Specimen processing liquid and use thereof

Abstract

The present invention addresses the problem of providing a means for detecting an oral / nasal infectious virus that makes it possible to perform correct positive / negative determination without generating a false positive even when nasal discharge, saliva, or the like is mixed in a specimen taken from a lesion. Provided is a kit which is for detecting an oral / nasal infectious virus and which includes a specimen processing liquid and a test cassette. The specimen processing liquid comprises 0.2-10 w / v% of a buffer, 0.02-1 w / v% of a calcium chelating agent, and magnesium ions in an amount equivalent to 0.002-0.1 w / v% of magnesium chloride. The test cassette has a sample drip part into which a sample solution that has been processed using the specimen processing liquid is dripped, an antibody holding part in which a first antiviral antibody that has been labeled is held, and a test line determination part which is disposed downstream of the sample drip part and the antibody holding part and in which a second antiviral antibody is immobilized.

Description

Specimen Treatment Solution and Use Thereof

[0001] The present invention relates to a specimen treatment solution for treating a specimen, a kit using the specimen treatment solution, and the like. The present invention can be used for treating a specimen collected from a lesion suspected of being infected with foot-and-mouth disease virus or the like.

[0002] Methods for detecting pathogens such as viruses using antigen-antibody reactions have been developed. This method generally uses an antibody to detect an antigen in a sample, and can specifically and rapidly detect a target antigen. However, in some cases, means for reducing the influence of components that interfere with the antigen-antibody reaction and means for eliminating unintended non-specific reactions are required.

[0003] For example, Patent Document 1 describes a specimen pretreatment reagent for immunoassay that contains hexadimethrine and is used for treating mucous membrane and / or mucus specimens from a living body. This document explains that the reagent can reduce the influence of components that interfere with antigen-antibody reaction even when a mucus specimen containing a large amount of acidic polysaccharide is used as a sample, and enables accurate detection of the target component in a biological sample. Patent Document 2 also discloses an immune reaction inhibition inhibitor that suppresses an immune reaction inhibition effect derived from body fluid in a sample in immunoassay, the immune reaction inhibition inhibitor being characterized by comprising a compound of any one of the following (1) and (2): (1) "R １ -SO ３ H" a sulfonic acid compound represented by or a salt thereof (in the formula, R １ is a linear C ５ to C ３０ alkyl group, a linear C ５ to C ３０ alkyl substituted linear C １ to C ３０ alkyl group, or a carbocyclic aromatic group having at least one linear C ５ to C ３０ alkyl group, and these groups may optionally have a substituent.), (2) "N ＋ -R ２ R ３ R ４ R ５The quaternary ammonium ion or its salt represented by " (wherein R ２ ~R ５ Each is an independently linear C １ ~C ３０ Alkyl alkyl group, or at least one linear C ５ ~C ３０ The document describes a carbocyclic aromatic group substituted with an alkyl group, which may have substituents. It explains that this immunosuppressant can efficiently suppress the decrease in antigen-antibody reaction even in bodily fluid samples such as saliva or test samples that may be contaminated with saliva.

[0004] On the other hand, foot-and-mouth disease is an acute febrile infectious disease caused by infection with the foot-and-mouth disease virus, which belongs to the Aftovirus genus of the Picornaviridae family. It is highly contagious and infects most even-toed ungulates, including livestock such as cattle, buffalo, pigs, sheep, and goats, as well as wild animals. Blisters form on the skin and mucous membranes around the mouth, hooves, and mammary glands of infected and diseased animals.

[0005] Products based on immunochromatography for detecting foot-and-mouth disease virus are commercially available. These products use epithelial tissue from lesions as specimens. The collected specimen is suspended in the specimen processing solution included with the product and dropped onto the sample application area of ​​the test cassette. After approximately 15 minutes, a positive / negative result is determined by visually inspecting the control line and test line. The package insert for this product (Non-Patent Literature 1) states, as pharmacological information, that when tested on nasal secretions, saliva, and feces, a false positive result was confirmed in one nasal secretion specimen from a Japanese Black cattle. It also states that the product reacts nonspecifically with blood, serum, nasal secretions, and saliva, which can lead to false positive results, and therefore, specimens should be collected while avoiding contamination with blood, pus, nasal secretions, or saliva as much as possible.

[0006] Japanese Patent Publication No. 2001-038903 (Patent No. 5334742), International Publication WO2018 / 181263 (Patent No. 6601932)

[0007] Veterinary medicine, veterinary biological preparation, NH ImmunoDetect (registered trademark), Foot-and-mouth disease, Approval directive number: Ministry of Agriculture, Forestry and Fisheries Directive 5, Animal Drugs No. 398, Created October 2023 (First Edition)

[0008] When using the above-mentioned products, since the sample is collected from the lesion, it is difficult to completely prevent contamination of the sample with nasal discharge, saliva, etc. Therefore, even if nasal discharge, etc., is present in the sample, a method is desirable that can correctly determine positive / negative results without producing false positives.

[0009] The inventors investigated how to solve the problem of false positives in the detection of foot-and-mouth disease virus by modifying the composition of the sample processing solution. They conducted tests using a composition with added hexadimethrin bromide, which is used for sample processing in Patent Document 1. Although the composition used in the experiment did not eliminate false positives, an improvement in measurement sensitivity was confirmed. Furthermore, by conducting tests with various changes to the buffer in the sample processing solution, they discovered that a specific buffer had the ability to eliminate false positives, thus completing the present invention.

[0010] The present invention provides the following: [1] A kit for detecting oral and nasal cavity infectious viruses, comprising the following sample processing solution and test cassette: a sample processing solution containing 0.2 to 10 w / v% buffering agent, 0.02 to 1 w / v% calcium chelating agent, and 0.002 to 0.1 w / v% equivalent of magnesium ions as magnesium chloride; and a test cassette having a sample application section for dropping a sample solution treated with the sample processing solution, an antibody holding section holding a labeled first antiviral antibody, and a test line determination section positioned downstream of the sample application section and the antibody holding section, on which a second antiviral antibody is immobilized. [2] A sample processing solution for processing lesional epithelium suspected to be infected with oral and nasal cavity infectious viruses as a sample, comprising 0.2 to 10 w / v% buffering agent, 0.02 to 1 w / v% calcium chelating agent, and 0.002 to 0.1 w / v% equivalent of magnesium ions as magnesium chloride. [3] The kit according to item 1, or the sample processing solution according to item 2, wherein the buffering agent comprises PIPES and HEPES. [4] The kit according to any one of items 1 to 3, or the sample processing solution, wherein the calcium chelating agent comprises glycol etherdiaminetetraacetic acid (EGTA). [5] The kit according to any one of items 1 to 4, or the sample processing solution, wherein the sample processing solution comprises a surfactant. [6] The kit according to any one of items 1 to 5, or the sample processing solution, wherein the sample processing solution comprises hexadimethrin bromide in an amount greater than 0.03 w / v% and less than or equal to 0.5 w / v%. [8] The kit according to any one of items 1 to 7, or the sample processing solution, wherein the pH of the sample processing solution is 3.0 to 11.0. [9] A kit or sample processing solution according to any one of items 1 to 8, wherein the pH of the sample processing solution is a pH effective for maintaining viral activity.

[10] A method for detecting oral and nasal infection viruses, comprising the following steps: Collect a sample of lesional epithelium suspected to be infected with an oral and nasal infection virus; Disintegrate the collected sample in a sample processing solution containing 0.2 to 10 w / v% buffer, 0.02 to 1 w / v% calcium chelating agent, and 0.002 to 0.1 w / v% equivalent magnesium ions as magnesium chloride to obtain a sample solution; Drop the obtained sample solution onto the sample application section of a test cassette having a sample application section, an antibody holding section containing a labeled first antiviral antibody, and a test line determination section located downstream of the sample application section and the antibody holding section, on which a second antiviral antibody is immobilized; Confirm the presence or absence of a predetermined line in the test line determination section.

[0011]

[11] A method for detecting oral and nasal infection viruses, comprising the following steps: A sample taken from lesional epithelium suspected to be infected with an oral and nasal infection virus is crushed in a sample processing solution containing 0.2 to 10 w / v% buffer, 0.02 to 1 w / v% calcium chelating agent, and 0.002 to 0.1 w / v% equivalent magnesium ions as magnesium chloride to obtain a sample solution; the obtained sample solution is dropped onto the sample application section of a test cassette having a sample application section, an antibody holding section containing a labeled first antiviral antibody, and a test line determination section located downstream of the sample application section and the antibody holding section, on which a second antiviral antibody is immobilized; and the presence or absence of a predetermined line is confirmed in the test line determination section.

[12] The method according to 10 or 11, wherein the buffer comprises PIPES and HEPES.

[13] The method according to any one of 10 to 12, wherein the calcium chelating agent comprises glycol etherdiaminetetraacetic acid (EGTA).

[14] A kit or sample processing solution according to any one of items 10 to 13, wherein the sample processing solution contains a surfactant.

[15] The method according to any one of items 10 to 14, wherein the sample processing solution contains hexadimethrin bromide.

[16] The method according to any one of items 10 to 15, wherein the sample processing solution contains hexadimethrin bromide in an amount greater than 0.03 w / v% and less than or equal to 0.5 w / v%.

[17] The method according to any one of items 10 to 16, wherein the pH of the sample processing solution is 3.0 to 11.0.

[18] The method according to any one of items 10 to 17, wherein the pH of the sample processing solution is a pH effective for maintaining viral activity.

[0012]

[21] A kit for use in a method for diagnosing oral and nasal viral infections, comprising the following sample processing solution and test cassette: A sample processing solution containing 0.2 to 10 w / v% buffer, 0.02 to 1 w / v% calcium chelating agent, and 0.002 to 0.1 w / v% equivalent of magnesium ions as magnesium chloride; and a test cassette having a sample application section for dropping the sample solution processed with the sample processing solution, an antibody holding section holding a labeled first antiviral antibody, and a test line determination section located downstream of the sample application section and the antibody holding section, on which a second antiviral antibody is immobilized.

[22] A sample processing solution for use in a method for diagnosing oral and nasal viral infections, comprising 0.2 to 10 w / v% buffer, 0.02 to 1 w / v% calcium chelating agent, and 0.002 to 0.1 w / v% equivalent of magnesium ions as magnesium chloride.

[23] The kit according to 21, or the sample processing solution according to 22, wherein the buffering agent comprises PIPES and HEPES.

[24] The kit according to any one of items 21 to 23, or the sample processing solution, wherein the calcium chelating agent comprises glycol etherdiaminetetraacetic acid (EGTA).

[25] The kit according to any one of items 21 to 24, or the sample processing solution, wherein the sample processing solution comprises a surfactant.

[26] The kit according to any one of items 21 to 25, or the sample processing solution, wherein the sample processing solution comprises hexadimethrin bromide in an amount greater than 0.03 w / v% and less than or equal to 0.5 w / v%.

[28] The kit according to any one of items 21 to 27, or the sample processing solution, wherein the pH of the sample processing solution is 3.0 to 11.0.

[29] A kit or sample processing solution according to any one of items 21 to 28, wherein the pH of the sample processing solution is a pH effective for maintaining viral activity.

[0013]

[31] Use of a kit including the following sample processing solution and test cassette in the manufacture of a product for detecting oral and nasal cavity infectious viruses: A sample processing solution containing 0.2 to 10 w / v% buffer, 0.02 to 1 w / v% calcium chelating agent, and 0.002 to 0.1 w / v% equivalent of magnesium ions as magnesium chloride; and a test cassette having a sample application section for dropping the sample solution processed with the sample processing solution, an antibody holding section holding a labeled first antiviral antibody, and a test line determination section located downstream of the sample application section and the antibody holding section, on which a second antiviral antibody is immobilized.

[32] Use of a sample processing solution containing 0.2 to 10 w / v% buffer, 0.02 to 1 w / v% calcium chelating agent, and 0.002 to 0.1 w / v% equivalent of magnesium ions as magnesium chloride in the manufacture of a product for processing lesional epithelium suspected to be infected with oral and nasal cavity infectious viruses as a sample.

[33] Use according to 31 or 32, wherein the buffer comprises PIPES and HEPES.

[34] Use according to any one of items 31 to 33, wherein the calcium chelating agent comprises glycol etherdiaminetetraacetic acid (EGTA).

[35] Use according to any one of items 31 to 34, wherein the sample processing solution comprises a surfactant.

[36] Use according to any one of items 31 to 35, wherein the sample processing solution comprises hexadimethrin bromide.

[37] Use according to any one of items 31 to 36, wherein the sample processing solution contains hexadimethrin bromide in an amount greater than 0.03 w / v% and less than or equal to 0.5 w / v%.

[38] Use according to any one of items 31 to 37, wherein the pH of the sample processing solution is 3.0 to 11.0.

[39] Use according to any one of items 31 to 38, wherein the pH of the sample processing solution is a pH effective for maintaining viral activity.

[0014] The present invention makes it possible to suppress false positives caused by contamination of a sample with saliva or nasal secretions when determining the presence or absence of foot-and-mouth disease virus and other viruses that infect the oral cavity and nasal cavity.

[0015] In certain aspects of the present invention, false positives can be suppressed without reducing detection sensitivity, or even while improving detection sensitivity, in determining the presence or absence of foot-and-mouth disease virus and other viruses that infect the oral and nasal cavities. Generally, preventing false positives often involves reducing detection sensitivity, such as by increasing the dilution ratio of the sample. However, since foot-and-mouth disease is a disease that requires the culling of all animals on farms where infected individuals occur and is highly contagious, a reduction in detection sensitivity is undesirable.

[0016] In certain aspects of the present invention, a test can be performed that suppresses false positives without inactivating the virus. Because the virus is not inactivated, the detected virus can be used to infect cells and confirm the characteristics of the virus.

[0017] Schematic diagram of the test cassette. (A) Perspective view, (B) Cross-sectional view. Photograph of the appearance of the test cassette used in the Examples section. Sensitivity of sample processing solutions of each composition. The vertical axis of the graph represents the density of the line when the test kit result is determined, which is read numerically by a machine; a higher number indicates better visibility (higher sensitivity). The detection limit was set to a value that can be confirmed by human vision (around 10). 1 × 10 is essential as the detection sensitivity of the kit. 5 TCID 50 When samples with concentrations of / mL were used, positive results were possible for all compositional treatment solutions.

[0018] The numerical range x to y represents a range that includes the values ​​x and y at both ends, unless otherwise specified. In relation to the present invention, the numerical value representing the concentration of the medium contained in the solution is a value based on the mass of the solute (w) and the volume of the solution (v) (e.g., w / v%), unless otherwise specified. In this specification, descriptions made for one embodiment also apply to other embodiments unless otherwise specified. For example, the following description of the sample processing solution in the first embodiment also applies to the sample processing solution in the second embodiment.

[0019] I. The first embodiment of the specimen processing solution relates to a specimen processing solution comprising at least a buffer, a calcium chelating agent, and magnesium ions. The specimen processing solution of this embodiment is for processing lesional epithelium, etc., suspected to be infected with oral and nasal viruses.

[0020] (Buffering agent) The sample processing solution of this embodiment contains a buffering agent. The buffering agent is capable of maintaining the aqueous solution within a predetermined pH range and typically includes a combination of a weak acid and its conjugate base (for example, alkali metal salts such as sodium or potassium, alkaline earth metal salts such as calcium or magnesium, or ammonium salts), or a combination of a weak base and its conjugate acid (for example, an amine compound and its hydrochloride salt). Examples of buffers include HEPES (2-[4-(2-Hydroxyethyl)-1-piperazinyl]ethanesulfonicacid) buffer, PIPES (Piperazine-1,4-bis(2-ethanesulfonicacid)) buffer, MES (2-Morpholinoethanesulfonicacid, monohydrate), Tris (2-Amino-2-hydroxymethyl-1,3-propanediol) buffer, carbonate buffers (sodium bicarbonate, sodium carbonate), phosphate buffers (phosphoric acid, potassium phosphate, sodium phosphate, disodium hydrogen phosphate, dipotassium hydrogen phosphate, dihydrogen phosphate, dipotassium dihydrogen phosphate, trisodium phosphate, tripotassium phosphate), citrate buffers (trisodium citrate), acetate buffers (sodium acetate, potassium acetate), borate buffers (sodium borate, sodium tetraborate), tartaric acid (sodium tartrate), and amino acid buffers (histidine, taurine, aspartic acid).

[0021] In the sample processing solution of this embodiment, one type of buffer may be used, or two or more types may be used.

[0022] In one embodiment, the buffer includes PIPES and HEPES. When PBS(-) is used as a buffer in the sample processing solution, the occurrence of false positives may not be suppressed. Also, when Tris is used in the composition used in the example of Patent Document 1, the occurrence of false positives may not be suppressed, and the sensitivity of the target measurement may be inferior compared to when other buffers are used. In contrast, when any of the group consisting of PIPES and HEPES is used as a buffer, the occurrence of false positives can be suppressed in various compositions.

[0023] The concentration of the buffer in the sample processing solution is not particularly limited, as long as it has buffering capacity and the sample processing solution can exert the desired effect. In one embodiment, the concentration of the buffer in the sample processing solution (the total concentration if multiple buffers are used) is 0.1 to 50 w / v%, regardless of the type or concentration of other components, and may be 0.2 to 10 w / v%, 0.4 to 5 w / v%, or 0.8 to 3 w / v%.

[0024] (Calcium chelating agent) The sample processing solution of this embodiment contains a calcium chelating agent. The calcium chelating agents that can be used in the sample processing solution of this embodiment include glycol etherdiaminetetraacetic acid (EGTA), ethylenediaminetetraacetic acid (EDTA), 1,2-bis(o-aminophenoxide)ethane-N,N,N',N'-tetraacetic acid (BAPTA), N'-(2-hydroxyethyl)ethylenediamine-N,N,N'-triacetic acid (HEDTA), and nitrilotriacetic acid (NTA). The buffering agent may be one type or two or more types.

[0025] In one preferred embodiment, the sample processing solution contains glycol etherdiaminetetraacetic acid (EGTA) as a calcium chelating agent.

[0026] The concentration of the calcium chelating agent in the sample treatment solution is not particularly limited as long as it has the calcium ion chelating ability and allows the sample treatment solution to exert the intended effect. In one embodiment, the concentration of the calcium chelating agent in the sample treatment solution (the total concentration when a plurality of buffer materials are used) is 0.01 to 5 w / v%, regardless of the type and concentration of other components, and may be 0.02 to 1 w / v%, 0.5 to 0.8 w / v%, or 0.1 to 0.5 w / v%.

[0027] (Magnesium ion) The sample treatment solution of the present embodiment contains magnesium ions.

[0028] The concentration of magnesium ions in the sample treatment solution is not particularly limited as long as the sample treatment solution can exert the intended effect. In one embodiment, magnesium ions are added to the sample treatment solution in the form of magnesium chloride. Regardless of the type and concentration of other components, the concentration of magnesium ions in the sample treatment solution, as magnesium chloride, is 0.001 to 0.2 w / v% equivalent, and may be 0.002 to 0.1 w / v% equivalent, 0.005 to 0.08 w / v% equivalent, or 0.01 to 0.05 w / v% equivalent.

[0029] (Surfactant) The sample treatment solution of the present embodiment may contain a surfactant. In one embodiment, the sample treatment solution contains a nonionic surfactant as the surfactant.

[0030] Structurally, nonionic surfactants are classified into ester types (such as glycerin fatty acid esters, sorbitan fatty acid esters, and sucrose fatty acid esters), ether types (such as polyoxyethylene alkyl ethers, polyoxyethylene alkylphenyl ethers, and polyoxyethylene polyoxypropylene glycol), ester ether types (such as fatty acid polyethylene glycol and fatty acid polyoxyethylene sorbitan), and alkanolamide types (such as fatty acid alkanolamides). In one embodiment, the sample treatment solution contains an ester ether type nonionic surfactant.

[0031] In one preferred embodiment, the sample treatment liquid comprises, as a nonionic surfactant, any one selected from Tween 20 (also known as Polysorbate 20, Polyoxyethylene (20) sorbitan monolaurate, Polyethylene glycol sorbitan monolaurate, polyoxyethylene sorbitan monolaurate), and Triton X-100 (also known as 4-(1,1,3,3-tetramethylbutyl)phenyl-polyethylene glycol, t-octylphenoxypolyethoxyethanol, polyethylene glycol tert-octylphenyl ether). Only one type of surfactant may be used, or two or more types may be used.

[0032] The concentration of the surfactant in the sample treatment liquid is not particularly limited as long as the sample treatment liquid can exert the intended effect. In one embodiment, regardless of the type and concentration of other components, the concentration of the surfactant in the sample treatment liquid is 0.001 to 10 w / v%, may be 0.005 to 5 w / v%, may be 0.01 to 4 w / v%, or may be 0.05 to 3 w / v%.

[0033] (Hexadimethrine bromide) The sample treatment liquid of the present embodiment may contain hexadimethrine bromide.

[0034] Hexadimethrine bromide (also known as 1,5-dimethyl-1,5-diazaundecamethylene polymethobromide, polybrene) is a polymer represented by (C 13 H 30 Br2N2) n . The molecular weight thereof is not particularly limited as long as the sample treatment liquid can exert the intended effect. In one embodiment, the molecular weight is 5,000 to 10,000, and in another embodiment, it is 800 to 5,000.

[0035] The concentration of hexadimetrin bromide in the sample processing solution is not particularly limited as long as the sample processing solution can exert the desired effect. However, from the viewpoint that the sample processing solution is used outdoors when processing samples collected from livestock such as cattle and is used at relatively low temperatures, it is preferable that the concentration does not become too high in viscosity. In one embodiment, from the viewpoint of effectiveness, the concentration of hexadimetrin bromide in the sample processing solution is 0.01 w / v% or higher, regardless of the type and concentration of other components, and may be greater than 0.03 w / v%, 0.05 w / v% or higher, or 0.07 w / v% or higher. Furthermore, from the viewpoint of not making the viscosity too high, the concentration of hexadimetrin bromide in the sample processing solution is 1 w / v% or less, regardless of the type and concentration of other components, and may be 0.5 w / v% or less, 0.4 w / v% or less, or 0.3 w / v% or less.

[0036] (Other components) Water can be used as the solvent for the sample processing solution in this embodiment. The sample processing solution in this embodiment may also contain components other than those listed above. Examples of other components include, for example, preservatives (e.g., sodium azide), agents for inhibiting nonspecific reactions (e.g., albumin), and R 1 Examples include sulfonic acid compounds represented by -SO3H or salts thereof (see Patent Document 2; for example, LAS, also known as sodium dodecylbenzenesulfonate, ammonium undecylbenzenesulfonate, salts of undecylbenzenesulfonic acid, ammonium undecan-1-ylbenzenesulfonate, ammonium undecylbenzenesulfonate (C11), etc.).

[0037] (pH) The pH of the sample processing solution in this embodiment is not particularly limited, as long as the target antigen contained in the sample does not lose its immunogenicity. In one embodiment, the pH of the sample processing solution is 3.0 to 11.0, for example, 4.0 to 10.0 or 5.0 to 9.0. In this pH range, the virus contained in the sample may be inactivated, but detection is still possible.

[0038] In another aspect, the pH of the sample processing solution is a pH effective for maintaining viral activity. A pH effective for maintaining viral activity is a pH at which the virus can be grown under appropriate conditions and its properties can be observed even after processing with the sample processing solution. Specifically, although it varies depending on the virus, in the case of foot-and-mouth disease virus, for example, it is generally known to be stable in the neutral range of pH 7.0 to 9.0 under low temperature conditions. The resistance of foot-and-mouth disease virus varies depending on the virus type and strain. For example, it is known that type C (Noville) is more susceptible to pH fluctuations than virus strains of type A and O. Furthermore, it is known that types A and Asia1 show the most resistance to heat, followed by types O and C, and then type SAT, in which order of decreasing resistance.

[0039] (False Positive Resolution Ability, Sensitivity) The sample processing solution of this embodiment has the ability to resolve false positives. The method described in Patent Document 1 is described as reducing the influence of components that interfere with the target antigen-antibody reaction contained in the biological sample solution, and hexadimethrin bromide is used in this document. According to the inventors' studies, simply adding hexadimethrin bromide to a sample processing solution of a conventional composition did not resolve false positives in the detection of foot-and-mouth disease virus, but the composition based on the present invention makes it possible to resolve false positives.

[0040] In one embodiment of the sample processing solution, false positives can be suppressed without reducing detection sensitivity, or while improving detection sensitivity.

[0041] One embodiment of the sample processing solution allows for testing that suppresses false positives without inactivating the virus. Because the virus is not inactivated, the detected virus can be used to infect cells and confirm the characteristics of the virus.

[0042] The sample processing solution of this embodiment is for processing lesional epithelium, etc., suspected to be infected with oral and nasal infection viruses such as foot-and-mouth disease virus, influenza virus, RSV, coronavirus, adenovirus, and metapneumovirus. In a particularly preferred embodiment, the sample processing solution is for processing lesional epithelium, etc., suspected to be infected with foot-and-mouth disease virus.

[0043] II. Kit The present invention provides, as a second embodiment, a kit for detecting oral and nasal infection viruses, comprising the following sample processing solution and test cassette: a sample processing solution comprising a buffer, a calcium chelating agent, and magnesium ions; and a test cassette having a sample application section for dropping a sample solution treated with the sample processing solution, an antibody holding section holding a labeled first antiviral antibody, and a test line determination section positioned downstream of the sample application section and the antibody holding section, on which a second antiviral antibody is immobilized.

[0044] (Device (test cassette) that uses immunochromatography as the measurement principle) In one preferred embodiment, the kit includes a device that uses immunochromatography as the measurement principle. The device that uses immunochromatography as the measurement principle is sometimes referred to as a test cassette.

[0045] Immunochromatography is an immunoassay method that utilizes the property of a liquid sample to flow along a membrane due to capillary action. Antigens in the sample move along the membrane, forming immune complexes with antibodies labeled with metal colloids or the like (labeled antibodies) that have been prepared in advance in the device. These antigens are then trapped by another antibody immobilized in the detection unit, forming a line which is then visually determined. In this embodiment, the labeled antibody corresponds to the first antiviral antibody that has been labeled. In this embodiment, the other antibody immobilized in the detection unit corresponds to the second antiviral antibody.

[0046] In the test cassette included in the kit, both the first and second antiviral antibodies are effective against specific oral and nasal infection viruses that the kit detects. The first and second antiviral antibodies can bind to the target simultaneously. Therefore, in one embodiment, the viral epitope recognized by the first antiviral antibody is different from the viral epitope recognized by the second antiviral antibody.

[0047] Figure 1 shows a schematic diagram of an example of a test cassette included in this embodiment. 1 is the sample application area, 2 is the antibody holding area (where the first antiviral antibody is held), 3 is the detection area, 31 is the test line (the line on which the second antiviral antibody is immobilized), 32 is the control line, 4 is the membrane, 5 is the absorbent pad, and 6 is the backing sheet.

[0048] Substances for antibody labeling include colored particles, enzymes, and radioisotopes, but it is preferable to use colored particles that can be detected visually without the need for special equipment. Examples of colored particles include metal nanoparticles such as gold and platinum (sometimes called gold colloid particles or platinum colloid particles), nonmetallic particles, and latex particles.

[0049] Test cassettes can be manufactured using commercially available materials by known methods. The material for the membrane of the test cassette (4 in the example in Figure 1) is not particularly limited as long as it can be used for immunochromatography, and examples include nitrocellulose, mixed nitrocellulose esters, polyvinylidene fluoride, nylon, etc. The material for the backing sheet of the test cassette (6 in the example in Figure 1) can be synthetic polymers such as nitrocellulose, mixed nitrocellulose esters, polyvinylidene fluoride, nylon polyethylene, polypropylene, polystyrene, acrylic resin, nylon, polyester, polycarbonate, polyacrylamide, and polyurethane, which are excellent in terms of ease of handling, stability, safety, moldability, and sterilization; natural polymers themselves such as cellulose, nitrocellulose, cellulose acetate, chitin, chitosan, and alginate, as well as crosslinked or modified structures thereof; and inorganic materials such as hydroxyapatite, glass, alumina, and titania.

[0050] (Vial containing sample processing solution) The kit includes a sample processing solution, which may be stored in a vial. The vial is preferably equipped with a function for grinding the sample. An example of such a vial is a finger masher tube (Zalstad).

[0051] (Other) In addition to the above, the kit may also include a solution for diluting the sample, and equipment for collecting the sample, such as a cotton swab and / or a suction catheter.

[0052] The kit is intended for detecting oral and nasal infection viruses such as foot-and-mouth disease virus, influenza virus, RSV, coronavirus, adenovirus, and metapneumovirus, but in a particularly preferred embodiment, it is intended for detecting foot-and-mouth disease virus.

[0053] III. Method for processing specimens, method for testing viruses using processed specimens, and method for diagnosing viral infections The present invention provides, as a third embodiment, a method for detecting oral and nasal infection viruses, comprising the following steps: Collect a lesional epithelium suspected to be infected with an oral and nasal infection virus as a specimen; Disintegrate the collected specimen in a specimen processing solution containing a buffer, a calcium chelating agent, and magnesium ions to obtain a sample solution; Drop the obtained sample solution onto the specimen portion of a test cassette having a specimen drop portion, an antibody holding portion holding a labeled first antiviral antibody, and a test line determination portion positioned downstream of the specimen drop portion and the antibody holding portion, on which a second antiviral antibody is immobilized; Confirm the presence or absence of a predetermined line in the test line determination portion.

[0054] The time from dropping the sample onto the sample application area of ​​the test cassette to checking for the presence or absence of a predetermined line in the test line determination area can be 5 to 30 minutes, or it may be 10 to 20 minutes.

[0055] (Viruses, etc.) The substances that can be detected in this embodiment are not particularly limited, but the method of this embodiment is preferably applicable to oral and nasal infection viruses. Examples of oral and nasal infection viruses include foot-and-mouth disease virus, influenza virus, parainfluenza virus, RSV, coronavirus, adenovirus, metapneumovirus, and herpesvirus. The diseases or conditions that are tested or diagnosed in this embodiment are not particularly limited, but are oral and nasal infections, specifically foot-and-mouth disease, influenza, parainfluenza virus infection, RSV infection, coronavirus infection, adenovirus infection, metapneumovirus infection, viral diarrhea, respiratory disease syndrome, and infectious rhinotracheitis. The animals that are tested or diagnosed in this embodiment are not particularly limited, but are artiodactyls, such as cattle, buffalo, pigs, sheep, and goats.

[0056] In a particularly preferred embodiment, the target for detection is the foot-and-mouth disease virus, the disease or condition being tested or diagnosed is foot-and-mouth disease, and the animal being tested or diagnosed is a cattle.

[0057] (Specimen) The specimen is the epithelium of the lesion site, such as oral epithelium, nasal cavity epithelium, or hoof epithelium, and may also be saliva, nasal secretions, blood, serum, or pus.

[0058] [Detection of Foot-and-Mouth Disease Virus] The current sample processing solution (Comparative Composition 1 in Table 1) was tested with the addition of hexadimethrin bromide (hereinafter, HB), which is used to eliminate false positives in the above patent (Comparative Composition 2 in Table 1), but the false positives were not eliminated. On the other hand, an effect of improving sensitivity was confirmed. Furthermore, even when the sample processing solution was prepared according to Example 1 of this patent (Comparative Composition 7 in Table 1) and the test was conducted, the false positives were not eliminated. Therefore, with the aim of increasing the specificity of the antigen-antibody reaction (elimination of false positives), the buffer of the sample processing solution was changed to PHEM (PIPES, HEPES, EGTA, MgCl2) and the test was conducted again.

[0059] (1) Tests to confirm the ability to eliminate false positives (i) Collection of bovine nasal secretions Nasal secretions were collected from healthy cows raised on farms in Japan by swabbing the inside of the nasal cavity with a cotton swab. The secretions were then diluted 10-fold with PBS.

[0060] (ii) Collection of bovine saliva Saliva was collected from healthy cows raised on farms in Japan by swabbing the inside of their mouths with cotton swabs. The saliva was then diluted 10-fold with PBS.

[0061] (iii) Testing and Confirmation of Results The bovine nasal secretions and saliva collected in (i) and (ii) were diluted 10-fold with each sample processing solution (compositions 1-10, comparative compositions 1-8) to prepare the sample solutions. 120 μL of these sample solutions were added to the sample application area of ​​a test cassette (veterinary drug "NH ImmunoDetect® Foot-and-Mouth Disease", approval order number: Ministry of Agriculture, Forestry and Fisheries Order 5 Animal Drug No. 398) using a micropipette, and the presence or absence of a test line was visually confirmed after 15 minutes.

[0062] (2) Test foot-and-mouth disease virus solution for sensitivity confirmation was diluted with each sample processing solution (composition 1 to 10, comparative composition 1 to 8) and 1 × 10 5 TCID 50 A virus solution was prepared at a concentration of / mL and used as the sample solution. 120 μL of these sample solutions was added to the sample application area of ​​a test cassette using a micropipette, and after 15 minutes, the concentration of the test line was read and quantified using a machine (machine name: C10066, manufacturer: Hamamatsu Photonics K.K.). The virus used was foot-and-mouth disease virus isolated from material from a case in Miyazaki Prefecture in April 2010, held by the Kodaira Overseas Disease Research Center, Animal Health Research Division, National Agriculture and Food Research Organization.

[0063] The composition and false-positive resolution capabilities of each sample processing solution are shown below.

[0064]

[0065]

[0066] Buffer composition PBS: NaCl (0.82% (w / v)), KCl (0.02% (w / v)), PO4 3-(0.096%(w / v)) PHEM: PIPES (1.8%(w / v)), HEPES (0.6%(w / v)), EGTA (0.24%(w / v)), MgCl2 (0.019%(w / v)) Tris: Polyblen (0.1%(w / v)), Tris-HCl (5.2%(w / v)), Triton X-100 (0.27%(w / v)), pH 9.3 (See example in Patent Document 1)

[0067] LAS: Linear dodecylbenzenesulfonate sodium (Fujifilm Wako Pure Chemical Corporation, Linear dodecylbenzenesulfonate sodium standard) BSA: Bovine serum albumin (Fujifilm Wako Pure Chemical Corporation, Albumin, derived from bovine serum (BSA), crystalline product) HB: Hexadimethrin bromide (Sigma-Aldrich, Hexadimethrin bromide, Molecular weight: 1122.6) TritonX: MP Biomedical, TritonX-100 Tween20: Fujifilm Wako Pure Chemical Corporation, Tween-20 (Polyoxyethylene (20) sorbitan monolaurate)

[0068] (3) Effects and Conclusions (i) Elimination of false positives and sensitivity When PBS(-) was used as a buffer, false positives were not eliminated in any of the compositions (Comparative compositions 1-6 in Table 1).

[0069] Next, following the example in Patent Document 1, we conducted tests using sample processing solutions prepared with Tris as a buffer (comparative compositions 7 and 8 in Table 1 and Figure 3), but the sensitivity was inferior compared to the results of the other sample processing solutions. Therefore, it was concluded that Tris does not have an effect on eliminating false positives, and that combining it with HB is not effective in improving sensitivity (Foot-and-mouth disease + Tris buffer + HB: decreased sensitivity).

[0070] When PHEM was used as a buffer, it was confirmed that it had the ability to eliminate false positives in all compositions (compositions 1-10 in Table 1). Among these, sensitivity was improved when Tween20 contained 0.05% or 0.1% HB, and in particular, the line density tended to be darker when HB was included, suggesting that Tween20 or HB has the effect of maintaining test sensitivity. Therefore, it was considered that combining PHEM with Tween20 or HB makes it possible to eliminate false positives while preventing a decrease in sensitivity.

[0071] Sensitivity was compared in Figure 3. The first test (compositions 4-7, comparative compositions 1-3) and the second test (compositions 1-3, 8-10, comparative compositions 4-8) in Figure 3 were conducted on different dates, and therefore the lots used were also different. To compare the compositions of the first and second tests under the same conditions, composition 4 from the first test was also tested on the same day as the composition from the second test, and a correction was made. The vertical axis of the graph represents the density of the line when the test kit result was determined, which was read by a machine and quantified; a higher number indicates better visibility (higher sensitivity). The detection limit was set to a value that can be confirmed by human visual acuity (around 10).

[0072] The kit's detection sensitivity is 1 × 10⁻¹⁰ 5 TCID 50 When samples with concentrations of / mL were used, positive results were obtainable for all compositional treatment solutions. Since the minimum visible line on the kit is approximately 10, the sensitivity for detecting foot-and-mouth disease appears to be sufficient for all compositions, including compositions 1-10 and comparative compositions 1-8.

[0073] 1. Sample application area 2. Antibody holding area 3. Detection area 31. Test line 32. Control line 4. Membrane 5. Absorbent pad 6. Backing sheet

Claims

1. A kit for detecting oral and nasal infection viruses, comprising the following sample processing solution and test cassette: A sample processing solution containing 0.2–10 w / v% buffer, 0.02–1 w / v% calcium chelating agent, and 0.002–0.1 w / v% equivalent of magnesium ions as magnesium chloride; and a test cassette having a sample application section for dropping the sample solution treated with the sample processing solution, an antibody holding section holding a labeled first antiviral antibody, and a test line determination section positioned downstream of the sample application section and antibody holding section, on which a second antiviral antibody is immobilized.

2. A sample processing solution for processing lesional epithelium suspected to be infected with oral and nasal viruses, containing 0.2–10 w / v% buffering agent, 0.02–1 w / v% calcium chelating agent, and 0.002–0.1 w / v% equivalent of magnesium ions as magnesium chloride.

3. The kit according to claim 1, or the sample processing solution according to claim 2, wherein the buffer comprises PIPES and HEPES.

4. The kit according to claim 1, or the sample processing solution according to claim 2, wherein the calcium chelating agent comprises glycol etherdiaminetetraacetic acid (EGTA).

5. The kit according to claim 1, or the sample processing solution according to claim 2, wherein the sample processing solution contains a surfactant.

6. The kit according to claim 1, or the sample processing solution according to claim 2, wherein the sample processing solution contains hexadimethrin bromide.

7. The kit according to claim 1, or the sample processing solution according to claim 2, wherein the sample processing solution contains hexadimethrin bromide in an amount greater than 0.03 w / v% and less than or equal to 0.5 w / v%.

8. The kit according to claim 1, or the sample processing solution according to claim 2, wherein the pH of the sample processing solution is 3.0 to 11.

0.

9. The kit according to claim 1, or the sample processing solution according to claim 2, wherein the pH of the sample processing solution is a pH effective for maintaining viral activity.

10. Method for detecting oral and nasal infection viruses, including the following steps: Collect a sample of epithelial tissue from a lesion suspected to be infected with an oral and nasal infection virus; Disintegrate the collected sample in a sample processing solution containing 0.2-10 w / v% buffer, 0.02-1 w / v% calcium chelating agent, and 0.002-0.1 w / v% equivalent magnesium ions as magnesium chloride to obtain a sample solution; Drop the obtained sample solution onto the sample application section of a test cassette having a sample application section, an antibody holding section containing a labeled first antiviral antibody, and a test line determination section located downstream of the sample application section and the antibody holding section, on which a second antiviral antibody is immobilized; Confirm the presence or absence of a predetermined line in the test line determination section.

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