Simple method of collecting mucus adhered to surface of nasal mucosa, and test kit for detecting target substance in sample

Abstract

To provide a quick, easy, and accurate method of analyzing a detection target substance in a sample, which involves easily and non-invasively collecting mucus adhered to a nasal mucosal surface using a nasal drop device, and using the collected mucus as a sample.SOLUTION: A method of detecting an infectious disease is provided, comprising introducing a lavage fluid into a nasal cavity of a subject suspected of having an infectious disease, and using the lavage fluid containing nasal mucosal surface mucus obtained by lavaging the nasal mucosal surface as a sample.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention provides a method for collecting mucus adhering to the surface of the nasal mucosa using a nasal drop device as a sample, and an analytical method for detecting an analyte in the sample obtained by said method. [Background technology]

[0002] Conventionally, when specimens are collected from the human body for conducting clinical tests, tests for infectious diseases, etc., various specimens have been used, such as blood, saliva, stool, biopsy tissue, corneal and conjunctival swabs, nasal swabs, pharyngeal swabs, nasal aspirates, nasal washes, and nasal mucus.

[0003] For example, in testing for infectious diseases such as influenza virus, nasal swabs, throat swabs, nasal aspirates, and nasal mucus are widely used, and the presence or absence of influenza virus antigens in the sample can be determined quickly and easily using a test kit that uses the collected sample, so samples are routinely collected and used for testing during influenza virus epidemics (see Patent Document 1).

[0004] Nasal swabs are generally collected using sterile cotton swabs for medical use. While collecting samples using cotton swabs is said to be minimally invasive, in order to obtain a sufficient sample for testing, the swab must be inserted into the nasopharynx and scraped hard to obtain a sufficient sample from the mucosa, which can sometimes cause severe pain and bleeding. In addition, when collecting pharyngeal swabs, the palatine tonsils and posterior pharyngeal wall must be scraped hard to obtain a sufficient amount of sample, which can sometimes cause a gag reflex and make it difficult to collect a sufficient amount of sample along with vomiting. In addition, nasal aspirates can only be used at some medical institutions that have dedicated suction devices, and depending on the patient's condition, there may be little nasal mucus, making it difficult to collect a sufficient amount of sample. Nasal lavage is performed by inserting a large amount of liquid into the nasal cavity with a syringe, and the liquid that flows out is collected using a suction device in the same way as with paper cups and nasal aspirates, but putting a large amount of liquid into the nasal cavity makes the patient feel short of breath, and in some cases the inserted liquid may flow from the nose into the throat, making it difficult to collect the liquid. Nasal mucus can only be used for patients who are old enough to blow their nose, and like nasal aspirates, it can be difficult to collect a sufficient amount of specimen depending on the patient's condition. For the reasons mentioned above, the five specimen types and collection methods used in influenza virus testing, nasal swabs, pharyngeal swabs, nasal aspirates, nasal washes, and nasal mucus, generally place a significant burden on patients. In addition, depending on the patient's condition, it may not be possible to obtain the required amount of specimen, which can affect the accuracy of the test. Therefore, there was a demand for a less invasive specimen collection method and test kit that can stably collect a certain amount of specimen.

[0005] In addition, the five types of specimens used in influenza virus testing and their collection methods involve a large amount of biological components, making them prone to non-specific reactions such as false positives, false negatives, or inability to determine a result. In particular, there was a problem with nasal washes, where the large amount of wash fluid dilutes the specimen, reducing its sensitivity. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] International Publication No. WO2005 / 121794 Summary of the Invention [Problem to be solved by the invention]

[0007] The present invention provides a method for analyzing analytes in a sample in a rapid, simple and highly accurate manner by using a nasal drop device to easily and non-invasively collect mucus adhering to the surface of the nasal mucosa and using this as a specimen. [Means for solving the problem]

[0008] As a result of extensive research, the inventors of the present application have found that samples can be collected non-invasively, simply and stably by spraying a predetermined amount of saline solution into the nasal cavity using a nasal drop device and collecting the adherent mucus by washing the surface of the nasal mucosa.They have also established a measurement system that provides sufficient sensitivity when using the collected sample to detect pathogens in the sample, discovering the usefulness of the sample as a clinical specimen and have completed the present invention, which enables the analysis of analytes in samples by simple and non-invasive sample collection.

[0009] The aspects of the present invention are as follows. [1] A method for detecting infectious diseases by adding a lavage fluid into the nasal cavity of a subject suspected of having an infectious disease to wash the nasal mucosal surface, and using the lavage fluid containing mucus adhering to the nasal mucosal surface as a sample. [2] The method of [1], in which the amount of irrigation solution added to the nasal cavity is 200 to 800 μL. [3] Method [1] or [2] in which the irrigation solution is added to the nasal cavity by spraying. [4] Any of the methods [1] to [3], wherein the infectious disease is an influenza virus infection. [5] Any of the methods [1] to [4] which are immunological detection methods. [6] Any of the methods [1] to [4] which are gene amplification methods. [7] A test kit for detecting an infectious disease that uses a lavage fluid containing mucus adhering to the nasal mucosal surface obtained by adding a lavage fluid into the nasal cavity of a subject suspected of having an infectious disease to wash the nasal mucosal surface as a sample, the test kit including a test reagent for detecting the infectious disease and a nasal drop device containing the lavage fluid for washing the nasal mucosa. [8] The test kit of [7], wherein the nasal drop device is a single-use nasal drop device, and the device contains 200 to 800 μL of irrigation solution to be added to the nasal cavity for one use. [9] A test kit according to [7] or [8], in which the method of adding irrigation fluid to the nasal cavity is spraying.

[10] A test kit for detecting influenza virus infection, any of [7] to [9].

[11] Any of the test kits [7] to

[10] that is an immunological test kit.

[12] Any of the test kits [7] to

[10] that is a gene amplification test kit.

[13] A method for collecting a specimen containing a lavage fluid that contains mucus adhering to the nasal mucosal surface by adding a lavage fluid into the nasal cavity of a subject suspected of having an infectious disease and washing the nasal mucosal surface.

[14] The method of

[13] , in which the amount of irrigation solution added to the nasal cavity is 200 to 800 μL.

[15] The method of

[13] or

[14] in which the irrigation solution is added to the nasal cavity by spraying.

[16] Any of the methods

[13] to

[15] , wherein the infectious disease is an influenza virus infection. Effect of the Invention

[0010] According to the method of the present invention, a method for diagnosing an infectious disease from a specimen containing adherent mucus obtained by washing the surface of the nasal mucosa could be established.

[0011] When the method of the present invention is used to diagnose an infectious disease using as a sample the mucus adherent to the surface of the nasal mucosa, the sensitivity of the diagnosis can be equal to or greater than that of the diagnosis using as a sample nasal swab, pharyngeal swab, nasal aspirate, nasal wash, and nasal mucus. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] The present invention will be described in detail below. The present invention is characterized in that a predetermined amount of washing solution such as physiological saline solution is sprayed into the nasal cavity using a nasal drip device, and the adherent mucus obtained by washing the surface of the nasal mucosa is collected as a sample. Here, the adherent mucus refers to the mucus that is adhered to the surface of the nasal mucosa.

[0013] For example, a nasal drip device pre-filled with cleaning solution is used to spray or drip a predetermined amount of cleaning solution into the nasal cavity of a subject who is suspected of having an infectious disease through a medical interview or other means. After spraying or dripping, the surface of the nasal mucosa is washed with the cleaning solution, and the cleaning solution containing the attached mucus that has flowed out near the entrance of the nose is collected as a sample. Various tests are performed using the sample thus obtained.

[0014] In the present invention, a nasal irrigation solution is sprayed into the nasal cavity, thereby washing the surface of the nasal mucosa with the irrigation solution, and the irrigation solution containing the adhering mucus that has flowed out near the entrance of the nose can be collected as a sample.

[0015] When a cleaning solution is poured into the nasal cavity using a dropper or a tool with a nozzle, the inside of the nasal cavity is cleaned locally, making it impossible to collect mucus from a wide area of ​​the nasal mucosa surface.

[0016] In contrast, by spraying a rinsing solution into the nasal cavity, mucus from a wide range of the nasal mucosa surface can be collected.

[0017] In addition, if cleaning solution is poured into the nasal cavity using a dropper or an instrument with a nozzle, the force of the solution will cause it to pass through the nasal cavity and flow into the throat, making it impossible to efficiently collect the cleaning solution.

[0018] In contrast, by spraying the cleaning liquid into the nasal cavity, it is possible to prevent the cleaning liquid from passing through the nasal cavity and flowing into the throat, and the cleaning liquid can be efficiently collected.

[0019] In addition, when a cleaning solution is poured into the nasal cavity using a dropper or an instrument with a nozzle, the force with which the cleaning solution is poured into the localized area of ​​the nasal cavity puts a strain on the cavity, and the subject (especially a child) often feels pain or shortness of breath.

[0020] In contrast, by spraying a cleaning solution into the nasal cavity, the burden inside the nasal cavity can be reduced, making the subject (especially a child) less likely to feel pain or shortness of breath.

[0021] In the present invention, the nasal drop device refers to a container filled with a cleaning solution such as a physiological saline solution. The nasal drop device of the present invention may be, but is not limited to, a commercially available nasal drop container, a spray device, a spray device for medical use, or the like.

[0022] The washing solution may be, for example, a physiological saline solution. The physiological saline may be, for example, a commercially available physiological saline solution prescribed in the Japanese Pharmacopoeia or a solution prepared according to the prescription of the Japanese Pharmacopoeia, but is not limited thereto. The washing solution may also be an isotonic solution such as Ringer's solution, lactate Ringer's solution, or glucose solution.

[0023] For rinsing the nasal cavity, 200 to 800 μL, preferably 400 to 700 μL, and more preferably 300 to 500 μL of the rinsing solution may be sprayed or dropped into the nasal cavity using a nasal drop device.

[0024] Methods for collecting the liquid containing the adherent mucus that has flowed out near the entrance of the nose as a specimen include collection methods using various collection tools, and as collection tools, tools capable of holding liquid, such as cotton swabs, swabs, brushes, tools with mesh tips, sponge-like tools, cloth-like absorbents, etc., can be used. The liquid containing the adherent mucus can be absorbed into these tools and collected.

[0025] In the present invention, the specimen collected in this manner is referred to as a simplified nasal mucosal surface adherent mucus specimen.

[0026] The specimen sample may be, for example, a liquid obtained by dissolving or suspending the specimen collected by the collection tool in a liquid. The specimen collected by the collection tool can be dissolved or suspended in the liquid by massaging, squeezing, or rubbing the part of the collection tool that has absorbed the liquid containing the attached mucus, for example, the cotton part at the tip of a cotton swab, in the liquid. A buffer solution can be used as the liquid for dissolving or suspending the specimen.

[0027] In the present invention, the specimen can be used directly as a sample without treatment such as concentration or culturing. The specimen may also be mixed with a buffer solution to prepare a sample. For example, a phosphate buffer solution can be used as the buffer solution, and it may contain a surfactant such as Tween 20 or serum albumin. For example, when performing an assay using the immunochromatography method, which is a lateral flow immunoassay method using a membrane, the specimen collected with a cotton swab is suspended in a buffer solution and used as a specimen sample.

[0028] In the present invention, the infectious diseases to be detected are infectious diseases caused by pathogens such as viruses, bacteria, protozoa, fungi, mycoplasma, rickettsia, and chlamydia, and among these, the purpose is to detect infectious diseases caused by viruses in particular. Examples of viruses include influenza viruses; coronaviruses such as SARS-CoV, MERS-CoV, and SARS-CoV-2; respiratory syncytial virus; adenovirus; and human metapneumovirus. Pathogens that cause these infectious diseases may be detected as the object to be detected.

[0029] Methods for detecting an analyte using the present invention include immunoassays (immunological assays), genetic testing, and isolation and culture / identification methods.

[0030] In the immunoassay, the virus in the sample is analyzed by utilizing an antibody-antigen reaction. Specifically, the analysis can be performed using an antibody specific to the antigen of the virus. The antibody against the antigen of the virus can be obtained by a known method. As the immunoassay, any method known to those skilled in the art can be used, such as immunostaining (including fluorescent antibody method, enzyme antibody method, heavy metal labeled antibody method, and radioisotope labeled antibody method), a method combining separation by electrophoresis with detection method using fluorescence, enzyme, radioisotope, etc. (including Western blot method and fluorescent two-dimensional electrophoresis method), enzyme-linked immunosorbent assay (ELISA), dot blotting method, latex agglutination method (LA: latex agglutination-turbidimetric immunoassay), and immunochromatography. In the present invention, "analysis" includes any of quantitative, semi-quantitative, and detection.

[0031] Among the above immunoassays, the sandwich method is preferred. The sandwich method itself is well known in the field of immunoassay, and can be carried out, for example, by immunochromatography or ELISA, which perform immunoassay in a lateral flow manner. All of these sandwich methods are well known, and the method of the present invention can be carried out by the well-known sandwich method.

[0032] In immunoassays using the sandwich method as the detection principle, any solid phase on which an antibody can be immobilized by known techniques can be used, and any known substance can be selected, such as a porous thin film (membrane) with capillary action, a particulate substance, a test tube, or a resin plate. In addition, substances that can be used to label an antibody include enzymes, radioisotopes, fluorescent substances, luminescent substances, colored particles, colloidal particles, and the like. Two or more types of antibodies may also be used. The two or more types of antibodies are preferably antibodies that are used in the sandwich method and recognize different epitopes from each other.

[0033] Among the immunoassays using the various materials mentioned above, immunochromatography, which is a lateral flow immunoassay using a membrane, is preferred, particularly from the viewpoint of simplicity and speed in clinical testing.

[0034] The lateral flow immunoassay according to the present invention can be performed using an immunoassay device that includes a support having a detection region where an antibody (antibody 1) that captures the substance to be measured (antigen) is immobilized, a label region having a mobile labeled antibody (antibody 2) labeled with an appropriate labeling substance such as colored polystyrene particles or gold colloid, a sample pad for dropping a specimen, an absorption band for absorbing the developed specimen liquid, and a backing sheet for bonding these components together. In this method, a specimen, which is a washing solution containing mucus adhering to the nasal mucosal surface obtained by adding a washing solution into the nasal cavity of a subject suspected of having an infectious disease to wash the nasal mucosal surface, is dropped onto the sample pad, and a complex of antibody 2 that can bind to a substance to be detected (labeled reagent) labeled with an appropriate labeling substance such as colored polystyrene particles or gold colloid is developed and moved on the solid support on which antibody 1 is immobilized, utilizing capillary action. As a result, a complex of the immobilized substance-analyte-labeled reagent is formed on the solid support, and the substance to be detected can be detected by detecting the signal of the labeled reagent emitted from the complex (in the case of gold colloid, the part of the solid support on which the substance capable of binding to the analyte is immobilized turns red). The immunoassay method can be carried out at 5 to 35°C, preferably at room temperature, and pretreatment with a specimen treatment solution may also be carried out within this temperature range.

[0035] The number of detection regions and the type of labeled antibody contained in the label region are not limited to one, and by using antibodies corresponding to multiple objects to be measured, two or more antigens can be detected using the same immunoassay device.

[0036] The present invention also includes an immunoassay reagent, which is the above-mentioned immunoassay instrument for analyzing an analyte in a specimen and detecting an infectious disease, and a test kit, which is an immunoassay kit including the immunoassay reagent and a nasal drop device.

[0037] When the number of the substance to be detected, such as a virus, is small, it may be cultured and multiplied by isolation culture and identification methods before analysis.

[0038] Examples of gene testing methods include gene amplification methods such as PCR (Polymerase Chain Reaction), LAMP (Loop Mediated Isothermal Amplification), TMA (Transcription Mediated Amplification), SDA (Strand Displacement Amplification), and ICAN (Isothermal and Chimeric Primer-initiated Amplification of Nucleic Acids). Among these, PCR can be preferably used. A washing solution containing mucus attached to the nasal mucosa surface obtained by adding a washing solution to the nasal cavity of a subject suspected of an infectious disease and washing the nasal mucosa surface is used as a sample, and the gene of a pathogen contained in the sample may be amplified. The PCR method is a method of amplifying a gene region surrounded by primers having a specific sequence in vitro using a Taq DNA polymerase reaction. The gene amplification reaction is performed by using a pair of primers, a forward primer and a reverse primer, and repeating a three-step thermal cycle of a) thermal denaturation of double-stranded DNA, ii) annealing of primers, and iii) extension reaction 30 to 40 times. Both methods are well known.

[0039] The present invention also includes a test kit that is a gene amplification kit including the above-mentioned gene amplification reagent for analyzing an analyte in a specimen and detecting an infectious disease, and the gene amplification reagent and a nasal drip device. The nasal drip device of the kit includes a washing solution to be added to the nasal cavity. The nasal drip device may be a disposable type, and a single-use type nasal drip device includes 200 to 800 μL, preferably 400 to 700 μL, and more preferably 300 to 500 μL of the washing solution to be added to the nasal cavity for one use. EXAMPLES

[0040] The present invention will be described in more detail below with reference to examples, but it is not limited to these examples.

[0041] [Example 1] 1. Preparation of nasal spray device A 20 mL nasal drop container (Kinkei Seisakusho) was filled with 10 mL of physiological saline (Otsuka Pharmaceutical) to prepare a nasal drop device. 2. Simple collection of mucus samples from the nasal mucosa Using a nasal drop device, 200μL, 300μL, 400μL, 500μL, 600μL, 700μL, and 800μL were sprayed onto three healthy adult volunteers, and the liquid that flowed out of the nasal entrance was collected by sucking it up with an Ex Swab 003T. This was used as a simple mucus specimen adhering to the nasal mucosa surface. 3. Sensory testing at the time of collection In 2., when collecting simple mucus samples adhering to the surface of the nasal mucosa, three healthy adult subjects were evaluated from three perspectives: ease of collection at each spray volume, breathlessness, and difficulty in flowing down the throat. 4.Comparative Consideration From the results in Table 1, although there are individual differences, the optimal spray volume was considered to be 300 to 500 μL. However, since this is for healthy adults, it may be different for children and may also vary depending on race, so it is not limited to this amount.

[0042] [Table 1]

[0043] [Example 2] 1. Simple SDS-PAGE analysis of mucus samples adhering to the nasal mucosa surface The simple mucus specimen attached to the surface of the nasal mucosa collected in Example 1, section 2, was suspended in 200 μL of physiological saline. A portion of this was taken, and each reagent was added to the final concentration of 62.5 mM Tris-HCl (pH 6.5), 10 (w / v)% glycerol, 2.3 (w / v)% SDS, and 0.05 (w / v)% BPB (dye), and the sample was subjected to heat denaturation treatment at 95° C. for 5 minutes, followed by standard SDS-PAGE. 2. Total protein analysis using a densitometry analyzer Analyze the CBB-stained SDS-PAGE gel from step 1 above using a densitometry analyzer (Bio-Rad), measure the density of all bands in each lane, and use the total density value as the density score. 3.Comparative Consideration The results of the density score are shown in Table 2. From the results in Table 2, it was revealed that, although there were individual differences, the amount of protein in the simple mucus specimen attached to the surface of the nasal mucosa collected with a spray volume of 400 to 700 μL was high. However, this is from healthy adults, and may vary in symptomatic individuals and children, and may also vary depending on race, so it is not limited to this.

[0044] [Table 2]

[0045] [Example 3] 1. SDS-PAGE analysis of nasal aspirate samples and simple mucus samples attached to the nasal mucosa Ten nasal aspirate samples were collected using Mentip P1503 (manufactured by Japan Cotton Swabs Co., Ltd.), suspended in 200 μL of physiological saline, and used as samples for SDS-PAGE. In addition, 400 μL of mucus specimens attached to the surface of the nasal mucosa of three healthy subjects collected in 2. of Example 1 were mixed to use as samples for SDS-PAGE. Each sample was separated, and each reagent was added to the final concentration of 62.5 mM Tris-HCl (pH 6.5), 10 (w / v) % glycerol, 2.3 (w / v) % SDS, and 0.05 (w / v) % BPB (dye), and the sample was subjected to heat denaturation treatment at 95 ° C for 5 minutes, and standard SDS-PAGE was performed. 2. Total protein analysis using a densitometry analyzer The CBB-stained gel of SDS-PAGE described above in 1 was analyzed using a densitometry analyzer (Bio-Rad), and the total density (density score) of all bands in each lane was measured. 3.Comparative Consideration The results of the density score are shown in Table 3. As can be seen from the results in Table 3, although there were differences between samples, there was no significant difference in the amount of protein that could be collected from the nasal aspirate sample and the simple mucus sample attached to the nasal mucosa surface when they were mixed together. This suggests that it is possible to test for influenza viruses using the simple mucus sample attached to the nasal mucosa surface of the present invention.

[0046] [Table 3]

[0047] [Example 4] 1. Preparation of anti-influenza A virus antibodies BALB / c mice were immunized with inactivated influenza A virus, and the spleens were removed from the mice after a certain period of time and fused with mouse myeloma cells (P3X63) by the method of Kohler et al. (Kohler et al., Nature, vol. 256, p495-497(1975)). The resulting fused cells (hybridomas) were maintained in a 37°C incubator, and the cells were purified (monocloned) while confirming the antibody activity of the supernatant by ELISA using a plate immobilized with an antigen extracted from Helicobacter pylori. The two obtained cell lines were intraperitoneally administered to pristane-treated BALB / c mice, and antibody-containing ascites was collected about two weeks later. IgG was purified from the ascites fluid by affinity chromatography using a protein A column, and two types of purified anti-influenza A virus antibodies were obtained.

[0048] 2. Generation of anti-influenza B virus antibodies BALB / c mice were immunized with inactivated influenza B virus, and the spleens were removed from the mice after a certain period of time and fused with mouse myeloma cells (P3X63) by the method of Kohler et al. (Kohler et al., Nature, vol. 256, p495-497(1975)). The resulting fused cells (hybridomas) were maintained in a 37°C incubator, and the cells were purified (monocloned) while confirming the antibody activity of the supernatant by ELISA using a plate immobilized with an antigen extracted from Helicobacter pylori. The two obtained cell lines were intraperitoneally administered to pristane-treated BALB / c mice, and antibody-containing ascites was collected about two weeks later. IgG was purified from the ascites fluid by affinity chromatography using a protein A column, and two types of purified anti-influenza B virus antibodies were obtained.

[0049] 3. Preparation of labeled anti-influenza A virus antibodies One of the anti-type A influenza virus antibodies was dialyzed against a 50 mM MES (2-Morpholinoethanesulfonic acid, monohydrate; Dojindo Chemical Industries, Ltd.) buffer solution (pH 6.0), and then diluted with the same buffer solution to OD280nm = 0.5 to prepare 10mL of the solution. Next, it was mixed with 10 (W / V)% blue polystyrene latex particles (particle size 0.45 μm, surface functional group is carboxyl group, functional group density 65 Å2 / COOH group; Magsphere Co., Ltd.) at a liquid volume ratio of 40:1 and reacted. Next, 1 (w / v)% EDAC (N-(3-Dimethlaminopropyl)-N'-ethylcarbodiimide hydrochloride; Sigma Co., Ltd.) was added to a final concentration of 0.1%, and the reaction was allowed to proceed for 2 hours. After washing, the particles were suspended in 20 mL of final suspension (5 mM Tris, 0.04 (w / v)% BSA (bovine serum albumin), 0.4 M trehalose, 0.2 (v / v)% Triton X-100) and subjected to an ultrasonic dispersion device (Olympus) to disperse the latex particles.

[0050] 4. Preparation of labeled anti-influenza B virus antibodies One of the anti-type B influenza virus antibodies was dialyzed against a 50 mM MES (2-Morpholinoethanesulfonic acid, monohydrate; Dojindo Chemical Industries, Ltd.) buffer solution (pH 6.0), and then diluted with the same buffer solution to OD280nm = 0.5 to prepare 10mL of the solution. Next, it was mixed with 10 (w / v)% blue polystyrene latex particles (particle size 0.45 μm, surface functional group is carboxyl group, functional group density 65 Å2 / COOH group; Magsphere Co., Ltd.) at a liquid volume ratio of 40:1 and reacted. Next, 1 (w / v)% EDAC (N-(3-Dimethlaminopropyl)-N'-ethylcarbodiimide hydrochloride; Sigma Co., Ltd.) was added to a final concentration of 0.1%, and the reaction was allowed to proceed for 2 hours. After washing, the particles were suspended in 20 mL of final suspension (5 mM Tris, 0.04 (w / v)% BSA (bovine serum albumin), 0.4 M trehalose, 0.2 (v / v)% Triton X-100) and subjected to an ultrasonic dispersion device (Olympus) to disperse the latex particles.

[0051] 5. Preparation of Latex Particle-labeled Antibody Dry Pads The latex particle-labeled anti-type A and type B influenza virus antibodies obtained in 3 and 4 above were mixed and sprayed over the entire surface of a 15 mm-wide reeled cellulose nonwoven fabric at a coating volume of 8 μL / cm using a positive pressure spray device (BioJet; BioDot Co., Ltd.). After spraying, the fabric was dried by blowing hot air at 50°C for 1 minute to prepare a latex particle-labeled antibody dry pad.

[0052] 6. Preparation of Membrane Immobilized Antibodies The purified anti-influenza A virus antibody prepared in 1 above that was not used for labeling was dialyzed against solid phase liquid (10 mM Tris-HCl (pH 8.0)), filtered through a 0.22 μm filter after dialysis, and diluted with the solid phase liquid to an OD280nm of 3.0 to prepare solid phase anti-influenza A virus antibody. The purified anti-influenza B virus antibody produced in 2 above that was not used for labeling was dialyzed against solid phase liquid (10 mM Tris-HCl (pH 8.0)), filtered through a 0.22 μm filter after dialysis, and diluted with the solid phase liquid to an OD280nm of 3.0 to prepare solid phase anti-influenza B virus antibody.

[0053] 7. Preparation of a lateral flow membrane assay device for influenza virus detection The membrane used was a nitrocellulose membrane (pore size 12 μm; Whatman) sheet (white) with a width of 3 cm and a length of 10 cm. A solid-phase anti-type A influenza virus antibody was applied linearly at a position 6 mm away from one end of the long axis (this end was the upstream end, and the opposite end was the downstream end) and a solid-phase anti-type B influenza virus antibody was applied linearly at a coating amount of 1 μL / cm using a positive pressure spray device (BioJet; BioDot Co.) at a position 8 mm away from the long axis end, and an anti-mouse IgG antibody was diluted to OD280nm=1.0 and applied linearly at a coating amount of 1 μL / cm using a positive pressure spray device (BioJet; BioDot Co.) at a position 13 mm away from one end of the long axis. After application, the membrane was dried by blowing hot air at 45°C for 30 minutes. Next, in order to fix the member and increase its strength, a plastic backing sheet (manufactured by BioDot) was adhered to the opposite side (this side is referred to as the lower side) of the antibody-coated surface of the membrane (this side is referred to as the upper side). Next, the latex particle-labeled antibody dry pad prepared in step 5 above was cut to a width of 15 mm and a length of 10 cm, and attached to the top surface of the membrane so that the upstream end of the membrane overlapped by 2 mm. Furthermore, a cellulose filter paper (Whatman) measuring 23 mm in width and 10 cm in length was attached to the top surface of the latex particle-labeled antibody dry pad so that it overlapped by 13 mm to create a sample drop application pad. Next, a cellulose filter paper (Whatman) measuring 30 mm in width and 10 cm in length was attached to the upper surface of the membrane so as to overlap the downstream end of the membrane by 5 mm to form a sample absorption pad. Next, the entire upper surface of the sample dropping pad was covered with a transparent plastic laminate (Adhesive Research) except for a 5 mm wide area at the upstream end. Finally, the membrane was cut along the longitudinal axis at 5 mm intervals to prepare a membrane assay device.

[0054] 8. Influenza virus detection Nasal swabs were taken using an influenza virus antigen kit, QuickNavi (trademark)-Flu2 (manufactured by Denka Seiken), to diagnose influenza virus infection. Five patients were diagnosed as positive (+) for influenza A virus, five patients were diagnosed as positive (+) for influenza B virus, and five patients were diagnosed as negative (-) for influenza virus based on overall findings. 400 μL of saline was sprayed into the nasal cavities of the subjects using the nasal drop device prepared in 1 of Example 1, and the liquid that flowed out at the entrance of the nose was collected by sucking it up with an Ex Swab 003T (manufactured by Denka Seiken), and this was used as a simple nasal mucosal surface adhering mucus specimen. The cotton ball of the cotton swab from which the specimen was collected was immersed in 0.2 mL of specimen suspension buffer (phosphate buffer (pH 7.4) containing 0.05 (w / v)% Tween 20 and 0.1 (w / v)% bovine serum albumin), and the material adhering to the tip was rubbed out and extracted into the specimen suspension buffer, which was used as the specimen sample. The sample drop pad side of the lateral flow membrane assay device for influenza virus detection prepared in 7. was immersed in the liquid in the specimen. After 10 minutes, the assay device was observed, and if color development was observed at the position where the anti-mouse IgG antibody was applied (control line), it was judged as valid, if color development was observed at the position where the solid-phase anti-type A influenza virus antibody was applied, it was judged as positive for type A influenza virus (+), if color development was observed at the position where the solid-phase anti-type B influenza virus antibody was applied, it was judged as positive for type B influenza virus (+), and if no color development was observed at either position, it was judged as negative (-). If no color development was observed at the position of the control line, it was judged as invalid.

[0055] 9.Comparative Consideration The assay results are shown in Table 4. The results in Table 4 confirm that the test results using the specimens according to the present invention are all consistent with the test results and overall findings obtained by collecting nasal swabs using QuickNavi-Flu2.

[0056] [Table 4]

[0057] [Example 5] 1. Sample collection for comparison of influenza virus load A nasal swab was collected using the influenza virus antigen kit QuickNavi (trademark)-Flu2 (manufactured by Denka Seiken) to diagnose influenza virus infection, and from one patient who was diagnosed as influenza A virus positive (+) in the overall findings, 400 μL of saline was sprayed using the nasal drop device prepared in 1. of Example 1, and the liquid that flowed out of the nasal entrance was absorbed by ExSwab 003T (manufactured by Denka Seiken) to collect a simple mucus specimen attached to the nasal mucosa surface, which was suspended in 200 μL of saline and used as a sample for qPCR. As a control, the remaining liquid from the nasal swab test of QuickNavi-Flu2 from the same patient was used as a sample. 2.Analysis by Real-time PCR Nucleic acids were extracted from the two samples in 1. using a QIAamp Viral RNA Mini Kit (nucleic acid extraction kit, manufactured by QIAGEN), and a specified PCR sample was added and measured using an Applied Biosystems QuantStudio (trademark) 3 PCR device (manufactured by Thermo Fisher Scientific). 3.Comparative Consideration The measurement results are shown in Table 5. From Table 5, the virus amount in the simple mucus specimen attached to the nasal mucosa surface was 1.39 x 10 6 copies / mL, whereas the viral load in the control nasal swab sample was 1.31x10 5The results were equivalent to 100 copies / mL. Compared to the conventional nasal swab specimen, the simplified nasal mucosal surface adhered mucus specimen contained a higher amount of virus, demonstrating that the simplified method allows for more stable collection of the virus than nasal swab specimens and can be used for clinical testing.

[0058] [Table 5] [Industrial Applicability]

[0059] The present invention can be used to detect respiratory infections.

Claims

1. A method for detecting infectious diseases using a lateral flow assay, in which a lavage fluid containing mucus adhering to the nasal mucosal surface obtained by adding a lavage fluid into the nasal cavity of a human subject suspected of having an infectious disease and washing the nasal mucosal surface is used as a sample.

2. The method according to claim 1, wherein the amount of washing solution added to the nasal cavity is 300 to 500 μL.

3. 3. The method according to claim 1 or 2, wherein the method of administering the irrigation solution into the nasal cavity is by spraying.

4. The method according to any one of claims 1 to 3, wherein the infectious disease is an influenza virus infection.

5. A test kit for detecting an infectious disease that uses as a sample a lavage fluid containing mucus adhering to the nasal mucosal surface obtained by adding a lavage fluid into the nasal cavity of a human subject suspected of having an infectious disease to wash the nasal mucosal surface, the test kit comprising a lateral flow assay test reagent for detecting an infectious disease, and a nasal drop device containing the lavage fluid for washing the nasal mucosa.

6. The test kit according to claim 5, wherein the nasal drop device is a single-use type nasal drop device, and the device contains 300 to 500 μL of the washing solution to be added to the nasal cavity per use.

7. 7. The test kit according to claim 5 or 6, wherein the method of adding the irrigation solution to the nasal cavity is spraying.

8. The test kit according to any one of claims 5 to 7, wherein the infectious disease is an influenza virus infection.

9. A method for collecting a sample containing a lavage fluid that contains mucus adhering to the nasal mucosal surface by adding a lavage fluid into the nasal cavity of a human subject suspected of having an infectious disease to wash the nasal mucosal surface.

10. The method according to claim 9, wherein the amount of washing solution added into the nasal cavity is 300 to 500 μL.

11. 11. The method according to claim 9 or 10, wherein the method of administering the irrigation solution into the nasal cavity is by spraying.

12. The method according to any one of claims 9 to 11, wherein the infectious disease is an influenza virus infection.

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