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

[0002] Conventionally, when collecting specimens from the human body for clinical testing, infectious disease testing, 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, pharyngeal swabs, nasal aspirates, and nasal discharge are widely used, and because 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, samples are routinely collected and used for testing during influenza virus epidemics (see Patent Document 1).

[0004] Nasal swabs are typically collected using sterile medical cotton swabs. While swab collection is considered minimally invasive, obtaining a sufficient sample for testing requires inserting the swab into the nasopharynx and vigorously scraping the mucosa, which can sometimes result in severe pain and bleeding. Furthermore, collecting pharyngeal swabs requires thorough scraping of the palatine tonsils and posterior pharyngeal wall, which can sometimes trigger a gag reflex and make it difficult to obtain a sufficient sample. Nasal aspirates are only available at select medical institutions equipped with specialized suction devices, and depending on the patient's condition, there may be little or no nasal mucus, making it difficult to obtain a sufficient sample. Nasal lavage involves inserting a large amount of liquid into the nasal cavity with a syringe, and the escaping liquid is collected using a suction device, similar to a paper cup or nasal aspirate. However, introducing a large amount of liquid into the nasal cavity can make the patient feel short of breath, and in some cases, the inserted liquid may leak down the nose and into the throat, making collection difficult. Nasal mucus can only be used on patients who are old enough to blow their nose, and like nasal aspirates, it can be difficult to collect a sufficient amount of sample depending on the patient's condition. For the reasons mentioned above, the five sample types and collection methods used in influenza virus testing - nasal swabs, throat swabs, nasal aspirates, nasal washes, and nasal mucus - generally place a significant burden on patients. Furthermore, depending on the patient's condition, it may be difficult to obtain the required sample volume, which can affect the accuracy of the test. Therefore, there was a need for a sample collection method and test kit that is less invasive and can stably collect a consistent amount of sample.

[0005] Furthermore, the five specimen types and collection methods used in influenza virus testing mentioned above involve large amounts of biological components, making them prone to non-specific reactions such as false positives, false negatives, or inability to determine a result. In particular, nasal washes have the problem of reducing sensitivity because the large amount of wash fluid dilutes the specimen. [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 quick, 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 use this as a sample. [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 that remains 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, and have discovered the usefulness of the sample as a clinical sample.They have completed the present invention, which makes it possible to analyze 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], wherein 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 is an immunological detection method. [6] Any of the methods [1] to [4], which are gene amplification methods. [7] A test kit for detecting infectious diseases 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 specimen, the test kit comprising a test reagent for detecting infectious diseases 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 rinsing solution to be added to the nasal cavity for one use. [9] A test kit according to [7] or [8], in which the nasal irrigation solution is administered by spraying.

[10] A test kit for any of [7] to [9], in which the infectious disease is an influenza virus infection.

[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 sample containing lavage fluid containing mucus adhering to the nasal mucosal surface by adding lavage fluid into the nasal cavity of a subject suspected of having an infectious disease to wash 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] Method

[13] or

[14] , in which the irrigation solution is administered into the nasal cavity by spraying.

[16] Any of the methods

[13] to

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

[0010] The method of the present invention has made it possible to establish a method for diagnosing infectious diseases from specimens containing adherent mucus obtained by washing the surface of the nasal mucosa.

[0011] When the method of the present invention is used to diagnose infectious diseases using the adherent mucus obtained by washing the surface of the nasal mucosa as a sample, infectious diseases can be diagnosed with sensitivity that is equal to or even better than when infectious diseases are diagnosed using nasal swabs, pharyngeal swabs, nasal aspirates, nasal washes, and nasal mucus as samples. DETAILED DESCRIPTION OF THE INVENTION

[0012] The present invention will be described in detail below. The present invention is characterized in that a predetermined amount of irrigation 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 nasal mucosal surface is collected as a sample. Here, the adherent mucus refers to the mucus adhering to the nasal mucosal surface.

[0013] For example, a patient suspected of having an infectious disease based on a medical interview or other factors is given a nasal drop device pre-filled with the solution, and a predetermined amount of the solution is sprayed or dripped into the patient's nasal cavity. After spraying or dripping, the mucosal surface of the nasal cavity is washed with the solution, and the wash containing the adhering mucus that has flowed out near the nasal entrance is collected as a sample. Various tests are performed using the sample obtained in this way.

[0014] In the present invention, the nasal mucosal surface is washed with the irrigation liquid by spraying the irrigation liquid into the nasal cavity, and the irrigation liquid containing the adhering mucus that flows out near the entrance of the nose can be collected as a sample.

[0015] If 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 mucosal surface.

[0016] In contrast, spraying a rinsing liquid into the nasal cavity allows collection of mucus from a wide range of the nasal mucosal surface.

[0017] Furthermore, if the cleaning solution is poured into the nasal cavity using a dropper or a tool 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, spraying the cleaning liquid into the nasal cavity can prevent the cleaning liquid from passing through the nasal cavity and flowing into the throat, allowing the cleaning liquid to be collected efficiently.

[0019] Furthermore, when irrigation solution is poured into the nasal cavity using a dropper or an instrument with a nozzle, the force with which the solution is poured locally into the nasal cavity puts strain on the cavity, and the subject (especially children) often feels pain or shortness of breath.

[0020] In contrast, spraying a nasal cleansing solution into the nasal cavity reduces the burden on the nasal cavity, making it less likely that the subject (especially a child) will 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 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 medical spray device, or the like.

[0022] Examples of irrigation solutions include physiological saline solutions. Physiological saline solutions that can be used include, but are not limited to, commercially available physiological saline solutions prescribed in the Japanese Pharmacopoeia and solutions prepared according to the prescriptions of the Japanese Pharmacopoeia. Isotonic solutions such as Ringer's solution, lactated Ringer's solution, and glucose solution can also be used as irrigation solutions.

[0023] For nasal cavity irrigation, 200 to 800 μL, preferably 400 to 700 μL, and more preferably 300 to 500 μL of irrigation 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 using various collection devices, and examples of such collection devices include devices that can hold liquid, such as cotton swabs, swabs, brushes, devices with mesh tips, sponge-like devices, and absorbent cloths. The liquid containing the adherent mucus can be collected by absorbing it into these devices.

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

[0026] The specimen sample can be prepared by dissolving or suspending the specimen collected with a collection device in a liquid. The specimen collected with a collection device can be dissolved or suspended in a liquid by rubbing, squeezing, or rubbing the part of the collection device that has absorbed the liquid containing the adherent mucus, such as the cotton tip of a cotton swab, into the liquid. A buffer solution can be used as the liquid for dissolving or suspending the specimen.

[0027] In the present invention, a specimen can be used directly as a sample without treatment such as concentration or culturing. Alternatively, the specimen may 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 immunochromatography, which is a lateral flow immunoassay using a membrane, a specimen collected with a cotton swab is suspended in a buffer solution and used as a specimen sample.

[0028] The infectious diseases to be detected in the present invention are those caused by pathogens such as viruses, bacteria, protozoa, fungi, mycoplasma, rickettsia, and chlamydia, and the present invention is particularly aimed at detecting viral infections. 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 can be detected as the test substance.

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

[0030] In immunoassays, viruses in a sample are analyzed using an antibody-antigen reaction. Specifically, analysis can be performed using antibodies specific to viral antigens. Antibodies against viral antigens can be obtained by known methods. Immunoassays can include any method known to those skilled in the art, such as immunostaining (including fluorescent antibody techniques, enzyme-linked antibody techniques, heavy metal-labeled antibody techniques, and radioisotope-labeled antibody techniques), methods combining electrophoretic separation with detection using fluorescence, enzymes, radioisotopes, etc. (including Western blotting and fluorescent two-dimensional electrophoresis), enzyme-linked immunosorbent assay (ELISA), dot blotting, latex agglutination-turbidimetric immunoassay (LA), and immunochromatography. In the present invention, "analysis" encompasses quantitative, semi-quantitative, and detection.

[0031] Among the above immunoassay methods, 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, particulate matter, a test tube, or a resin plate. Furthermore, substances that can be used to label the antibody include enzymes, radioisotopes, fluorescent substances, luminescent substances, colored particles, and colloidal particles. Two or more types of antibodies may also be used. Preferably, the two or more types of antibodies used in the sandwich method recognize different epitopes.

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

[0034] The lateral flow immunoassay of the present invention can be performed using an immunoassay device comprising a support having a detection region where an antibody (antibody 1) that captures the analyte (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 colloidal gold, a sample pad for adding a drop of sample, an absorption band for absorbing the developed sample liquid, and a backing sheet for bonding these components together. In this method, a sample is added dropwise to the sample pad, which is a lavage fluid containing mucus adhering to the nasal mucosal surface obtained by adding a lavage fluid to the nasal cavity of a subject suspected of having an infectious disease to wash the nasal mucosal surface. Capillary action is then used to develop and migrate a complex of antibody 2, which can bind to the analyte (labeled reagent) labeled with an appropriate labeling substance such as colored polystyrene particles or colloidal gold, onto the solid support on which antibody 1 is immobilized. As a result, a complex of the immobilized substance, analyte, and labeled reagent is formed on the solid support, and the analyte 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). This immunoassay method can be performed at 5 to 35°C, preferably at room temperature, and pretreatment with a specimen treatment solution can also be performed 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 sample and detecting an infectious disease, and a test kit, which is an immunoassay kit including the immunoassay reagent and a nasal drop device.

[0037] If the number of the substance to be detected, such as a virus, is small, it may be cultured and increased using the isolation and identification method before analysis.

[0038] Genetic 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 is particularly suitable. A nasal lavage solution containing mucus adhering to the nasal mucosal surface of a subject suspected of infectious disease is administered into the nasal cavity to wash the surface. The lavage solution is used as a sample, and the pathogen genes contained in the sample are amplified. PCR utilizes the Taq DNA polymerase reaction to amplify in vitro a gene region surrounded by primers with specific sequences. The gene amplification reaction uses a pair of primers (forward and reverse) and involves 30 to 40 cycles of three steps: a) thermal denaturation of double-stranded DNA, ii) primer annealing, and iii) extension. Both methods are well known.

[0039] The present invention also encompasses a test kit that is a gene amplification kit comprising the above-described gene amplification reagent for analyzing an analyte in a sample and detecting an infectious disease, and the gene amplification reagent and a nasal drop device. The nasal drop device of the kit contains a nasal irrigation solution to be added to the nasal cavity. The nasal drop device may be a disposable type, and a single-use nasal drop device contains 200 to 800 μL, preferably 400 to 700 μL, and more preferably 300 to 500 μL of irrigation solution to be added to the nasal cavity in one dose. [Example]

[0040] The present invention will be explained in more detail below by way of examples, but is not limited to these examples.

[0041] [Example 1] 1. Preparation of nasal instillation 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 specimens adhering to the nasal mucosa Three healthy adults were sprayed with 200μL, 300μL, 400μL, 500μL, 600μL, 700μL, and 800μL using a nasal drop device, and the liquid that flowed out of the nasal entrance was collected by sucking it up with ExSwab 003T, and this was used as a simple mucus specimen attached to the nasal mucosal surface. 3. Sensory test at the time of collection 2. When collecting simple mucus samples adhering to the surface of the nasal mucosa, three healthy adults 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 is thought to be 300 to 500 μL. However, since this is for healthy adults, it may differ for children and may also differ 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 mucosal surface The simple mucus specimen attached to the nasal mucosa surface collected in Example 1, section 2, was suspended in 200 μL of physiological saline. A portion of this was taken, and reagents were added to the final concentrations 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). The sample was then heat-denatured at 95°C for 5 minutes and subjected to 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 measurements are shown in Table 2. The results in Table 2 reveal that, although there are individual differences, the amount of protein in the simple mucus specimens attached to the nasal mucosal surface collected with a spray volume of 400 to 700 μL is high. However, these are from healthy adults, and the results may differ in symptomatic individuals and children, and may also differ depending on race, so the results are not limited to these.

[0044] [Table 2]

[0045] [Example 3] 1. SDS-PAGE analysis of nasal aspirate samples and simple nasal mucosal surface adherent mucus samples Ten nasal aspirate samples were collected using a Mentip P1503 (manufactured by Nippon Cotton Swab Co., Ltd.) and suspended in 200 μL of saline to prepare samples for SDS-PAGE. Additionally, 400 μL of simple nasal mucus samples collected from three healthy volunteers in section 2 of Example 1 were mixed to prepare samples for SDS-PAGE. Each sample was aliquoted, and the following reagents were added to final concentrations: 62.5 mM Tris-HCl (pH 6.5), 10 (w / v)% glycerol, 2.3 (w / v)% SDS, and 0.05 (w / v)% BPB (dye). Heat denaturation was performed at 95°C for 5 minutes, and standard SDS-PAGE was performed. 2. Total protein analysis using a densitometry analyzer The CBB-stained gel from SDS-PAGE (1) above 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 measurements are shown in Table 3. The results in Table 3 show that, although there were differences between samples, there was no significant difference in the amount of protein that could be collected from nasal aspirate samples and simple nasal mucosal surface-adherent mucus samples when compared under the respective mixing conditions. This suggests that influenza virus testing can be performed using the simple nasal mucosal surface-adherent mucus sample 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 then housed for a certain period of time. Spleens were then removed and fused with mouse myeloma cells (P3X63) using the method of Kohler et al. (Nature, vol. 256, pp. 495-497 (1975)). The resulting fused cells (hybridomas) were maintained in a 37°C incubator, and the antibody activity of the supernatant was confirmed by ELISA using plates coated with an antigen extracted from Helicobacter pylori. Purification (monoclonalization) of the cells was then performed. The two cell lines obtained were then intraperitoneally administered to pristane-treated BALB / c mice, and antibody-containing ascites fluid was collected approximately 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. Preparation of anti-influenza B virus antibodies BALB / c mice were immunized with inactivated influenza B virus, and their spleens were removed and fused with mouse myeloma cells (P3X63) by the method of Kohler et al. (Nature, vol. 256, pp. 495-497 (1975)). The resulting fused cells (hybridomas) were maintained in a 37°C incubator, and the antibody activity of the supernatant was confirmed by ELISA using plates coated with antigen extracted from Helicobacter pylori. The two obtained cell lines were then intraperitoneally administered to pristane-treated BALB / c mice, and antibody-containing ascites fluid was collected approximately 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-influenza A virus antibodies was dialyzed against 50 mM MES (2-Morpholinoethanesulfonic acid, monohydrate; Dojindo Laboratories) buffer (pH 6.0) and diluted with the same buffer to an OD280nm of 0.5 to prepare a 10mL solution. This solution was then mixed with 10% (w / v) blue polystyrene latex particles (particle size 0.45 μm, surface functional groups carboxyl, functional group density 65 Å / COOH; Magsphere) at a volume ratio of 40:1 and reacted. Next, 1% (w / v) EDAC (N-(3-Dimethlaminopropyl)-N'-ethylcarbodiimide hydrochloride; Sigma) was added to a final concentration of 0.1%, and the mixture was allowed to react for 2 hours. After washing, the particles were suspended in 20 mL of the 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-influenza B virus antibodies was dialyzed against 50 mM MES (2-Morpholinoethanesulfonic acid, monohydrate; Dojindo Laboratories) buffer (pH 6.0) and diluted with the same buffer to an OD280nm of 0.5 to prepare a 10mL solution. This solution was then mixed with 10% (w / v) blue polystyrene latex particles (particle size 0.45 μm, surface functional groups carboxyl, functional group density 65 Å / COOH; Magsphere) at a volume ratio of 40:1 and reacted. Next, 1% (w / v) EDAC (N-(3-Dimethlaminopropyl)-N'-ethylcarbodiimide hydrochloride; Sigma) was added to a final concentration of 0.1%, and the mixture was allowed to react for 2 hours. After washing, the particles were suspended in 20 mL of the 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 steps 3 and 4 above were mixed and sprayed onto the entire surface of a 15 mm-wide reel of cellulose nonwoven fabric using a positive pressure sprayer (BioJet; BioDot) at a coating rate of 8 μL / cm. 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 solution (10 mM Tris-HCl (pH 8.0)), filtered through a 0.22 μm sieve after dialysis, and diluted with the solid phase solution to an OD280nm of 3.0 to prepare solid phase anti-influenza A virus antibody. The purified anti-influenza B virus antibody prepared in 2 above that was not used for labeling was dialyzed against solid phase solution (10 mM Tris-HCl (pH 8.0)), filtered through a 0.22 μm sieve after dialysis, and diluted with the solid phase solution 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 was a 3 cm wide x 10 cm long white nitrocellulose membrane (pore size 12 μm; Whatman). A solid-phase anti-type influenza A virus antibody was applied linearly at a position 6 mm from one end of the long axis (this end was designated the upstream end, and the opposite end the downstream end) using a positive pressure sprayer (BioJet; BioDot) at a coating volume of 1 μL / cm. A solid-phase anti-type influenza B virus antibody was applied linearly at a position 8 mm from the long axis (this end was designated the upstream end, and the opposite end the downstream end) using a positive pressure sprayer (BioJet; BioDot). Anti-mouse IgG antibody, diluted to OD280nm = 1.0, was applied linearly at a position 13 mm from the long axis using a positive pressure sprayer (BioJet; BioDot) at a coating volume of 1 μL / cm. After application, the membrane was dried by blowing hot air at 45°C for 30 minutes. Next, 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 bottom side) of the antibody-coated surface of the membrane (this side is referred to as the top side). Next, the latex particle-labeled antibody dry pad prepared in 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 23 mm wide and 10 cm long cellulose filter paper (Whatman) 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 into 5 mm sections along the longitudinal axis to prepare membrane assay devices.

[0054] 8. Influenza virus detection Nasal swabs were collected using the influenza virus antigen kit QuickNavi (trademark)-Flu2 (manufactured by Denka Seiken) to diagnose influenza virus infection. Five patients were diagnosed as influenza A virus positive (+) based on overall findings, five patients were diagnosed as influenza B virus positive (+), and five patients were diagnosed as influenza virus negative (-). 400 μL of saline was sprayed into the nasal cavity of each subject 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). 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 massaged 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 test sample. 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 considered valid. If color development was observed at the position where the solid-phase anti-type A influenza virus antibody was applied, it was considered 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 considered positive for type B influenza virus (+). If color development was not observed at either position, it was considered negative (-). If color development was not observed at the control line, it was considered 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 influenza virus load comparison Nasal swabs were collected using the influenza virus antigen kit QuickNavi™-Flu2 (manufactured by Denka Seiken) to diagnose influenza virus infection. From one patient who tested positive for influenza A virus (+) based on overall findings, 400 μL of saline was sprayed using the nasal drop device prepared in Example 1-1. The liquid that flowed out of the nasal entrance was absorbed by an ExSwab 003T (manufactured by Denka Seiken) to collect a simple nasal mucosal surface mucus specimen. This was suspended in 200 μL of saline and used as a sample for qPCR. As a control, the remaining liquid from the QuickNavi-Flu2 nasal swab test of 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™ 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 method of nasal swab specimens, the amount of virus was higher in the simple nasal mucosal surface adhered mucus specimen, demonstrating that the virus can be collected more stably than with nasal swabs 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 an infectious disease using a lateral flow assay, in which 300 to 500 μL of lavage fluid is sprayed into the nasal cavity of a human subject suspected of having an infectious disease to wash the nasal mucosal surface, and the lavage fluid containing the adhered mucus that flows out near the nasal entrance is collected, and the resulting lavage fluid containing the adhered mucus on the nasal mucosal surface is used as a sample.

2. 2. The method of claim 1, wherein the infectious disease is an influenza virus infection.

3. A test kit for detecting an infectious disease, which involves spraying 300 to 500 μL of a lavage fluid into the nasal cavity of a human subject suspected of having an infectious disease to wash the nasal mucosal surface, and then collecting the lavage fluid containing the adhering mucus that has flowed out near the entrance of the nose, and using the resulting lavage fluid containing the mucus adhering to the nasal mucosal surface as a specimen, the test kit comprising a lateral flow assay test reagent for detecting an infectious disease, and a single-use nasal drop device containing a lavage fluid for washing the nasal mucosa, the device containing 300 to 500 μL of lavage fluid to be added to the nasal cavity for one use.

4. The test kit according to claim 3, wherein the infectious disease is an influenza virus infection.

5. A method for collecting a specimen containing rinsing fluid containing mucus adhering to the nasal mucosal surface by spraying 300 to 500 μL of rinsing fluid into the nasal cavity of a human subject suspected of having an infectious disease, and then collecting the rinsing fluid containing the adhering mucus that flows out near the nasal entrance.

6. The method of claim 5, wherein the infectious disease is an influenza virus infection.

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