Method for virus detection

Abstract

To a technique that enables simple detection of a virus from a specimen that can be collected in a short time with low invasiveness.SOLUTION: A method for virus detection according to one embodiment of the present invention is a method for detecting a target virus to be detected, the method including: a step of bringing a specimen, which is an oral rinse liquid discharged after rinsing the oral cavity, into contact with a porous carrier holding an antibody against the target virus; and a step of detecting the target virus through an antigen-antibody reaction between the target virus in the specimen flowing through the porous carrier and the antibody.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a virus detection method for quickly and easily detecting a virus. [Background technology]

[0002] With the spread of upper respiratory tract infections caused by viruses such as SARS-CoV-2, antigen testing methods such as immunochromatography are being used to quickly and easily detect infection. Nasopharyngeal swabs are commonly used as specimens for upper respiratory tract infection testing, but their invasiveness poses a problem. Saliva samples are also known as less invasive specimens than nasopharyngeal swabs. However, collecting saliva can take a long time and can be difficult, especially in elderly people who produce less saliva.

[0003] In contrast to this, gargle fluid is known as a specimen that is less invasive and can be collected in a relatively short time (Non-Patent Documents 1 and 2). [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] McLennan K. et.al., Diagn. Microbiol. Infect. Dis., (2022), 104(1):115732. [Non-patent document 2] Niko K. et.al., J. Clin. Med.,(2021), 10, 5751. Summary of the Invention [Problem to be solved by the invention]

[0005] Although Non-Patent Documents 1 and 2 detect the SARS-CoV-2 virus from gargle fluid samples, no information has been obtained as to whether gargle fluid samples are useful for simple antigen testing methods such as immunochromatography.

[0006] The present invention relates to a technology that enables simple detection of viruses from specimens that are minimally invasive and can be collected in a short time. [Means for solving the problem]

[0007] A virus detection method according to one embodiment of the present invention is a method for detecting a target virus that is a detection target, comprising: A step of contacting a specimen consisting of the mouthwash expelled after rinsing the oral cavity with a porous carrier that retains antibodies against the target virus; and detecting the target virus through an antigen-antibody reaction between the target virus and the antibody in the sample flowing through the porous carrier. [Effects of the Invention]

[0008] According to the present invention, viruses can be detected simply and easily from specimens that can be collected in a short time with minimal invasiveness. [Brief explanation of the drawings]

[0009] [Figure 1] 1 is a flowchart illustrating a virus detection method according to an embodiment of the present invention. [Figure 2] 1A and 1B are schematic diagrams showing an example of a membrane carrier used in the immunochromatography method according to the virus detection method, in which (A) is a plan view and (B) is a cross-sectional view. DETAILED DESCRIPTION OF THE INVENTION

[0010] [Summary of the Invention] The present invention relates to a method for easily detecting a target virus from a sample that is minimally invasive and can be collected in a short time. More specifically, the present invention uses a sample made of mouthwash to detect the target virus through an antigen-antibody reaction that utilizes the fluidity of the sample in a porous carrier. Details are described below.

[0011] [Target virus] In the present invention, the target virus is a virus to be detected, and may be any virus that can be detected in saliva or the oral cavity, but is preferably a virus that is transmitted through saliva droplets. Such viruses include, for example, at least one selected from influenza virus, respiratory syncytial virus, coronavirus, adenovirus, parainfluenza virus, mumps virus, rubella virus, and human metapneumovirus. In particular, the target virus preferably includes SARS-CoV-2, which causes acute respiratory disease (COVID-19) and belongs to the coronavirus family. The target virus may be one type only, or may include multiple types.

[0012] [Specimen] In the present invention, the sample consists of mouthwash. In the present invention, mouthwash is the liquid expelled after rinsing the oral cavity. The act of producing mouthwash, including rinsing the oral cavity, is also referred to as "mouthwashing." By using mouthwash as a sample, a sample containing viruses present in saliva and / or the mucous membranes in the oral cavity can be obtained. The duration of the mouthwashing is preferably 5 seconds or more, more preferably 10 seconds or more, from the viewpoint of containing a sufficient amount of viruses. Furthermore, the duration of the mouthwashing is preferably 20 seconds or less, more preferably 15 seconds or less, from the viewpoint of reducing the burden on the subject. Furthermore, the amount of mouthwash liquid to be placed in the mouth is preferably 5 mL or more, more preferably 10 mL or more, from the viewpoint of containing a sufficient amount of viruses, and is preferably 20 mL or less, more preferably 15 mL or less.

[0013] Furthermore, in the present invention, the mouthwash includes gargling in addition to rinsing the oral cavity, and the mouthwash is preferably a liquid that is spit out after rinsing the oral cavity and gargling. By using the mouthwash as a liquid that is spit out after rinsing the oral cavity and gargling, it is possible to obtain a sample containing not only viruses present in saliva and / or the mucous membranes in the oral cavity, but also viruses present in the mucous membranes of the pharynx at the back of the oral cavity. The gargling time in the mouthwash is preferably 3 seconds or more from the viewpoint of containing a sufficient amount of viruses. Furthermore, from the viewpoint of reducing the burden on the subject, the gargling time is preferably 20 seconds or less, more preferably 10 seconds or less, and even more preferably 5 seconds or less.

[0014] The mouthwash to be placed in the mouth is not particularly limited as long as it does not affect the test process, etc., and does not harm the health of the subject. However, from the viewpoint of low-cost and simple processing, for example, drinking water or purified water is preferable, and from the viewpoint of further suppressing the influence of solutes on the antigen-antibody reaction, purified water is more preferable. Drinking water is drinkable water that meets drinking water quality standards, such as tap water. Purified water is water that has been purified from tap water, etc., such as deionized water and distilled water. Note that the mouthwash is not limited to these, and may also contain sodium chloride, surfactants and / or buffer solutions, etc.

[0015] [Virus detection method] A virus detection method according to one embodiment of the present invention includes a step (S1) of contacting a sample with a porous carrier and a step (S2) of detecting a target virus. This example is shown in Figure 1. The virus detection method according to the present invention may use immunochromatography, or may be any other virus detection method that utilizes the fluidity of a porous carrier and an antigen-antibody reaction.

[0016] (Contacting step between sample and porous carrier (S1)) In this step, a sample consisting of mouthwash is brought into contact with a porous carrier that holds antibodies to the target virus. In one embodiment of the present invention, the sample used is at least a portion of the mouthwash spat out of the subject's mouth. Furthermore, from the viewpoint of ensuring reliable virus detection in the subsequent detection step (S2), the sample used in this step is preferably 100 μL or more, more preferably 150 μL or more, and preferably 1 mL or less, more preferably 0.5 mL or less.

[0017] Furthermore, in this step, the specimen may be diluted with a specimen dilution solution. The specimen dilution solution is not particularly limited as long as it assists the flow of the specimen through the porous carrier and suppresses nonspecific reactions caused by the antibody, but may be, for example, an aqueous solution containing a buffer solution, a salt such as sodium caseinate, a surfactant, a polymer compound, an antibacterial agent, a chelating agent, etc. The specimen dilution solution may be added to the porous carrier as a mixed solution containing the specimen, or may be added to the specimen contact portion of the porous carrier before contact between the specimen and the porous carrier.

[0018] The porous carrier may be made of one or more materials as long as it is configured to allow the sample to flow (permeate). Examples of materials constituting the porous carrier include cellulose-based materials, glass fibers, and synthetic resins (polypropylene, polyethylene, polyurethane, polyvinylidene fluoride, ethylene vinyl acetate, acrylonitrile, polytetrafluoroethylene, nylon, etc.). Examples of materials constituting the porous carrier include fiber assemblies and other porous substrates. The porous carrier is preferably made of a fiber assembly such as a nonwoven fabric, a woven fabric, or a cellulose membrane, as this makes it easier to control the fluidity of the sample. A cellulose membrane is preferred because it is highly hydrophilic. The cellulose membrane referred to here is a membrane whose main component (accounting for 50% or more by mass of the total) is a cellulose-based fiber made of cellulose and / or a cellulose derivative (nitrocellulose, cellulose acetate, etc.).

[0019] The porous carrier can take on various shapes depending on the flow direction of the sample, and examples thereof include a membrane-like (sheet-like) shape that allows flow on the surface or in the thickness direction of the carrier, and a cylindrical or truncated cone-like shape that allows flow inside the carrier. As will be described later, from the viewpoint of causing the antigen-antibody reaction to occur on the surface of the carrier and making it easier to recognize visually, it is preferable that the porous carrier be configured as a membrane-like carrier. Here, "membrane-like" refers to a shape that has a main surface and a thickness perpendicular to the main surface, and the maximum thickness dimension is smaller than the minimum dimension of the main surface.

[0020] The method for contacting the porous carrier with the sample is not particularly limited, and for example, the sample-containing liquid may be dropped onto at least a portion of the porous carrier, or at least a portion of the porous carrier may be immersed in the sample-containing liquid. In one embodiment of the present invention, it is preferable to contact the sample with a portion of the porous carrier, from the viewpoint of causing a detectable antigen-antibody reaction at a predetermined position on the porous carrier by the flow of the sample.

[0021] The term "antibody retained on a porous carrier" refers to an antibody that is retained on the porous carrier without being largely detached during distribution, storage, and detection. Such an antibody may be immobilized so as not to move from the porous carrier, or may be arranged so as to be mobile on the surface or inside of the porous carrier in association with the flow of the sample.

[0022] The antibody against the target virus is an antibody that recognizes and specifically binds to the target virus. The antibody against the target virus is not particularly limited, but preferably includes a labeled antibody bound to a labeling substance from the viewpoint of facilitating detection by the antigen-antibody reaction described below. Furthermore, the antibody against the target virus is not limited to one type, and may include two or more types of antibodies that differ in the presence or absence of labeling, the retention form in the porous carrier, etc.

[0023] The labeling substance is not particularly limited, but from the viewpoint of enabling visual determination of virus detection, it includes at least one selected from, for example, metal particles such as gold, silver, platinum, etc., metal oxide particles such as iron oxide, non-metal particles such as sulfur, synthetic polymers such as latex particles, and other insoluble labeling substances. Of these, from the viewpoint of enabling visual determination of detection, the labeling substance is preferably metal particles or metal oxide particles that exhibit a color according to their particle size, and is preferably, for example, gold colloid particles.

[0024] The antibody against the target virus may be retained on the sample flow path in the porous carrier, but from the viewpoint of facilitating visual recognition of detection by antigen-antibody reaction, it is preferable that it be retained on the surface of the porous carrier. In one embodiment, the position of the antibody against the target virus on the porous carrier is appropriately selected from a position away from the sample contact area, a position adjacent to the sample contact area, a position within the sample contact area, etc.

[0025] (Target virus detection step (S2)) In this step, the target virus is detected by an antigen-antibody reaction between the target virus and an antibody in the sample flowing through the porous carrier.

[0026] Although the method of causing the sample to flow through the porous carrier may be a method utilizing pressure or gravity, in one embodiment of the present invention, it is preferable to cause the sample to flow through the porous carrier by utilizing capillary action, which allows the sample to flow spontaneously regardless of the flow direction.

[0027] The method for detecting an antigen-antibody reaction may be a direct method in which a signal from a labeling substance directly bound to the antigen-antibody complex is detected, or an indirect method in which an antibody (secondary antibody) is further bound to the antibody (primary antibody) bound to the antigen, and the signal from the labeling substance bound to the secondary antibody is detected. Furthermore, the method for detecting the signal from the labeling substance can be appropriately selected depending on the labeling substance, but for example, when the labeling substance is metal particles or metal oxide particles, visual detection is possible.

[0028] As described above, according to the virus detection method of one embodiment of the present invention, by using a sample made of mouthwash, the invasiveness of sample collection can be kept significantly lower than that of conventional nasopharyngeal swabs, and the sample collection time can be shortened compared to saliva samples, etc. Furthermore, as shown in the examples described below, mouthwash has less interference with antigen-antibody reactions than saliva samples, and can increase the virus detection sensitivity.

[0029] Furthermore, by using a porous carrier that holds antibodies against a target virus and detecting the target virus through an antigen-antibody reaction between the target virus and the antibody in a sample flowing through the porous carrier, the contact efficiency between the target virus in the sample and the antibody can be increased, which makes it possible to omit or simplify incubation processes such as shaking for the antigen-antibody reaction, and to easily detect the target virus.

[0030] In order to more reliably achieve the above-mentioned effects, it is preferable to use immunochromatography for detecting the target virus in one embodiment of the present invention. Specific examples using immunochromatography will be described below.

[0031] [Example of target virus detection using immunochromatography] 2(A) and (B) show examples of membrane-like carriers used in immunochromatography. The membrane-like carrier 10 shown in these figures includes a first end 10a and a second end 10b, which are ends in the flow direction X of the sample, and extends, for example, along the flow direction X of the sample as a whole. The membrane-like carrier 10 further includes a sample contact section 11, a detection section 12, and a first antibody 15 and a second antibody 16 as antibodies against the target virus. The membrane-like carrier 10 may be contained in a housing (not shown). Note that the configuration of the membrane-like carrier used in immunochromatography is not limited to the example shown in FIGS. 2(A) and (B).

[0032] The specimen contact section 11 is a site that comes into contact with the specimen, and is arranged on the first end 10a side. The "first end 10a side" of the membrane-like carrier 10 refers to the side on which the first end 10a is present when the membrane-like carrier 10 is divided into two equal parts in the flow direction. In the example shown in FIGS. 2(A) and 2(B), the specimen contact section 11 forms the first end 10a. From the viewpoints of promoting antigen-antibody reactions and suppressing nonspecific reactions, the specimen contact section 11 may appropriately contain reagents such as buffer solutions, salts such as sodium caseinate, surfactants, polymer compounds, antibacterial agents, and chelating agents.

[0033] The first antibody 15 is a labeled antibody bound to a labeling substance 15a, and is held in the specimen contact section 11 or between the specimen contact section 11 and the detection section 12. The first antibody 15 is held on the membrane-like carrier 10 in a state in which it can be released as the specimen flows.

[0034] The second antibody 16 is a capture antibody immobilized on the detection section 12. In other words, the second antibody 16 is configured so as to be able to maintain a state of being immobilized on the membrane-like carrier 10 even when liquid flows.

[0035] Furthermore, the membrane-like carrier 10 may have a control section 13 arranged between the detection section 12 and the second end 10b, and a third antibody 17 that is fixed to the control section 13 and is directed against the first antibody 15. Although the first antibody 15, the second antibody 16, and the third antibody 17 are shown schematically in Figures 2(A) and (B), these antibodies cannot actually be confirmed visually.

[0036] More specifically, in the example shown in Fig. 2(B), the membrane-like carrier 10 has a spreading layer 20, a sample contact layer 21, a labeled antibody retention layer 22, and an absorption layer 23. In the membrane-like carrier 10, the sample contact layer 21, the labeled antibody retention layer 22, the spreading layer 20, and the absorption layer 23 are arranged in this order along the flow direction X. The spreading layer 20 is the longest layer in the flow direction X, and is the main layer through which the sample spreads. The sample contact layer 21, the labeled antibody retention layer 22, and the absorption layer 23 are all configured as pad-shaped porous carriers that are shorter in the flow direction X than the spreading layer 20.

[0037] The specimen contact layer 21 is a layer including the specimen contact portion 11 and forms the first end portion 10a. For example, a downstream portion of the specimen contact layer 21 is connected to the labeled antibody retention layer 22, and an upstream portion of the specimen contact layer 21 protrudes from the labeled antibody retention layer 22 in the direction opposite to the flow direction X.

[0038] The labeled antibody holding layer 22 holds the first antibody 15. In the example shown in Fig. 2(A), the labeled antibody holding layer 22 is arranged so as to overlap the specimen contact layer 21 and the spreading layer 20 in a plan view seen from the thickness direction. In the example shown in Fig. 2(B), the labeled antibody holding layer 22 is arranged below the specimen contact layer 21 and on the first end 20a of the spreading layer 20.

[0039] The spreading layer 20 includes a first end 20a located upstream in the flow direction X and a second end 20b located downstream. The spreading layer 20 includes a detection unit 12 and a control unit 13 between the first end 20a and the second end 20b. The control unit 13 is located between the detection unit 12 and the second end 20b.

[0040] The absorbent layer 23 is disposed on the second end 20b of the spreading layer 20, and absorbs excess liquid that reaches the second end 20b. The absorbent layer 23 also forms the second end 10b of the membrane-like carrier 10.

[0041] 2(A) and (B), a method for detecting a target virus by immunochromatography will be described. First, a sample is brought into contact with the sample contact section 11 (S1). The sample is preferably contacted by dropping the sample onto the sample contact layer 21, but the membrane-like carrier 10 may be placed vertically in a container containing the sample, and the sample contact section 11 may be immersed in the sample. This causes the sample to begin to flow in the flow direction X due to capillary action.

[0042] The target virus in the sample that has reached the labeled antibody retention layer 22 binds to the first antibody 15 to form a complex, and then reaches the spreading layer 20 and continues to flow in the flow direction X. The complex of the target virus and the first antibody 15 that has reached the detection unit 12 binds to the second antibody 16, forming a complex of the first antibody 15, the target virus, and the second antibody 16. As a result, many complexes of the first antibody 15, the target virus, and the second antibody 16 are arranged on the detection unit 12, and a signal is emitted by the labeling substance 15a of the first antibody 15. For example, if the labeling substance 15a is a gold colloid label, a detection line can be visually determined on the detection unit 12, and the target virus can be detected (detection step (S2)).

[0043] On the other hand, the first antibody 15 that has flowed beyond the detection section 12 binds to the third antibody 17 on the control section 13. As a result, a large number of complexes of the first antibody 15 and the third antibody 17 are arranged on the control section 13, and a signal is emitted by the labeling substance 15a of the first antibody 15. For example, if the labeling substance 15a is a gold colloid label, a control line can be visually determined on the control section 13.

[0044] Furthermore, the liquid that reaches the second end 20b of the spreading layer 20 is absorbed by the absorbent layer .

[0045] As described above, the immunochromatography method using the membrane-like carrier 10 makes it possible to detect target viruses simply by bringing the specimen into contact with the membrane-like carrier 10. Furthermore, the immunochromatography method using a specimen made of mouthwash makes it possible to easily detect target viruses from specimens that are minimally invasive and can be collected in a short time.

[0046] Although the embodiments of the present invention have been described above, the present invention is not limited to the above-described embodiments, and it goes without saying that various modifications can be made within the scope of the gist of the present invention. [Example]

[0047] <Test Example 1: Examination of SARS-CoV-2 viral load by collecting mouthwash> In Test Example 1, the amount of virus in mouthwash and saliva samples collected from patients infected with SARS-CoV-2 was examined.

[0048] [Collection of mouthwash samples] (Collection using mouthwash method A) The subjects took 15 ml of distilled water (Otsuka Pharmaceutical Factory, Inc.) into their mouths, gargled for 5 seconds, and then rinsed their mouths for 5 seconds. After rinsing, the mouthwash was immediately dispensed into a 100 ml centrifuge tube (Iwaki).

[0049] (Saliva sample collection) A funnel (Otsuka Techno Co., Ltd.) was inserted into a 5 ml tube (Eppendorf), and saliva collected in the oral cavity was expelled using the spitting method. Collection was terminated when 1 ml or more of saliva had accumulated or when saliva had been expelled for 5 minutes.

[0050] (Those for whom samples are to be collected) Saliva samples and mouthwash samples using Method A were collected from patients diagnosed with SARS-CoV-2 infection by qualitative or quantitative testing of nasopharyngeal swab samples.

[0051] [Evaluation of viral load in specimens] (Example A1) The viral load in Example A1 was evaluated as follows.

[0052] The SARS-CoV-2 Direct Detection RT-qPCR Kit (Takara Bio Inc.) was used to quantify the copy number of the viral N1 / N2 gene region in mouthwash collected using Method A. First, 8 μl of thoroughly mixed mouthwash and 2 μl of Solution A provided with the kit were mixed in a 96-well PCR plate (Invitrogen) and heated at 95°C for 5 minutes in a thermal cycler. Next, 25 μl of RT-qPCR Mix, 5 μl of Primer / Probe Mix (SARS-CoV-2), 1 μl of ROX Reference Dye / Dye II, and 9 μl of RNase-free H2O were added per sample. After mixing thoroughly by pipetting, a sticker (Invitrogen) was attached to the 96-well plate. Measurements were performed using a QuantStudio 7 Flex (Applied Biosystems) to detect Cy5 fluorescence. The RNA positive control included in the kit was diluted 10-fold, and a calibration curve was created from the Ct values ​​of the control samples at six concentrations. The viral load in the mouthwash was then calculated based on this calibration curve. The results are shown in Table 1.

[0053] (Comparative Example A1) The viral load was measured in the same manner as in Example A1, except that saliva samples were used. The results are shown in Table 1.

[0054] [Table 1]

[0055] (result) It was found that the number of virus copies in the mouthwash obtained by mouthrinse method A was equivalent to that in the saliva samples. Furthermore, while saliva samples took up to 5 minutes to collect, all mouthrinse samples could be collected in 10 seconds. These results demonstrate that collecting mouthrinse is a useful method for rapidly obtaining large amounts of SARS-CoV-2 concentrations equivalent to those obtained by existing sample collection methods.

[0056] <Test Example 2: Effect of the use of mouthwash samples on immunochromatographic antigen detection> In Test Example 2, the test line detection values ​​of the immunochromatographic test using mouthwash samples and saliva samples collected from healthy subjects were examined.

[0057] [Those who will be subject to sample collection] Saliva samples and mouthwash samples were collected using Method A from healthy individuals without symptoms such as fever, sore throat, or taste disorders.

[0058] [Evaluation of specimen suitability for immunochromatographic detection using antigen test kits] Example B1 The immunochromatographic detection of Example B1 was evaluated as follows.

[0059] 140 μl of mouthwash collected using Method A was added to the sample extraction solution provided with the Adtest SARS-CoV-2 NEO (Adtec Co., Ltd.), and the sample extraction tube was mixed thoroughly by rubbing with the fingers. Next, 0.98 μl of a solution of His-tagged purified SARS-CoV-2 nucleocapsid protein (Sino Biological) diluted with PBS to 250 μg / ml was added and mixed thoroughly by pipetting. The filter nozzle provided with the kit was inserted into the sample extraction tube, and three drops of the prepared sample were dispensed onto the sample dispenser section of the included test card according to the kit's instructions. After 5 minutes, the colored detection line was quantified using an Immunochromato Reader (Hamamatsu Photonics).

[0060] (Comparative Example B1) The immunochromatographic detection line value was measured in the same manner as in Example B1, except that saliva samples were used.

[0061] (Comparative example B2) The immunochromatographic detection line value was measured in the same manner as in Example B1, except that distilled water was used as the sample.

[0062] [Table 2]

[0063] (result) The color of the detection line value for saliva samples was lighter than that for distilled water samples, but the color of the detection line value for mouthwash samples was equivalent to that of distilled water.These results demonstrate that mouthwash samples do not interfere with immunochromatographic detection as much as existing samples, and that mouthwash is a significant sample for immunochromatographic testing.

[0064] <Test Example 3: Examination of SARS-CoV-2 viral load by collecting rinse fluid> In Test Example 3, the amount of virus in the rinse solution collected from patients infected with SARS-CoV-2 was examined.

[0065] [Collection of rinse fluid sample] (Collection using mouthwash method B) The subjects were asked to take 15 ml of distilled water (Otsuka Pharmaceutical Factory, Inc.) into their mouths and rinse their mouths for 10 seconds. After rinsing, the rinse was immediately expelled into a 100 ml centrifuge tube (Iwaki).

[0066] (Those for whom samples are to be collected) Rinse samples were collected using mouthwash method B from patients diagnosed with SARS-CoV-2 infection by qualitative or quantitative testing of nasopharyngeal swab specimens.

[0067] [Evaluation of viral load in specimens] Example C1 The viral load of Example C1 was evaluated as follows.

[0068] The SARS-CoV-2 Direct Detection RT-qPCR Kit (Takara Bio Inc.) was used to quantify the copy number of the viral N1 / N2 gene region in the rinse solution collected using Method B. First, 8 μl of thoroughly mixed rinse solution and 2 μl of Solution A provided with the kit were mixed in a 96-well PCR plate (Invitrogen) and heated at 95°C for 5 minutes in a thermal cycler. Next, 25 μl of RT-qPCR Mix, 5 μl of Primer / Probe Mix (SARS-CoV-2), 1 μl of ROX Reference Dye / Dye II, and 9 μl of RNase-free H2O were added per sample. After mixing thoroughly by pipetting, a sticker (Invitrogen) was attached to the 96-well plate. Measurements were performed using a QuantStudio 7 Flex (Applied Biosystems) to detect Cy5 fluorescence. The RNA positive control included in the kit was diluted 10-fold, and a standard curve was created from the Ct values ​​of the control samples at six concentrations. The viral load in the rinse solution was then calculated based on this standard curve. The results are shown in Table 3.

[0069] [Table 3]

[0070] Table 4 shows the detection sensitivity of commercially available antigen test kits that detect SARS-CoV-2 antigens using immunochromatography. The minimum detection sensitivity of each product was converted to virus copy number based on the value listed in the package insert. Specifically, the conversion from N antigen concentration (pg / ml) to virus copy number (copies / ml) was performed by referring to the non-patent document (Pollock, NR et al., J. Clin. Microbiol., (2021), Mar., 19;59(4):e03077-20), where an N antigen concentration of 1.5 pg / ml is converted to 1 x 10 4 The TCID was calculated as copies / ml. 50For conversion from / mL to viral copy number (copies / ml), please refer to the non-patent literature (Joshua MD et.al., https: / / doi.org / 10.1101 / 2021.12.15.21267691) and calculate 5.8 × 10 3 TCID 50 / mL to 1.3 x 10 6 Calculated as copies / ml.

[0071] [Table 4]

[0072] (result) The rinse obtained using mouthwash method B contained virus at levels above the minimum detection sensitivity of some commercially available antigen test kits. Therefore, it was found that the collected rinse can be used for antigen testing of SARS-CoV-2 using immunochromatography. Furthermore, because collection of rinse is easier and takes less time than mouthwash that involves gargling, it was found that rinse is a useful specimen for detecting the virus using immunochromatography.

[0073] <Test Example 4: Effect of using rinse samples on immunochromatographic antigen detection> In Test Example 4, the test line detection values ​​of the immunochromatographic test using rinse samples and saliva samples collected from healthy subjects were examined.

[0074] [Those who will be subject to sample collection] Saliva samples and mouth rinse samples using mouthwash method B were collected from 15 healthy individuals without symptoms such as fever, sore throat, or taste disorders.

[0075] [Evaluation of specimen suitability for immunochromatographic detection using antigen test kits] Example D1 The immunochromatographic detection of Example D1 was evaluated as follows.

[0076] 140 μl of rinse fluid collected using mouthwash method B was added to the sample extraction solution provided with the Adtest SARS-CoV-2 NEO (Adtec Co., Ltd.), and the sample extraction tube was mixed thoroughly by rubbing with the fingers. Next, 0.98 μl of a solution of His-tagged purified SARS-CoV-2 nucleocapsid protein (Sino Biological) diluted with PBS to 160 μg / ml was added and mixed thoroughly by pipetting. The filter nozzle provided with the kit was inserted into the sample extraction tube, and three drops of the prepared sample were dispensed onto the sample dispenser section of the included test card according to the kit's instructions. After 5 minutes, the colored detection line was quantified using an Immunochromato Reader (Hamamatsu Photonics).

[0077] (Comparative Example D1) The immunochromatographic detection line value was measured in the same manner as in Example D1, except that a saliva sample was used.

[0078] (Comparative example D2) The immunochromatographic detection line value was measured in the same manner as in Example D1, except that phosphate buffered saline (PBS) was used as the sample.

[0079] [Table 5]

[0080] (result) The color of the detection line value for saliva samples was lighter than that for PBS samples, but the color of the detection line value for rinse samples was equal to or greater than that for PBS. These results demonstrate that rinse samples are less likely to interfere with immunochromatographic detection than existing samples, making rinse samples a useful sample for immunochromatographic testing. [Explanation of symbols]

[0081] 10...Membrane-like carrier (porous carrier) 11...Sample contact part 12...Detection unit 13...Control section 15…First antibody 16…Second antibody

Claims

1. A method for detecting a target virus to be detected, comprising: A sample consisting of the mouthwash expelled after rinsing the oral cavity is brought into contact with a porous carrier that retains antibodies against the target virus, The target virus is detected by an antigen-antibody reaction between the target virus and the antibody in the sample flowing through the porous carrier. Virus detection methods.

2. The mouthwash is a liquid that is expelled after rinsing the oral cavity and gargling. The virus detection method according to claim 1.

3. the porous carrier is configured as a membrane-like carrier for detecting the target virus by immunochromatography, the membrane-like carrier has a first end and a second end which are ends in the flow direction of the sample, the antibody includes a labeled antibody bound to a labeling substance, The sample is brought into contact with a sample contact portion disposed on the first end side of the membrane-like carrier, In a detection section located between the sample contact section and the second end of the membrane-like carrier, a signal due to the labeling substance of the labeled antibody bound to the target virus is detected, thereby detecting the target virus. The virus detection method according to claim 1.

4. The target virus is a virus that is transmitted through saliva droplets. The virus detection method according to claim 1.

5. The target virus includes SARS-CoV-2. The virus detection method according to claim 4.

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