hydrochloride is, for example, more than 0 mmol / L, 500 mmol / L or less, 8.8 to 264 mmol / L, 35.2 to 228.8 mmol / L, 88 to 193.6 mmol / L, 105.6 to 176 mmol / L, 123.2 to 158.4 mmol / L because the inhibitory effect of lysozyme on nucleic acid amplification can be more effectively suppressed. By setting the concentration of the guanidine hydrochloride to 228.8 mmol / L or less, the nucleic acid amplification reaction specific to the target nucleic acid can be more promoted. Further, in the coexistence step, by setting the concentration of the guanidine hydrochloride to 88 mmol / L or more, the inhibitory effect of lysozyme on nucleic acid amplification can be more effectively suppressed.

[0062] In the coexistence step, the molar concentration (C L ) of lysozyme and the molar concentration (C G ) of the guanidine or its salt, and the concentration ratio (C L :C G ) are, for example, 1:1×10 5 to 1:1×10 9 , 5:1×10 5 to 1:5×10 9 , 1:1×10 6 to 1:1×10 9 , 1:5×10 6~1:1×10 9 、 or 1:1×10 7 ~1:1×10 9 and can more effectively promote the nucleic acid amplification reaction specific to the target nucleic acid and more effectively suppress the nucleic acid amplification inhibitory effect by lysozyme. Therefore, preferably, 1:1×10 6 ~1:1×10 9 、 1:5×10 6 ~1:1×10 9 、 or 1:1×10 7 ~1:1×10 9 .

[0063] When the guanidine is guanidine thiocyanate, the concentration ratio (C L :C G ) is 1:1×10 5 ~1:1×10 9 、 5:1×10 5 ~1:5×10 9 、 1:1×10 6 ~1:1×10 9 、 1:1.43×10 6 ~1:1×10 9 、 1:1.79×10 6 ~1:1×10 9 、 1:2.38×10 6 ~1:1×10 8 、 1:3.58×10 6 ~1:1×10 8 、 1:7.17×10 6 ~1:1×10 8 、 or 1:7.1×10 7 ~1:1×10 8 and can more effectively promote the nucleic acid amplification reaction specific to the target nucleic acid and more effectively suppress the nucleic acid amplification inhibitory effect by lysozyme. Therefore, preferably, 1:1.79×10 6 ~1:1×10 9 、 1:2.38×10 6 ~1:1×10 8 、 1:3.58×10 6 ~1:1×10 8 、 1:7.17×10 6 ~1:1×10 8 、 or 1:7.1×10 7~1:1×10 8 is as follows.

[0064] When the guanidine is guanidine hydrochloride, the concentration ratio (C L :C G ) is 1:1×10 5 ~1:1×10 9 , 5:1×10 5 ~1:5×10 9 , 1:1×10 6 ~1:1×10 9 , 1:2.86×10 6 ~1:1×10 9 , 1:3.58×10 6 ~1:1×10 9 , 1:4.77×10 6 ~1:1×10 9 , 1:7.15×10 6 ~1:1×10 9 , 1:1.43×10 7 ~1:1×10 9 , or 1:1.42×10 8 ~1:1×10 9 ; it can more effectively promote the nucleic acid amplification reaction specific to the target nucleic acid and more effectively suppress the nucleic acid amplification inhibitory effect of lysozyme. Therefore, preferably, it is 1:1.43×10 7 ~1:1×10 9 , or 1:1.42×10 8 ~1:1×10 9 .

[0065] The suppression method of the present disclosure may include a decomposition step of decomposing the cell wall of microorganisms in the sample by bringing the sample into contact with the lysozyme. The decomposition conditions (for example, temperature, time, pH) in the decomposition step can be appropriately set according to, for example, the content of microorganisms in the sample and the origin of the lysozyme. As a specific example, the decomposition temperature is, for example, 30 to 60°C. The decomposition time is, for example, 5 to 30 minutes.

[0066] The guanidine, for example, does not inhibit the degradation of the cell wall by the lysozyme. Therefore, the degradation step may be carried out in the presence of the guanidine or a salt thereof. In this case, the suppression method of the present disclosure may carry out the degradation step in the coexistence step or in addition to the coexistence step. When carrying out the degradation step in the coexistence step, the coexistence step may also be referred to as a degradation step, for example.

[0067] When the suppression method of the present disclosure includes, for example, the degradation step, it is preferably free of an isolation step of isolating the nucleic acid of the microorganism after the degradation. The suppression method of the present disclosure suppresses, for example, the inhibitory action of the lysozyme on nucleic acid amplification as described above. Therefore, according to the suppression method of the present disclosure, for example, even when the sample contains lysozyme, nucleic acid amplification can be carried out without performing a removal treatment of the lysozyme, for example, isolation of nucleic acid derived from the sample.

[0068] The suppression of the nucleic acid amplification inhibition can also be said, for example, that the nucleic acid amplification is promoted. The nucleic acid amplification is, for example, nucleic acid amplification specific to a target nucleic acid. The suppression of the nucleic acid amplification inhibition can be evaluated by, for example, quantitative PCR (qPCR) according to Example 2 described below. The evaluation is, for example, when the relative amount of the amplification product in a sample containing the test substance and lysozyme is 1.5 times or more, 2 times or more, 4 times or more, 8 times or more, or 16 times or more compared to the relative amount of the amplification product in a control sample under the same conditions except that the test substance is not included, the test substance can be evaluated as capable of suppressing the nucleic acid amplification inhibition.

[0069] <Nucleic Acid Amplification Method> In another aspect, the present disclosure provides a nucleic acid amplification method in which the inhibitory effect of lysozyme on nucleic acid amplification is suppressed. The nucleic acid amplification method of the present disclosure (hereinafter, also referred to as "amplification method") includes a step of amplifying a target nucleic acid in the sample in the coexistence of a target sample, lysozyme, and guanidine or a salt thereof. In the amplification method of the present disclosure, in the amplification step, since the lysozyme and the guanidine or a salt thereof coexist, the inhibitory effect of the nucleic acid amplification reaction by the lysozyme can be suppressed. Therefore, according to the amplification method of the present disclosure, for example, even when the lysozyme is included, nucleic acid amplification can be carried out without performing a removal treatment of the lysozyme, for example, isolation of nucleic acid derived from the sample. Therefore, according to the amplification method of the present disclosure, for example, for a sample containing a microorganism in which cell wall lysis treatment by the lysozyme is performed, amplification of the target nucleic acid can be suitably carried out.

[0070] In the amplification step, the target nucleic acid in the sample is amplified in the coexistence of lysozyme and guanidine or a salt thereof. The coexistence can be prepared, for example, in the same manner as the coexistence system in the suppression method of the present disclosure. In this case, in the amplification step, for example, a nucleic acid amplification reagent used for amplifying the target nucleic acid is added to the coexistence system to prepare an amplification reaction system. Further, when the sample is a sample containing lysozyme without addition of the guanidine or a salt thereof, in the amplification step, for example, the guanidine or a salt thereof and a nucleic acid amplification reagent used for amplifying the target nucleic acid are added to the sample to prepare an amplification reaction system. Next, in the amplification step, for example, a nucleic acid amplification reaction is carried out on the amplification reaction system to amplify the target nucleic acid in the sample.

[0071] The nucleic acid amplification reaction may be a DNA amplification reaction for amplifying the target nucleic acid using DNA as a template, or an RNA amplification reaction for amplifying the target nucleic acid using RNA as a template. Examples of the DNA amplification reaction include a polymerase chain reaction (PCR) method, a loop-mediated isothermal amplification (LAMP) method, an isothermal chimeraprimer nucleic acid amplification (ICAN) method, a strand displacement amplification (SDA) method, and the like. Examples of the RNA amplification reaction include a transcription-mediated amplification (TMA) method, a nucleic acid sequence-based amplification (NASBA) method, and the like.

[0072] The reaction conditions (e.g., temperature, time, and number of cycles) of the nucleic acid amplification reaction can be appropriately set according to, for example, the type of the nucleic acid amplification reaction, the type of the DNA polymerase, the type of the sample, and the like.

[0073] The target nucleic acid can be, for example, any nucleic acid region in the nucleic acid contained in the sample. The nucleic acid contained in the sample may be DNA or RNA. Examples of the target nucleic acid include conserved genes such as 16S rRNA gene, 23S rRNA gene, 5S rRNA gene, and protein elongation factor Tu gene, and species-specific gene sequences.

[0074] The amplification method of the present disclosure may include a detection step of detecting the amplified target nucleic acid (amplification fragment) after or during the amplification step. The detection of the amplification fragment can be carried out by, for example, a fluorescence detection method, electrophoresis using a gel such as an agarose gel or an acrylamide gel, a nucleic acid chromatography method, a quencher-mediated fluorescence detection method, Southern blotting, Northern blotting, or the like. The detection of the amplification fragment may be, for example, the detection of the base sequence of the amplification fragment. The detection of the base sequence of the amplification fragment can be carried out by, for example, short-read sequencing such as Sanger sequencing, sequencing by the Sequence-By-Synthesis (SBS) method, pyrosequencing; long-read sequencing such as single-molecule-real-time (SMRT) sequencing, nanopore sequencing; and the like.

[0075] The detection step may be carried out in parallel with the amplification step, that is, the detection step may perform detection in real time during the implementation of the amplification step. In this case, the detection step can be carried out by a fluorescence detection method using an intercalating fluorescent dye or a fluorescently labeled probe or the like.

[0076] The intercalator fluorescent dye is, for example, a dye that binds to double-stranded DNA synthesized by the nucleic acid amplification and emits fluorescence upon irradiation with excitation light. When using the intercalator fluorescent dye, in the detection step, the amount of amplified fragment can be measured by measuring the intensity of the fluorescence.

[0077] Examples of the fluorescently labeled probe include TaqMan (registered trademark) probe, Molecular Beacon, cycling probe, etc. The TaqMan probe is an oligonucleotide modified with a fluorescent dye at the 5'-end and a quencher substance at the 3'-end. Examples of the fluorescent dye include FAM, ROX, Cy5, Alexa dye, etc. Examples of the quencher include TAMRA (registered trademark), Non Fluorescent Quencher (NFQ), etc.

[0078] According to the nucleic acid amplification method of the present disclosure, for example, a sample containing a microorganism including the cell wall, such as a fecal sample, etc., can also suitably amplify the target nucleic acid. Therefore, the nucleic acid amplification method of the present disclosure can be suitably used, for example, as a method for detecting or examining the microorganism.

Examples

[0079] Hereinafter, the present disclosure will be described in detail using examples, but the present disclosure is not limited to the embodiments described in the examples. Also, unless otherwise specified, commercially available reagents and kits were used according to the attached protocol. In the following description, "mol / L" may also be denoted as "M".

[0080] [Example 1] It was confirmed that the inhibitory effect of lysozyme on the nucleic acid amplification reaction can be suppressed by adding guanidine thiocyanate.

[0081] (1) PCR As PCR reaction solutions, four types of reaction solutions were prepared: a reaction solution without adding lysozyme and thiocyanic acid (control), a reaction solution with added lysozyme (LZ-added group), a reaction solution with added guanidine thiocyanate (GuSCN-added group), and a reaction solution with added lysozyme and guanidine thiocyanate (LZ+GuSCN-added group, example). Specifically, the PCR reaction solution for each sample was prepared by mixing the reaction reagents shown in Table 1 below. For the control, water (8.8 μL) was added without adding lysozyme and guanidine thiocyanate to the PCR reaction solution. In the LZ-added group, the final concentration of lysozyme in the PCR reaction solution was 88 μg / mL. In the GuSCN-added group, the final concentration of guanidine thiocyanate in the PCR reaction solution was 22 mM. In the LZ+GuSCN-added group, the final concentrations of lysozyme and guanidine thiocyanate in the PCR reaction solution were 88 μg / mL and 22 mM, respectively. For each of the above PCR reaction solutions, PCR was performed using a QuantStudio (registered trademark) 5 real-time PCR system (manufactured by Thermo Fisher Scientific Inc.). The reaction conditions for PCR were: after treatment at 95°C for 60 seconds, 40 cycles were repeated with 95°C for 15 seconds and 60°C for 60 seconds as one cycle.

[0082]

Table 1

[0083] · Primer set for 16S rRNA gene Forward primer (Forward primer 16S) 5’-CGGTGAATACGTTCYCGG-3’ (SEQ ID NO: 1) (Y = C, T) Reverse primer (Reverse primer 16S) 5’-AAGGAGGTGATCCRGCCGCA-3’ (SEQ ID NO: 2) (R = A, G)

[0084] (2) Electrophoresis and measurement of nucleic acid concentration For the PCR product obtained in Example 1(1) above, electrophoresis was performed using High Sensitivity D1000 ScreenTape (manufactured by Agilent Technologies, Cat No.: 5067-5584), High Sensitivity D1000 Reagent Kit (manufactured by Agilent Technologies, Cat No.: 5067-5585), and an automatic electrophoresis apparatus Agilent TapeStation System (manufactured by Agilent Technologies, G2991BA). Also, using the automatic electrophoresis apparatus, the nucleic acid concentration in each lane was measured from each band after electrophoresis, and the nucleic acid concentration in the PCR product was calculated. The measurement results of the nucleic acid concentration are shown in Table 2 below. Also, the results of the electrophoresis are shown in Figure 1.

[0085] [Table 2]

[0086] Table 2 above is a table showing the nucleic acid concentration of the PCR products obtained with each PCR reaction solution. As shown in Table 2 above, in the LZ addition group, nucleic acid could not be detected, and an inhibitory effect on nucleic acid amplification was confirmed. In contrast, the LZ+GuSCN addition group had a nucleic acid concentration comparable to that of the control. From the above, it was found that the inhibitory effect of lysozyme on the nucleic acid amplification reaction can be suppressed by guanidine thiocyanate.

[0087] Figure 1 is a photograph showing the results of electrophoresis. In Figure 1, each lane shows, from left to right, a molecular weight marker (A1(L)), a PCR product of the control (control, F1), a PCR product of the LZ addition group (LZ, G1), a PCR product of the GuSCN addition group (GuSCN, H1), and a PCR product of the LZ+GuSCN addition group (LZ+GuSCN, A2). Also, in lanes F1, H1, and A2 of Figure 1, the triangles on the right side of each lane indicate PCR products specific to the primer set for the 16S rRNA gene (SEQ ID NO: 1, SEQ ID NO: 2). As shown in Figure 1, no amplification of specific PCR products was confirmed in the LZ addition group. In contrast, amplification of specific PCR products was confirmed in the LZ+GuSCN addition group.

[0088] From the above, it was found that lysozyme inhibits the primer-specific nucleic acid amplification reaction, while guanidine thiocyanate can suppress the inhibitory effect of lysozyme on the primer-specific nucleic acid amplification reaction.

[0089] [Example 2] It was confirmed that the inhibitory effect of the nucleic acid amplification reaction caused by lysozyme can be suppressed by adding guanidine hydrochloride (guanidine hydrochloride), which is another guanidine salt.

[0090] As PCR reaction solutions, four types of reaction solutions were prepared: a control, an LZ-added group, an LZ + GuSCN-added group, and a reaction solution to which lysozyme and guanidine hydrochloride were added (LZ + GuHCl-added group, Example). The PCR reaction solutions for each sample were prepared in the same manner as in Example 1(1) above. In the LZ-added group, the final concentration of lysozyme in the PCR reaction solution was 44 μg / mL. In the LZ + GuSCN-added group, the final concentrations of lysozyme and guanidine thiocyanate in the PCR reaction solution were 44 μg / mL and 22 mM, respectively. In the LZ + GuHCl-added group, the final concentrations of lysozyme and guanidine hydrochloride in the PCR reaction solution were 44 μg / mL and 44 mM, respectively. For each of the above PCR reaction solutions, PCR was performed using the thermal cycler of Example 1(1) above. The reaction conditions for PCR were: after treatment at 95°C for 60 seconds, 40 cycles were repeated with 95°C for 15 seconds and 60°C for 60 seconds as one cycle. After the cycle treatment, the temperature was raised from 60°C to 95°C at 6°C / min for melting curve analysis. The measurement wavelengths at the end of each cycle and in the melting curve analysis were 520 nm / 470 nm (fluorescence wavelength / excitation wavelength). The fluorescent dye used was SYBR (registered trademark) Green dye. By the melting curve analysis, it was confirmed that the PCR products in each PCR reaction solution were primer-specific PCR products. These results are shown in Figure 2.

[0091] Figure 2 is a graph showing the amplification curves during PCR of each PCR reaction solution. In Figure 2, the horizontal axis represents the number of cycles, and the vertical axis represents the intensity of the fluorescence signal. As shown in Figure 2, in the LZ addition group, compared with the control, the rise of the amplification curve during the exponential amplification phase of the specific PCR product was slow. In contrast, in the LZ + GuSCN addition group and the LZ + GuHCl addition group, compared with the LZ addition group, the rise of the amplification curve during the exponential amplification phase of the specific PCR product was fast. From the above, it was found that lysozyme inhibits the primer-specific nucleic acid amplification reaction, while guanidine thiocyanate and guanidine hydrochloride can each suppress the inhibitory effect of lysozyme on the primer-specific nucleic acid amplification reaction. Also, in both guanidine thiocyanate and guanidine hydrochloride, since the inhibitory effect of lysozyme on the primer-specific nucleic acid amplification reaction can be suppressed, it was found that guanidine is important for suppressing the inhibitory effect of lysozyme on the primer-specific nucleic acid amplification reaction, that is, the amplification reaction of the target nucleic acid.

[0092] [Example 3] It was confirmed that guanidine can suppress the inhibitory effect of lysozyme on the nucleic acid amplification reaction even at different lysozyme concentrations.

[0093] As PCR reaction solutions, three types of reaction solutions were prepared: the LZ addition group, the LZ + GuSCN addition group, and the LZ + GuHCl addition group. The PCR reaction solutions for each sample were prepared in the same manner as in Example 1(1) above. In the LZ addition group, the final concentration of lysozyme in the PCR reaction solution was set to a predetermined concentration (0, 4.4, 44, 88, 132, 176, or 220 μg / mL). Also, in the LZ + GuSCN addition group, the final concentration of lysozyme in the PCR reaction solution was the same as that in the LZ addition group. In the LZ + GuSCN addition group, the final concentration of guanidine thiocyanate in the PCR reaction solution was set to 22 mM. In the LZ + GuHCl addition group, the final concentration of lysozyme in the PCR reaction solution was the same as that in the LZ addition group. In the LZ + GuHCl addition group, the final concentration of guanidine hydrochloride in the PCR reaction solution was set to 44 mM. For each of the above PCR reaction solutions, PCR was performed using the thermal cycler of Example 1(1) above. The reaction conditions for the PCR were the same as in Example 2. In addition, by the melting curve analysis, it was confirmed that the PCR products in each PCR reaction solution were primer-specific PCR products. The measurement results of the Ct (Cycle Threshold) values of the amplification curves of the PCR products in each PCR reaction solution are shown in Table 3 below.

[0094]

Table 3

[0095] Table 3 above is a table showing the Ct values during PCR of each PCR reaction solution. When the Ct value is 30 or less, it was evaluated that the amplification reaction occurred particularly preferably in each PCR reaction. As shown in Table 3, in the LZ addition group (H2O), when the lysozyme concentration was 44 μg / mL, the Ct value became higher than 17, and when the lysozyme concentration exceeded 44 μg / mL, detection became impossible. On the other hand, in the LZ+GuSCN addition group and the LZ+GuHCl addition group, an amplification reaction occurred even at a lysozyme concentration at which detection was impossible in the LZ addition group. Further, in the LZ+GuSCN addition group, when the lysozyme concentration was 220 μg / mL or less, and in the LZ+GuHCl addition group, when the lysozyme concentration was 44 μg / mL or less, the amplification reaction occurred particularly preferably.

[0096] From the above, it was found that lysozyme inhibits the primer-specific nucleic acid amplification reaction, whereas guanidine thiocyanate and guanidine hydrochloride can each suppress the inhibitory effect of lysozyme on the primer-specific nucleic acid amplification reaction. Further, it was found that guanidine thiocyanate has a higher inhibitory effect on the nucleic acid amplification inhibitory effect of lysozyme than guanidine hydrochloride.

[0097] [Example 4] It was confirmed that the inhibitory effect of the nucleic acid amplification reaction caused by lysozyme can be suppressed by adding guanidine thiocyanate.

[0098] As PCR reaction solutions, four types of reaction solutions, namely a control, an LZ addition group, a GuSCN addition group, and an LZ+GuSCN addition group, were prepared. The PCR reaction solutions for each sample were prepared in the same manner as in Example 1 above. In the LZ addition group, the final concentration of lysozyme in the PCR reaction solution was 88 μg / mL. In the GuSCN addition group, the final concentration of guanidine thiocyanate in the PCR reaction solution was 22 mM. In the LZ+GuSCN addition group, the final concentrations of lysozyme and guanidine thiocyanate in the PCR reaction solution were 88 μg / mL and 22 mM, respectively.

[0099] For each of the above PCR reaction solutions, PCR was performed using the thermal cycler of Example 1(1). The reaction conditions for the PCR were the same as those in Example 2. In addition, by the melting curve analysis, it was confirmed that the PCR products in each PCR reaction solution were primer-specific PCR products (n = 3 for each group). These results are shown in Fig. 3. Also, taking the average value of the Ct values in the control PCR reaction solution as 1, relative values were calculated for the Ct values of the other samples. These results are shown in Fig. 3.

[0100] Fig. 3 is a graph showing the amplification curves during PCR of each PCR reaction solution. In Fig. 3, the horizontal axis represents the number of cycles, and the vertical axis represents the intensity of the fluorescence signal. As shown in Fig. 3, in the LZ addition group, no amplification curve of the exponential amplification phase of the specific PCR product was confirmed. In contrast, in the LZ+GuSCN addition group, the rise of the amplification curve of the exponential amplification phase of the specific PCR product was confirmed.

[0101] Fig. 4 is a graph showing the relative values of the Ct values of the amplification curves during PCR of each PCR reaction solution. As shown in Fig. 4, in the LZ addition group, the Ct value was 0. In contrast, in the LZ+GuSCN addition group, the Ct value recovered and became comparable to that of the control.

[0102] From the above, it was found that lysozyme inhibits the primer-specific nucleic acid amplification reaction, while guanidine thiocyanate can suppress the inhibitory effect of lysozyme on the primer-specific nucleic acid amplification reaction.

[0103] [Example 5] Using human fecal specimens, it was confirmed that the inhibitory effect on the nucleic acid amplification reaction caused by lysozyme treatment can be suppressed by adding guanidine thiocyanate.

[0104] As PCR reaction solutions for human fecal samples, four types of PCR reaction solutions were prepared: a control (H2O), a reaction solution with lysozyme added (LZ-added group), a reaction solution with guanidine storage solution added (GuSCN storage solution-added group), and a reaction solution with both lysozyme and guanidine storage solution added (LZ + GuSCN storage solution-added group). The composition of the guanidine storage solution was 35.2 mM GuSCN, 8.8 mM Tris-HCl, 0.352 mM EDTA, and 0.0000088% Bromothymol blue. Specifically, first, 1 mL of the guanidine storage solution was added to 1 mg of the human fecal sample, and then lysozyme was added so that the final concentration was 0.2 mg / mL to prepare a human fecal sample solution. The human fecal sample solution was shaken under the conditions of 50 °C for 15 minutes to lyse the bacterial cells in the feces. Next, the lysed human fecal sample solution was mixed with the reaction reagents in the compounding amounts shown in Table 4 below to prepare a PCR reaction solution containing the human fecal sample. In the LZ-added group, the final concentration of lysozyme in the PCR reaction solution was 88 μg / mL. In the GuSCN storage solution-added group, the final concentration of guanidine thiocyanate in the PCR reaction solution was 35.2 mM. In the LZ + GuSCN storage solution-added group, the final concentrations of lysozyme and guanidine thiocyanate in the PCR reaction solution were 88 μg / mL and 35.2 mM, respectively. For each of the above PCR reaction solutions, PCR was performed using the thermal cycler of Example 1(1). The reaction conditions for the PCR were the same as those in Example 2. In addition, by the melting curve analysis, it was confirmed that the PCR products in each PCR reaction solution were primer-specific PCR products. These results are shown in Figure 5.

[0105]

Table 4

[0106] FIG. 5 is a graph showing the amplification curves during PCR of the PCR reaction solutions of each human fecal sample. In FIG. 5, the horizontal axis represents the number of cycles, and the vertical axis represents the intensity of the fluorescence signal. As shown in FIG. 5, in the LZ addition group, the rise of the amplification curve during the exponential amplification phase of the specific PCR product was slow. In contrast, in the GuSCN preservation solution addition group and the LZ+GuSCN preservation solution addition group, the rise of the amplification curve during the exponential amplification phase of the specific PCR product was faster compared to the LZ addition group.

[0107] From the above, it was found that in nucleic acid amplification using human fecal samples, lysozyme inhibits the primer-specific nucleic acid amplification reaction, while guanidine thiocyanate can suppress the inhibitory effect of lysozyme on the primer-specific nucleic acid amplification reaction.

[0108] [Example 6] It was confirmed that heat-denatured lysozyme inhibits the nucleic acid amplification reaction. It was also confirmed that the inhibitory effect of the nucleic acid amplification reaction caused by heat-denatured lysozyme can be suppressed by adding guanidine thiocyanate.

[0109] As PCR reaction solutions, five types of reaction solutions were prepared: a control (H2O), a reaction solution with heat-denatured lysozyme added (dLZ addition group), a reaction solution with heat-denatured lysozyme and guanidine storage solution added (dLZ + storage solution addition group), a reaction solution with guanidine storage solution added (storage solution addition group), a reaction solution with lysozyme added (LZ addition group), and a reaction solution with lysozyme and guanidine storage solution added (LZ + storage solution addition group). The heat-denatured lysozyme was prepared by heat-treating 0.3 mg / mL of lysozyme at 95°C for 30 minutes to cause heat denaturation. Specifically, the PCR reaction solution for each sample was prepared by mixing the reaction reagents shown in Table 5 below. In the dLZ addition group, dLZ + storage solution addition group, LZ addition group, and LZ + storage solution addition group, the final concentration of heat-denatured lysozyme or lysozyme in the PCR reaction solution was 132 μg / mL. Also, in the storage solution addition group, dLZ + storage solution addition group, and LZ + storage solution addition group, the final concentration of guanidine thiocyanate in the PCR reaction solution was 35.2 mM. For each of the above PCR reaction solutions, PCR was performed using the thermal cycler of Example 1(2). The reaction conditions for the PCR were the same as those in Example 2. By the melting curve analysis, it was confirmed that the PCR products in each PCR reaction solution were primer-specific PCR products. These results are shown in Figure 6.

[0110]

Table 5

[0111] Figure 6 is a graph showing the amplification curves during PCR for each PCR reaction solution. In Figure 6, the horizontal axis represents the number of cycles, and the vertical axis represents the intensity of the fluorescence signal. As shown in Figure 6, the LZ addition group and the dLZ addition group were undetectable. On the other hand, specific PCR products were confirmed in the dLZ + storage solution addition group and the LZ + storage solution addition group.

[0112] From the above, it was found that heat-denatured lysozyme inhibits primer-specific nucleic acid amplification reactions. That is, it was found that lysozyme has inhibitory activity on nucleic acid amplification reactions even when denatured as a protein. In addition, it was found that guanidine thiocyanate can suppress the inhibitory effect on nucleic acid amplification reactions caused by heat-denatured lysozyme, that is, it suppresses the inhibitory activity of lysozyme on nucleic acid amplification reactions through a pathway independent of the enzyme activity of lysozyme.

[0113] [Reference Example 1] It was confirmed that the inhibitory effect on nucleic acid amplification reactions caused by lysozyme occurs without depending on DNA polymerase. In addition, it was confirmed that nucleic acid amplification reactions can be carried out when guanidine thiocyanate or guanidine hydrochloride is added without depending on DNA polymerase.

[0114] The PCR reaction solution for each sample was prepared in the same manner except that a commercially available master mix reagent for each PCR was used instead of the qPCR mix. The final concentration of lysozyme in the PCR reaction solution was set to a predetermined concentration (0, 8.8, 17.6, 35.2, 52.8, 70.4, or 88 μg / mL). In addition, the final concentration of guanidine thiocyanate in the PCR reaction solution was set to a predetermined concentration (0, 17.6, 35.2, 52.8, or 70.4 mM). The final concentration of guanidine hydrochloride in the PCR reaction solution was set to a predetermined concentration (0, 88, 105.6, 123.2, 140.8, or 158.4 mM). For each of the above PCR reaction solutions, PCR was performed using the thermal cycler of Example 1(2). The reaction conditions for PCR were carried out according to each master mix reagent for PCR. In addition, it was confirmed by the melting curve analysis that the PCR products in each PCR reaction solution were primer-specific PCR products. The measurement results of the CT values of the amplification curves of the PCR products in each PCR reaction solution are shown in Tables 6 to 8 below.

[0115]

Table 6

[0116] Table 6 above is a table showing the Ct values during PCR of each PCR reaction solution. As shown in Table 6 above, even when using each commercially available master mix reagent for PCR, that is, in any DNA polymerase, it was confirmed that the Ct value decreased according to the lysozyme concentration, and the inhibitory effect of the nucleic acid amplification reaction occurred due to lysozyme.

[0117]

Table 7

[0118] Table 7 above is a table showing the Ct values during PCR of each PCR reaction solution. As shown in Table 7 above, even when using each commercially available master mix reagent for PCR, that is, in any DNA polymerase, it was confirmed that the nucleic acid amplification reaction occurred in the presence of guanidine thiocyanate.

[0119]

Table 8

[0120] Table 8 above is a table showing the Ct values during PCR of each PCR reaction solution. As shown in Table 8 above, even when using each commercially available master mix reagent for PCR, that is, in any DNA polymerase, it was confirmed that the nucleic acid amplification reaction occurred in the presence of guanidine hydrochloride.

[0121] As described above, the present disclosure has been described with reference to the embodiments and examples, but the present disclosure is not limited to the above embodiments and examples. Various changes that can be understood by those skilled in the art can be made to the configuration and details of the present disclosure within the scope of the present disclosure.

[0122] <Supplementary Note> Some or all of the above embodiments and examples can be described as follows in the supplementary note below, but are not limited thereto. <Composition for use in suppressing nucleic acid amplification inhibition by lysozyme> (Supplementary Note 1) A composition for use in suppressing nucleic acid amplification inhibition by lysozyme, comprising guanidine or a salt thereof. (Appendix 2) The composition according to Appendix 1, wherein the salt of guanidine is selected from the group consisting of guanidine hydrochloride, guanidine thiocyanate, guanidine nitrate, guanidine phosphate, guanidine carbonate, guanidine sulfamate, and guanidine hydrobromide. (Appendix 3) The composition according to Appendix 2, wherein the salt comprises at least one of the guanidine hydrochloride and the guanidine thiocyanate. (Appendix 4) The composition according to any one of Appendices 1 to 3, wherein the lysozyme comprises egg white lysozyme. (Appendix 5) Comprising a unit packaging form per test, The composition according to any one of Appendices 1 to 4, wherein the unit contains 0.1 to 100 μmol of the guanidine or a salt thereof as a test sample amount. (Appendix 6) Comprising a unit packaging form per test, The composition according to any one of Appendices 1 to 5, wherein the unit contains 1 to 100 μL of a solution containing the guanidine or a salt thereof at a concentration of 0.1 to 100 mol / L as a test sample amount. <Kit for use in nucleic acid amplification> (Appendix 7) A kit for use in nucleic acid amplification, comprising guanidine or a salt thereof, lysozyme, and a nucleic acid amplification reagent. (Appendix 8) Including a container for storing a sample, The kit according to Appendix 7, wherein the guanidine or a salt thereof is disposed in the container. (Appendix 9) The kit according to Appendix 7 or 8, wherein the nucleic acid amplification reagent is selected from the group consisting of DNA polymerase, primer, substrate, and buffer solution. (Appendix 10) Comprising a unit packaging form per test, The kit according to any one of Appendices 7 to 9, wherein the unit contains 0.1 to 100 μmol of the guanidine or a salt thereof as one test sample amount. (Appendix 11) Comprising a unit packaging form per test, The kit according to any one of Appendices 7 to 10, wherein the unit contains 1 to 100 μL of a liquid containing the guanidine or a salt thereof at a concentration of 0.1 to 100 mol / L as one test sample amount. (Method for Inhibiting Inhibition of Nucleic Acid Amplification by Lysozyme) (Appendix 12) A method for inhibiting inhibition of nucleic acid amplification by lysozyme, comprising a coexistence step of coexisting a target sample, lysozyme, and guanidine or a salt thereof. (Appendix 13) The inhibition method according to Appendix 12, comprising a decomposition step of decomposing the cell wall of microorganisms in the sample by bringing the sample into contact with the lysozyme. (Appendix 14) The inhibition method according to Appendix 13, wherein the microorganism includes Gram-positive bacteria. (Appendix 15) The inhibition method according to Appendix 13 or 14, wherein the decomposition is carried out in the presence of the guanidine or a salt thereof. (Appendix 16) The inhibition method according to any one of Appendices 13 to 15, not including an isolation step of isolating the nucleic acid of the microorganism after the decomposition. (Appendix 17) The inhibition method according to any one of Appendices 12 to 16, wherein the concentration of the guanidine or a salt thereof is more than 0 mmol / L and 500 mmol / L or less. (Appendix 18) The inhibition method according to any one of Appendices 12 to 17, wherein the concentration of the lysozyme is more than 0 μg / L and 270 μg / L or less. (Appendix 19) The molar concentration (C L ) of the lysozyme and the molar concentration (C G ) of the guanidine or a salt thereof, and the concentration ratio (C L :C G ) is 1:1×10 5 ~1:1×109 The suppression method according to any one of Appendices 12 to 18. (Appendix 20) The suppression method according to Appendix 18 or 19, wherein the concentration of the lysozyme is the concentration of exogenous lysozyme. (Appendix 21) The suppression method according to any one of Appendices 12 to 20, wherein the sample contains a biological sample. (Appendix 22) The suppression method according to Appendix 21, wherein the biological sample is a biological sample containing bacteria. <Nucleic acid amplification method> (Appendix 23) A nucleic acid amplification method including an amplification step of amplifying a target nucleic acid in the sample in the coexistence of a target sample, lysozyme, and guanidine or a salt thereof. (Appendix 24) The nucleic acid amplification method according to Appendix 23, including a decomposition step of decomposing the cell wall of microorganisms in the sample by bringing the sample into contact with the lysozyme. (Appendix 25) The nucleic acid amplification method according to Appendix 24, wherein the microorganism includes Gram-positive bacteria. (Appendix 26) The nucleic acid amplification method according to Appendix 24 or 25, wherein the decomposition is carried out in the presence of the guanidine or a salt thereof. (Appendix 27) The nucleic acid amplification method according to any one of Appendices 24 to 26, not including an isolation step of isolating the nucleic acid of the microorganism after the decomposition. (Appendix 28) The nucleic acid amplification method according to any one of Appendices 23 to 27, including a detection step of detecting the amplified target nucleic acid. (Appendix 29) The nucleic acid amplification method according to any one of Appendices 23 to 28, wherein the concentration of the guanidine or a salt thereof is more than 0 mmol / L and 500 mmol / L or less. (Appendix 30) The nucleic acid amplification method according to any one of Appendices 23 to 29, wherein the concentration of the lysozyme is more than 0 μg / L and 270 μg / L or less. (Appendix 31) The molar concentration (C L ) of the lysozyme and the molar concentration (C G ) of the guanidine or a salt thereof, and the concentration ratio (C L :C G ) are from 1:1×10 5 to 1:1×10 9 . The nucleic acid amplification method according to any one of Supplementary Notes 23 to 30. (Supplementary Note 32) The concentration of the lysozyme is the concentration of exogenous lysozyme. The nucleic acid amplification method according to Supplementary Note 30 or 31. (Supplementary Note 33) The sample contains a biological sample. The nucleic acid amplification method according to any one of Supplementary Notes 23 to 32. (Supplementary Note 34) The biological sample is a biological sample containing bacteria. The nucleic acid amplification method according to Supplementary Note 33.

Industrial Applicability

[0123] As described above, according to the present disclosure, the inhibitory effect of lysozyme on nucleic acid amplification can be suppressed. Therefore, the present disclosure is extremely useful, for example, in the field of inspection.

Claims

1. A composition for use in suppressing the inhibition of nucleic acid amplification by lysozyme, comprising guanidine or a salt thereof.

2. The composition according to claim 1, wherein the salt of guanidine is selected from the group consisting of guanidine hydrochloride, guanidine thiocyanate, guanidine nitrate, guanidine phosphate, guanidine carbonate, guanidine sulfamate, and guanidine hydrobromide.

3. The composition according to claim 2, wherein the salt comprises at least one of the guanidine hydrochloride and the guanidine thiocyanate.

4. The composition according to claim 1 or 2, wherein the lysozyme comprises egg white lysozyme.

5. Consisting of a unit packaging form per test, The composition according to claim 1 or 2, wherein the unit contains 0.1 to 100 μmol of the guanidine or a salt thereof as a test sample amount per test.

6. Consisting of a unit packaging form per test, The composition according to any one of claims 1 to 5, wherein the unit contains 1 to 100 μL of a liquid containing the guanidine or a salt thereof at a concentration of 0.1 to 100 mol / L as a test sample amount per test.

7. A kit for nucleic acid amplification, comprising guanidine or a salt thereof, lysozyme, and a nucleic acid amplification reagent.

8. Including a container for storing a sample, The kit according to claim 7, wherein the guanidine or a salt thereof is disposed in the container.

9. A nucleic acid amplification method, comprising an amplification step of amplifying a target nucleic acid in a sample in the coexistence of the sample, lysozyme, and guanidine or a salt thereof.

10. The nucleic acid amplification method according to claim 9, comprising a decomposition step of decomposing the cell wall of microorganisms in the sample by contacting the sample with the lysozyme.

11. The nucleic acid amplification method according to claim 9 or 10, wherein the decomposition is carried out in the presence of the guanidine or a salt thereof.

12. The nucleic acid amplification method according to claim 9 or 10, wherein the concentration of the guanidine or a salt thereof is more than 0 mmol / L and 500 mmol / L or less.

13. The nucleic acid amplification method according to claim 9 or 10, wherein the concentration of the lysozyme is more than 0 μg / L and 270 μg / L or less.

14. The molar concentration of the lysozyme (C Ｌ ) and the molar concentration of the guanidine or its salt (C Ｇ ) and the concentration ratio (C Ｌ :C Ｇ ) is 1:1 x 10 ５ ~1:1 x 10 ９ The method for amplifying nucleic acid according to claim 9 or 10,

15. The nucleic acid amplification method according to claim 9 or 10, wherein the sample comprises a biological sample.

16. The nucleic acid amplification method according to claim 15, wherein the biological sample is a biological sample containing bacteria.

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