Method for detecting tinea fungus and kit for detecting tinea fungus

Abstract

To provide a method for detecting Trichophyton and a kit for detecting Trichophyton.SOLUTION: A method for detecting Trichophyton in a biological sample includes the steps of: (a) bringing a support body having a non-polar surface holding a biological sample or a substance derived from a biological sample into contact with a first reagent containing a substrate of keratinase; and (b) detecting the substrate of keratinase after the step (a).SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present disclosure relates to a method for detecting tinea fungus, a kit for detecting tinea fungus, and the like. [Background technology]

[0002] Tinea is an infectious disease caused by tinea fungi (dermatophyte fungi). Trichophytons secrete a proteolytic enzyme (keratinase) that uses keratin as a substrate, decomposing the keratin and using it as a nutrient source for growth. Therefore, tinea fungi are prone to infect areas where keratin is abundant, such as the stratum corneum that covers the surface of the skin, nails that are formed by the transformation of the stratum corneum, and hair. Depending on the area infected by the tinea fungus, tinea is called tinea pedis (also known as athlete's foot), tinea cruris (also known as jock itch), tinea capitis (also known as tinea cruris), tinea unguium, tinea manus, tinea corporis (also known as tinea cruris), tinea facialis, etc.

[0003] Although the treatment methods for tinea are different from those for other skin diseases, there are many skin diseases (e.g., eczema, dermatitis, and dyshidrotic eczema) that present with symptoms similar to those of tinea, making it difficult to clearly distinguish and diagnose tinea from other skin diseases based on symptoms.

[0004] For this reason, the KOH direct microscopic method is commonly used to diagnose tinea, in which scales are collected from the subject's skin with tweezers or the like, dissolved in caustic potash (KOH), and then observed under a microscope. However, the KOH direct microscopic method requires considerable skill due to the difficulty of collecting samples and determining whether or not tinea fungi are present. There is also a method in which the collected scales are cultured and then the presence or absence of tinea fungi is confirmed, but this takes a considerable amount of time to obtain results.

[0005] The present inventors have previously developed a method for detecting Trichophyton by using a support having a polar surface to visualize keratinase secreted by Trichophyton when it invades the stratum corneum (Patent Document 1). [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Special Publication No. 2017-061618 Summary of the Invention [Problem to be solved by the invention]

[0007] The objective of the present invention is to provide a method and kit for detecting tinea fungi. [Means for solving the problem]

[0008] The present inventors have found that keratinase can be visualized (tinea fungi can be detected) by using a support having a non-polar surface, and have made further improvements.

[0009] The present disclosure encompasses, for example, the subject matter described in the following sections: Section 1. A method for detecting Trichophyton in a biological sample, comprising: (a) a support having a non-polar surface that supports a biological sample or a substance derived from a biological sample; contacting the keratinase with a first reagent comprising a substrate for the keratinase; (b) after step (a), detecting a substrate for said keratinase; A method comprising: Section 2. Before step (a), a support having a non-polar surface that holds the biological sample or a substance derived from the biological sample; contacting the tissue with a second reagent containing a substrate for keratinase; The method of claim 1, further comprising: Section 3. Item 3. The method according to Item 1 or 2, wherein the support is a polyvinylidene fluoride membrane. Section 4. Item 4. The method according to any one of Items 1 to 3, wherein the substrate of the keratinase in the first reagent and / or the second reagent is at least one selected from the group consisting of keratin, casein, and bovine serum albumin. Section 5. Item 5. The method according to any one of Items 1 to 4, wherein the step (b) is a step of detecting a labeling substance that binds to or is bound to a substrate of the keratinase. Section 6. Item 6. The method according to any one of Items 1 to 5, wherein the first reagent is supported on a substrate. Section 7. A kit for detecting tinea fungus, comprising a support having a non-polar surface. Section 8. Item 8. The kit according to Item 7, further comprising a first reagent comprising a substrate for keratinase. Section 9. Item 10. The kit of item 8, further comprising a second reagent comprising a substrate for keratinase. Section 10. Item 10. The kit according to any one of Items 7 to 9, wherein the support is a polyvinylidene fluoride membrane. Section 11. Item 11. The kit according to any one of Items 8 to 10, wherein the substrate of the keratinase in the first reagent and / or the second reagent is at least one selected from the group consisting of keratin, casein, and bovine serum albumin. Section 12. Item 12. The kit according to any one of Items 8 to 11, further comprising a labeling substance that binds to or is bound to a substrate of the keratinase. Section 13. Item 13. The kit according to any one of Items 8 to 12, wherein the first reagent is supported on a substrate. [Effects of the Invention]

[0010] Methods and kits for detecting tinea fungi are provided. [Brief explanation of the drawings]

[0011] [Figure 1] Fluorescence micrographs of nitrocellulose and PVDF membranes, and growth of Trichophyton on an agar sheet are shown. [Figure 2] Fluorescence micrographs are shown for the cases where various concentrations of keratinase were used. [Figure 3]Fluorescence micrographs are shown for the cases where the treatment time with 1% keratin aqueous solution was 10 or 60 minutes. [Figure 4] Fluorescence micrographs are shown for the cases where a 1% keratin aqueous solution, a 1% casein aqueous solution, and a 1% bovine serum albumin (BSA) aqueous solution were used. [Figure 5] This shows a fluorescence micrograph of a skin specimen from the sole of a patient suspected of having tinea fungus infection. DETAILED DESCRIPTION OF THE INVENTION

[0012] Each embodiment included in the present disclosure will be described in further detail below. The present disclosure includes a method for detecting Trichophyton in a biological sample. In this specification, the detection method may be referred to as the "detection method of the present disclosure."

[0013] The biological sample is not particularly limited as long as it can be parasitized by Trichophyton. Examples include skin, nails, etc. More specifically, examples of skin include skin from the feet, groin, head, hands, face, and other body parts. Examples of substances derived from biological samples include keratinase, tinea fungus, etc., which may be contained in the biological sample. These may be used alone or in combination of two or more.

[0014] The subject from which the biological sample is collected is not particularly limited as long as it can be parasitized by Trichophyton, including, for example, humans; mammals other than humans, such as dogs, cats, cows, horses, and pigs; and birds, such as chickens and parakeets.

[0015] "Tinea fungus" is a dermatophyte that causes tinea. The form of tinea fungus is not particularly limited, and may be in the form of fungal fragments such as spores, hyphae, or conidia. Examples of tinea fungi include the genus Trichophyton, the genus Microsporum, the genus Epidermophyton, and the sexual form (Arthroderma) thereof. Examples of anthropophilic tinea fungi include Trichophyton rubrum, Trichophyton mentagrophytes, Trichophyton concentricum, Trichophyton schoenleinii, Trichophyton tonsurans, Trichophyton violaceum, and Epidermophyton floccosum. Examples of zoophilic tinea fungi that are found in dogs, cats, etc. include Microsporum canis, Microsporum gallinae, Trichophyton equinum, Trichophyton mentagrophytes, and Trichophyton verrucosum. Among these, representative tinea fungi that cause tinea include, for example, Trichophyton rubrum, Trichophyton mentagrophytes, Trichophyton tonsurans, Epidermophyton floccosum, and Microsporum canis, and it is preferable to detect these.

[0016] The detection method of the present disclosure preferably includes a step of contacting a support having a non-polar surface that holds a biological sample or a substance derived from a biological sample with a first reagent containing a substrate for keratinase. In this specification, this step may be referred to as "step (a)."

[0017] As used herein, a non-polar surface refers to a surface that does not have polar groups, such as nitro, sulfo, sulfate, phosphate, hydroxyl, carboxyl, amino, secondary amino (e.g., methylamino), and tertiary amino (e.g., dimethylamino) groups. Examples of the support having a non-polar surface include polyvinylidene fluoride films, polyolefin resin films such as polyethylene, polypropylene, and polystyrene, etc. Among these, polyvinylidene fluoride films are preferred.

[0018] A support having a non-polar surface is preferably hydrophilized before retaining a biological sample or a substance derived from a biological sample. Hydrophilization methods are known in the art and can be carried out using any technique, such as methanol treatment.

[0019] The support may be in the form of a film (sheet), for example. The support is preferably capable of maintaining a solid state under conditions in which the enzymatic reaction of keratinase proceeds. The support is preferably in a wet state. For example, the support is preferably in a wet state with a solvent such as water; physiological saline; or a pH buffer solution such as phosphate buffer, Tris-HCl buffer, Tris-EDTA buffer, bicarbonate buffer, or sodium borate buffer. The solvent may contain ethylenediaminetetraacetic acid (EDTA). The concentration of EDTA may be, for example, 0.01 mM to 1 M, or 0.05 mM to 100 mM. The support is preferably flexible, since the shape of the support can be deformed to match the shape of the biological sample, facilitating the operation of contacting the non-polar surface of the support with the biological sample. The support may be liquid permeable. An example of an embodiment in which the support is liquid permeable is an embodiment in which the support has liquid permeable holes (for example, through holes with a diameter of 0.2 to 0.45 μm). The size of the support is not particularly limited and can be adjusted appropriately depending on the size of the biological sample to be held.

[0020] The support may be laminated on a substrate. When the support is laminated on a substrate, it is preferably laminated on the substrate so that the non-polar surface of the support is exposed. Examples of the substrate include filter paper, nonwoven fabric, cotton fabric, film, a substrate having a pressure-sensitive adhesive layer laminated on one side, or a combination thereof.

[0021] Examples of methods for retaining a biological sample or a substance derived from a biological sample on a support having a non-polar surface include contacting the non-polar surface of the support with the biological sample. The biological sample may be a biological sample after collection from a subject, or a biological sample before collection from a subject. A biological sample can be collected from a subject by contacting the non-polar surface of the support with the biological sample before collection from the subject. Furthermore, for example, by contacting the non-polar surface of the support with a biological sample and allowing the biological sample-derived substances contained in the biological sample to be adsorbed and transferred to the support having a non-polar surface, the biological sample-derived substances can be retained on the support having a non-polar surface. In other words, the detection method of the present disclosure may include a step of retaining a biological sample or a substance derived from a biological sample on a support having a non-polar surface, more specifically, a step of contacting the non-polar surface of the support with the biological sample.

[0022] The time for retaining a biological sample or a substance derived from a biological sample on a support having a non-polar surface, more specifically, the time for contacting the non-polar surface of the support with the biological sample, is not particularly limited, but can be, for example, about 1 minute to 24 hours. It may also be, for example, about 1 to 15 minutes. For example, when the non-polar surface of the support is to be directly contacted with an affected area such as the subject's foot for a long period of time (e.g., overnight), a humidity-retaining sheet (e.g., filter paper) containing physiological saline or an appropriate buffer solution may be placed on the side opposite the skin-contacting surface of the support, followed by a drying prevention sheet (e.g., film) to prevent drying, and the sheet may be fixed to the affected area with medical tape or the like.

[0023] The substrate for keratinase contained in the first reagent is not particularly limited as long as it can be decomposed by keratinase. Examples include keratin, casein, bovine serum albumin, gelatin, collagen, etc. Among these, keratin, casein, or bovine serum albumin is preferred, and keratin is more preferred. These may be used alone or in combination of two or more.

[0024] The content of the keratinase substrate contained in the first reagent is not particularly limited and can be set appropriately, for example, up to 100% by mass.

[0025] The first reagent contains a keratinase substrate and may further contain other components, such as bases, carriers, solvents, dispersants, emulsifiers, buffers, stabilizers, excipients, binders, disintegrants, lubricants, thickeners, antioxidants, preservatives, coating agents, and colorants. These components may be used singly or in combination of two or more.

[0026] The first reagent can be prepared by a conventional method by combining a substrate for keratinase and, if necessary, other components.

[0027] The first reagent may be in the form of a liquid (for example, a solution, a suspension, etc.), a paste, or a solid (for example, a powder, a granule, etc.).

[0028] The first reagent may be held on a substrate, which may be in the form of, for example, a film (sheet) or a plate. Examples of substrates on which the first reagent is held include slide glasses; nitrocellulose membranes; nylon membranes; polyvinylidene fluoride membranes; cellulose ester membranes such as cellulose acetate; polyester membranes such as polyethylene terephthalate; and polyolefin resin membranes such as polyethylene, polypropylene, and polystyrene.

[0029] When the first reagent is held on a substrate, the binding mode of the first reagent (keratinase substrate) to the surface of the substrate is not particularly limited. For example, by applying an appropriate coating treatment to the surface of the substrate, the first reagent (keratinase substrate) can be adsorbed onto the coated surface of the substrate. Examples of coating agents used in the coating treatment include collagen, silane, silicone, lysine, silica gel, alumina, polyamide resin, etc.

[0030] Other binding modes include, for example, specific interactions between streptavidin or avidin and biotin, hydrophobic interactions, magnetic interactions, polar interactions, formation of covalent bonds (e.g., amide bonds, disulfide bonds, thioether bonds, etc.), crosslinking with a crosslinking agent, etc. To enable these binding modes, the surface of the base material or the substrate can be appropriately chemically modified using known techniques.

[0031] In addition to the specific interaction between streptavidin or avidin and biotin, specific interactions such as maltose-binding protein / maltose, polyhistidine peptide / metal ions such as nickel and cobalt, glutathione-S-transferase / glutathione, calmodulin / calmodulin-binding peptide, ATP-binding protein / ATP, nucleic acid / complementary nucleic acid, receptor protein / ligand, enzyme / substrate, antibody / antigen, and IgG / protein A can also be utilized.

[0032] When utilizing the interaction between avidin or streptavidin and biotin, for example, a substrate to which biotin has been introduced can be bound to a substrate coated with avidin or streptavidin.

[0033] When covalent bonding is utilized, covalent bonding can be achieved by utilizing functional groups on the substrate or base material. Examples of functional groups capable of forming covalent bonds include carboxyl groups, amino groups, and hydroxyl groups. For example, when carboxyl groups are present on the surface of the substrate, the carboxyl groups can be activated with carbodiimides such as 1-ethyl-3-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and then reacted with amino groups on the substrate to form amide bonds between the substrate and the base material. Furthermore, when amino groups are present on the surface of the substrate, the amino groups can be converted to carboxyl groups using a cyclic acid anhydride such as succinic anhydride, and then reacted with amino groups on the substrate to form amide bonds between the substrate and the base material.

[0034] When crosslinking with a crosslinking agent is used, various crosslinking agents capable of reacting with functional groups of the base material and substrate can be used. Examples of crosslinking agents include multifunctional reagents such as bifunctional and trifunctional reagents. Examples of multifunctional reagents include N-succinimidyl (4-iodoacetyl)aminobenzoate (SIAB), dimaleimide, dithio-bis-nitrobenzoic acid (DTNB), N-succinimidyl-S-acetyl-thioacetate (SATA), N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), and 6-hydrazinonicotinide (HYNIC).

[0035] The method for contacting the support having a non-polar surface holding the biological sample or a substance derived from the biological sample with the first reagent containing a keratinase substrate in step (a) is not particularly limited. Examples include a method in which the first reagent containing a keratinase substrate is added to the non-polar surface of the support holding the biological sample or a substance derived from the biological sample; or, when the first reagent is held on a substrate, a method in which the non-polar surface of the support holding the biological sample or a substance derived from the biological sample is brought into contact with the surface of the substrate on which the first reagent (keratinase substrate) is held.

[0036] In step (a), the contact of the support having a non-polar surface that holds the biological sample or a substance derived from the biological sample with the first reagent containing a substrate for keratinase is preferably carried out under conditions that allow the enzymatic reaction of keratinase to proceed. The temperature may be, for example, about 20 to 60°C, or about 25 to 50°C. The pH may be, for example, about 5 to 10, or about 7 to 9.

[0037] The pH can be adjusted using a pH buffer solution, such as Tris-HCl buffer solution, Tris-EDTA buffer solution, bicarbonate buffer solution, sodium borate buffer solution, and phosphate buffer solution. The pH of the pH buffer solution may be, for example, about 5 to 10, or about 7 to 9.

[0038] The detection method of the present disclosure may further include, for example, a step of contacting with a pH buffer solution. This step may be included before step (a), or after step (a) and before step (b). The pH buffer may be contacted with the support, more specifically, the non-polar surface of the support on which the biological sample or a substance derived from the biological sample is supported, or the non-polar surface of the support before the biological sample or a substance derived from the biological sample is supported, or, if the first reagent is supported on a substrate, the pH buffer may be contacted with the substrate, more specifically, the surface of the substrate on which the first reagent (keratinase substrate) is supported.

[0039] The detection method of the present disclosure may further include, for example, a step of contacting with a chelating agent. Examples of chelating agents include ethylenediaminetetraacetic acid (EDTA) and glycoletherdiaminetetraacetic acid (EGTA). The chelating agent can activate keratinase. The chelating agent may be contained in the aforementioned pH buffer solution, or in a solvent separate from the pH buffer solution. Examples of the solvent that can be used include water and Tris buffer solution. The concentration of the chelating agent may be, for example, 0.01 mM to 1 M, or 0.05 mM to 100 mM. This step may be included before step (a), or after step (a) and before step (b). The pH buffer may be contacted with the support, more specifically, the non-polar surface of the support on which the biological sample or a substance derived from the biological sample is supported, or the non-polar surface of the support before the biological sample or a substance derived from the biological sample is supported, or, if the first reagent is supported on a substrate, the pH buffer may be contacted with the substrate, more specifically, the surface of the substrate on which the first reagent (keratinase substrate) is supported.

[0040] In step (a), the contact time between the support having a non-polar surface that holds the biological sample or a substance derived from the biological sample and the first reagent containing the keratinase substrate can be, for example, about 30 minutes to 24 hours, or may be, for example, about 1 to 3 hours.

[0041] When keratinase is contained in a biological sample or a material derived from a biological sample, in step (a), a support having a non-polar surface that holds the biological sample or a material derived from the biological sample is contacted with a first reagent containing a substrate for the keratinase, whereby the keratinase substrate contained in the first reagent is decomposed by the keratinase contained in the biological sample or material derived from the biological sample held on the non-polar surface of the support.

[0042] The detection method of the present disclosure preferably includes, after step (a), a step of detecting a substrate of keratinase. In this specification, this step may be referred to as "step (b)."

[0043] Methods for detecting a keratinase substrate include, for example, a method of detecting a labeled substance that binds to or is bound to a keratinase substrate; a method of binding a labeled antibody that recognizes a keratinase substrate to a keratinase substrate and detecting a labeled substance that labels the antibody; a protein staining method, etc.

[0044] The detection of the keratinase substrate may be carried out on a support, more specifically, on the non-polar surface of the support on which the biological sample or a substance derived from the biological sample is supported, or, if the first reagent is supported on a substrate, on the substrate, more specifically, on the surface of the substrate on which the first reagent (keratinase substrate) is supported.

[0045] When the method for detecting a keratinase substrate is, for example, a method for detecting a labeled substance that binds to or is bound to the keratinase substrate, the keratinase substrate contained in the first reagent may have a labeled substance bound thereto. Alternatively, the detection method of the present disclosure may include, after step (a) and before step (b), a step of binding a labeling substance to the (remaining) keratinase substrate contained in the first reagent.

[0046] The labeling substance is not particularly limited, but examples thereof include fluorescent dyes (e.g., fluoresceins, rhodamines, coumarins, pyrenes, cyanines, etc.), radioactive substances (e.g., 13 C. 3 H, etc.), enzymes (for example, alkaline phosphatase, peroxidase, etc.), colored particles (for example, metal colloid particles, colored latex, etc.), etc.

[0047] Methods for binding a labeling substance are known in the art, and any method can be used.

[0048] For example, when a method for detecting a keratinase substrate involves binding a labeled antibody that recognizes the keratinase substrate to the keratinase substrate and detecting a labeled substance that labels the antibody, methods for binding a labeled antibody to a keratinase substrate are known in the art and can be carried out using any technique.

[0049] Methods for detecting a labeling substance are known in the art, and any method can be used.

[0050] A portion where the labeled substance is detected, or a portion where the detection intensity of the labeled substance is comparable to the detection intensity of a portion of the non-polar surface of the support that does not retain a biological sample or a substance derived from a biological sample, or the detection intensity when a support that does not retain a biological sample or a substance derived from a biological sample is used, can be determined to be a portion where the keratinase substrate has not been decomposed.It can be determined that keratinase is not present in the portion where the keratinase substrate has not been decomposed. Areas where no labeled substance is detected, or areas where the detection intensity of the labeled substance is lower than the detection intensity of areas on the non-polar surface of the support that do not retain a biological sample or a substance derived from a biological sample, or the detection intensity when a support that does not retain a biological sample or a substance derived from a biological sample is used, can be determined to be areas where the keratinase substrate has been decomposed.Keratinase can be determined to be present in areas where the keratinase substrate has been decomposed. In other words, when a portion where the substrate of keratinase has been decomposed is confirmed, it can be determined that Trichophyton is present in the biological sample.

[0051] When the method for detecting a keratinase substrate is, for example, a protein staining method, examples of the protein staining method include Coomassie brilliant blue staining, silver staining, gold colloid staining, and fluorescent staining (e.g., Sypro Rubby manufactured by BioRad), and can be performed according to conventional methods. Protein-stained areas, or areas where the staining intensity is comparable to the staining intensity of areas of the non-polar surface of the support that do not retain a biological sample or a substance derived from a biological sample, or areas where a support that does not retain a biological sample or a substance derived from a biological sample is used, can be determined to be areas where the keratinase substrate has not been decomposed. It can be determined that keratinase is not present in areas where the keratinase substrate has not been decomposed. Areas where protein is not stained, or areas where the staining intensity is lower than the staining intensity of areas on the non-polar surface of the support that do not retain a biological sample or a substance derived from a biological sample, or the staining intensity when a support that does not retain a biological sample or a substance derived from a biological sample is used, can be determined to be areas where the keratinase substrate has been decomposed.It can be determined that keratinase is present in areas where the keratinase substrate has been decomposed. In other words, when a portion where the substrate of keratinase has been decomposed is confirmed, it can be determined that Trichophyton is present in the biological sample.

[0052] It should be noted that a portion where the keratinase substrate has disappeared due to degradation by keratinase can be clearly distinguished from a portion where the keratinase substrate has disappeared due to other factors, based on its area, shape, continuity, etc. For example, in a portion where the keratinase substrate has disappeared due to factors other than degradation by keratinase, the remaining keratinase substrate may be scattered, and the disappearance of the keratinase substrate may occur discontinuously. In contrast, in a portion where the keratinase substrate has disappeared due to degradation by keratinase, the remaining keratinase substrate may not be scattered, and the disappearance of the keratinase substrate may occur continuously.

[0053] The detection method of the present disclosure may include, prior to step (b), a step of washing the support that has been contacted with the first reagent, or, if the first reagent is held on a substrate, the substrate. This removes degradation products and the like resulting from degradation of the keratinase substrate, making it easier to detect the keratinase substrate. For example, water can be used for washing.

[0054] The detection method of the present disclosure may further include, prior to step (a), a step of contacting a support having a non-polar surface that holds the biological sample or a substance derived from the biological sample with a second reagent containing a substrate for keratinase. In this specification, this step may be referred to as "step (a')." By further including a step of contacting a support having a non-polar surface that holds a biological sample or a substance derived from a biological sample with a second reagent that contains a substrate for keratinase before step (a), it is expected that the adhesion of the first reagent that contains a substrate for keratinase to the non-polar surface of the support will be improved.

[0055] The substrate for keratinase contained in the second reagent is not particularly limited as long as it can be decomposed by keratinase. Examples include keratin, casein, bovine serum albumin, gelatin, collagen, etc. Among these, keratin, casein, or bovine serum albumin is preferred, and keratin is more preferred. These may be used alone or in combination of two or more.

[0056] The content of the keratinase substrate in the second reagent is not particularly limited. For example, it can be appropriately set within the limit of the concentration at which the substrate can be dissolved. For example, it may be about 0.1 to 5% by mass, or about 0.2 to 2% by mass.

[0057] The second reagent contains a substrate for keratinase and may further contain other components, such as bases, carriers, solvents, dispersants, emulsifiers, buffers, stabilizers, excipients, binders, disintegrants, lubricants, thickeners, antioxidants, preservatives, coating agents, and colorants. These components may be used singly or in combination of two or more.

[0058] The second reagent can be prepared by a conventional method by combining a substrate for keratinase and, if necessary, other components.

[0059] The second reagent may be in the form of a liquid (for example, a solution, a suspension, etc.), a paste, or a solid (for example, a powder, a granule, etc.), among which a liquid form is preferred.

[0060] The method for contacting the support having a non-polar surface holding the biological sample or a substance derived from the biological sample with the second reagent containing a keratinase substrate in step (a') is not particularly limited. Examples include a method in which the second reagent containing a keratinase substrate is added to the non-polar surface of the support holding the biological sample or a substance derived from the biological sample; or a method in which the support holding the biological sample or a substance derived from the biological sample is immersed in the second reagent (keratinase substrate).

[0061] In step (a'), the contact time between the support having a non-polar surface that holds the biological sample or a substance derived from the biological sample and the second reagent containing the keratinase substrate can be, for example, about 1 to 120 minutes, preferably 5 to 60 minutes, and more preferably 5 to 45 minutes. By setting the contact time within this range, for example, it is expected that the detection sensitivity will be improved. The temperature may be, for example, about 0 to 60°C, or about 25 to 45°C. The pH may be, for example, about 5 to 10, or about 7 to 9.

[0062] According to the detection method of the present disclosure, tinea fungus can be detected in a biological sample. Therefore, the detection method of the present disclosure is useful for diagnosing tinea in a subject from which a biological sample has been collected. In other words, the detection method of the present disclosure can assist in diagnosing tinea in a subject from which a biological sample has been collected.

[0063] The present disclosure also encompasses a kit for detecting tinea fungi. In this specification, the kit may be referred to as the "kit of the present disclosure."

[0064] The kit of the present disclosure includes a support having a non-polar surface. The above description can be applied to the support having a non-polar surface.

[0065] The kit of the present disclosure may further include a first reagent containing a substrate for keratinase. The above-mentioned description of the first reagent may be used.

[0066] The kit of the present disclosure may further include a second reagent containing a substrate for keratinase. The above-mentioned description of the second reagent may be used.

[0067] The kit of the present disclosure may further comprise a labeling substance that binds to or is bound to the keratinase substrate. The above description of the labeling substance can be used.

[0068] The kit of the present disclosure allows collection of a biological sample or a substance derived from a biological sample for detecting tinea fungus. Furthermore, the kit of the present disclosure is suitable for use in detecting tinea fungus in a biological sample. Therefore, the kit of the present disclosure is useful for diagnosing tinea in a subject from whom a biological sample has been collected. In other words, the kit of the present disclosure can assist in the diagnosis of tinea in a subject from whom a biological sample has been collected.

[0069] It should be noted that in this specification, the term "comprising" includes "consisting essentially of" and "consisting of." Furthermore, the present disclosure encompasses all arbitrary combinations of the constituent elements described in this specification.

[0070] Furthermore, the various characteristics (properties, structures, functions, etc.) described in each embodiment of the present disclosure above may be combined in any way to specify the subject matter encompassed by the present disclosure, i.e., the present disclosure encompasses all subject matter consisting of any combination of the combinable characteristics described herein. [Example]

[0071] The contents of the present disclosure will be specifically explained using the following examples. However, the present disclosure is not limited to these examples. In the following, unless otherwise specified, experiments were performed under atmospheric pressure and room temperature conditions. Furthermore, unless otherwise specified, "%" means "% by mass."

[0072] Preparation of FITC-labeled keratin-coated glass slides Powdered keratin (derived from sheep wool, Nakarai Desk) was dissolved in 0.1 M Tris-HCl buffer (pH 9.0) to prepare a 0.2% solution. FITC-labeled keratin (2 mg / ml) was obtained using the Fluorescein Labeling Kit-NH2 (Dojindo). 0.05 ml of FITC-labeled keratin, 0.2 ml of 1 M Tris-HCl buffer (pH 9.0), and 1.75 ml of 10 mL of EDTA were mixed, and 1 ml of this mixture was added to each well of a collagen-coated, 2-well chamber slide (Matsunami Glass). The slide was then left at room temperature for at least 1 hour in the dark. The chamber was then removed, washed with purified water, and dried to obtain a slide coated with FITC-labeled keratin.

[0073] Measurement of keratinase activity A 1.5 cm square membrane (polyvinylidene fluoride (PVDF) membrane or nitrocellulose membrane, BIO-RAD) moistened with enzyme reaction buffer (0.1 M Tris-HCl buffer (pH 8.3) containing 10 mM EDTA) was attached with the keratinase-transferred side of the membrane to a glass slide coated with FITC-labeled keratin. A 1.5 cm square piece of filter paper moistened with enzyme reaction buffer was placed on top of the membrane and placed in a humidity chamber at 37°C for overnight or longer. After the reaction was complete, the filter paper and membrane were removed, and the glass surface was washed with purified water and dried. The dried slide was observed under a fluorescence microscope (Keyence BZ-9000) and fluorescent images were obtained. Keratinase activity was observed as a decrease in FITC-induced fluorescence.

[0074] Preparation of powdered keratin-containing agar sheets 600 mg of powdered keratin was pulverized and suspended in diethyl ether for sterilization. After the diethyl ether was completely evaporated, the suspension was suspended in 5 ml of sterilized water, and 10 ml of sterilized agar solution (5 ml of 1 M phosphate buffer (pH 7.4), 1.125 g of agar, 45 ml of purified water) was added and mixed, and poured into a 9 cm diameter sterilized petri dish to solidify. 1.5 cm square pieces were cut from the solidified agar to obtain a powdered keratin-containing agar sheet.

[0075] Preparation of Trichophyton spore suspension The tinea fungi used were Trichophyton rubrum IFM 66221 and T. mentagrophytes IFM 65887, both provided by the Chiba University Medical Mycology Research Center. Each tinea fungus was cultured on Sabouraud dextrose agar medium supplemented with chloramphenicol. After culture, the fungal surface was scraped with a platinum loop together with a sterilized spore preparation solution (15% glycerin, 0.1% Tween 20, 1 / 15 M phosphate buffer (pH 7.4)), and the spore solution was obtained by filtering through cotton.

[0076] Hydrophilic treatment of PVDF membrane The PVDF membrane cut into 1.5 cm squares was immersed in methanol, and then immersed overnight or more in 1 / 15 M phosphate buffer (pH 7.4) containing 1 mg / ml chloramphenicol that had been filtered through a 0.45 μm membrane filter to perform a hydrophilization treatment.

[0077] Experiment 1 An agar sheet containing powdered keratin was placed on a sterile glass slide, and 1 μl of spore suspension of T. rubrum IFM 66221 was inoculated near the center of the agar sheet. A sterilized cover glass was placed on top, and the agar sheet was placed in a moist Petri dish and cultured at 25°C for 7 days. A hydrophilized PVDF membrane or nitrocellulose membrane was then placed on top of the agar sheet and cultured at 25°C for an additional 8 days. After culture, the membrane was recovered and washed with enzyme reaction buffer, and keratinase activity was measured using the method described above.

[0078] As shown in Figure 1(A), when a PVDF membrane was used, keratinase activity was observed at the location corresponding to the site of bacterial growth, but when a nitrocellulose membrane was used, no keratinase activity was observed.

[0079] In addition, spores of T. mentagrophytes IFM 65887 were inoculated in the same manner and cultured at 25°C for 6 days. After that, a hydrophilized PVDF or nitrocellulose membrane was placed on top of the agar sheet and cultured for another day at 25°C. After the culture, the membrane was recovered, washed with enzyme reaction buffer, and the keratinase activity was measured.

[0080] As shown in Figure 1(B), as with T. rubrum, keratinase activity was observed at the site where the fungus had grown when the PVDF membrane was used, but no keratinase activity was observed when the nitrocellulose membrane was used.

[0081] Experiment 2 In Experiment 1, keratinase activity was detected on the PVDF membrane but not on the nitrocellulose membrane. We assumed that the reason for this was the difference in detection sensitivity, and measured the detection sensitivity of keratinase derived from Bacillus licheniformis (purchased from Wako Pure Chemical Industries, Ltd.) using the PVDF membrane. The detection sensitivity of the same enzyme when using a nitrocellulose membrane was 5 ng.

[0082] Keratinase derived from B. licheniformis was dissolved at 10 mg / ml in 1 M phosphate buffer (pH 7.4) containing 1 M KCl, and this was serially diluted with 1 / 15 M phosphate buffer (pH 7.4) containing 0.1% Tween 20. 1 μl of diluted keratinase was dropped onto a hydrophilized PVDF membrane to allow it to soak into the membrane. After blotting the keratinase onto the PVDF membrane, the surface of the PVDF membrane was treated with a 1% aqueous solution of keratin dissolved in 0.1 M Tris-HCl buffer (pH 8.3) for 10 minutes, and keratinase activity was measured as described above.

[0083] As shown in Figure 2, the detection sensitivity of keratinase was 0.2 ng.

[0084] Experiment 3 Next, we investigated the treatment time of the 1% keratin aqueous solution. Using the same method as in Experiment 2, we compared the keratinase activity when the treatment time was 10 minutes or 60 minutes.

[0085] As shown in FIG. 3, keratinase activity was observed after 60 minutes of treatment, similar to that observed after 10 minutes of treatment.

[0086] Experiment 4 Next, the effects of treating the PVDF membrane with proteins other than keratin were compared with those of keratin. Casein and BSA were used as non-keratin proteins. The PVDF membrane was blotted with keratinase in the same manner as in Experiment 2, and treated with 1% keratin solution, 1% casein solution, and 1% BSA solution in 0.1 M Tris-HCl buffer (pH 8.3) for 10 minutes each, and then the keratinase activity was measured.

[0087] As shown in Figure 4, the keratinase activity of the blotted areas was similar for all three protein treatments. However, no circular dark areas were observed in the area blotted with only the diluted enzyme solution (blank, no keratinase) treated with the keratin aqueous solution, whereas slight circular dark areas were observed in the area treated with the casein aqueous solution and the BSA aqueous solution. This indicates that keratin is the most suitable of the three, namely, keratin.

[0088] Experiment 5 Next, we investigated the possibility of detecting keratinase activity in skin samples. A skin sample from the sole of a patient suspected of having a tinea fungus infection was placed on a hydrophilized PVDF membrane, sandwiched between filter paper soaked in 1 / 15 M phosphate buffer (pH 7.4) containing 1 mg / ml chloramphenicol, and left in a refrigerator overnight. The PVDF membrane and skin sample were then collected, and the PVDF membrane was treated with a 1% keratin solution for 10 minutes. Keratinase activity was then measured using the method described above. The skin specimens were examined under a microscope using the KOH method (using Zoom (Hisamitsu Pharmaceutical)) to confirm the presence or absence of tinea fungi.

[0089] As shown in Figure 5, keratinase activity was detected in the skin specimens in which Trichophyton was detected by the KOH method.

Claims

1. A method for detecting Trichophyton in a biological sample, comprising: (a) a support having a non-polar surface that holds a biological sample or a substance derived from a biological sample; contacting the keratinase with a first reagent comprising a substrate for the keratinase; (b) after step (a), detecting a substrate for the keratinase; Including, Before step (a), a support having a non-polar surface that holds the biological sample or a substance derived from the biological sample; contacting the keratinase with a second reagent containing a substrate for the keratinase; The method further comprises:

2. The method of claim 1 , wherein the support is a polyvinylidene fluoride membrane.

3. The method according to claim 1 or 2, wherein the substrate of the keratinase in the first reagent and / or the second reagent is at least one selected from the group consisting of keratin, casein, and bovine serum albumin.

4. The method according to any one of claims 1 to 3, wherein the step (b) is a step of detecting a labeled substance that binds to or is bound to a substrate of the keratinase.

5. The method according to any one of claims 1 to 4, wherein the first reagent is supported on a substrate.

6. a support having a non-polar surface, further comprising a first reagent comprising a substrate for keratinase; A kit for detecting tinea fungus, further comprising a second reagent comprising a substrate for keratinase.

7. The kit of claim 6, wherein the support is a polyvinylidene fluoride membrane.

8. The kit according to claim 6 or 7, wherein the substrate of the keratinase in the first reagent and / or the second reagent is at least one selected from the group consisting of keratin, casein, and bovine serum albumin.

9. The kit according to any one of claims 6 to 8, further comprising a labeling substance that is bound to or attached to a substrate of the keratinase.

10. The kit according to any one of claims 6 to 9, wherein the first reagent is supported on a substrate.

Images