Simple method for collecting mucus adhering to the nasal mucosa surface and test kit for detecting an analyte in the sample

Abstract

The present invention provides a method for non-invasively and easily removing adherent mucous membranes on the nasal mucosa by using a nasal drop device. By collecting a liquid and using it as a sample, detection of analytes in the sample can be performed quickly, easily, and with high accuracy. The present invention aims to provide a method for analyzing objects. A washing solution is added to the nasal cavity of a subject suspected of having an infectious disease to wash the nasal mucosal surface. A method for detecting infectious diseases using a washing solution containing mucus adhering to the nasal mucosal surface obtained by the above method as a specimen. Law.

Description

[Technical Field]

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

[0002] Conventionally, when collecting specimens from the human body for clinical testing, infectious disease testing, etc., Species include blood, saliva, stool, biopsy tissue, corneal and conjunctival swabs, nasal swabs, pharyngeal swabs, Various specimens are used, including nasal aspirates, nasal washes, and nasal mucus.

[0003] For example, in testing for infectious diseases such as influenza virus, nasal swabs are Throat swabs, nasal aspirates, and nasal mucus are widely used, and tests using the collected samples are The kit allows for quick and easy determination of the presence or absence of influenza virus antigens in samples. Therefore, specimens are routinely collected and used for testing during influenza virus epidemics. (See Patent Document 1).

[0004] Nasal swabs are generally collected using sterile medical cotton swabs. While collection is said to be minimally invasive, it is necessary to reach the nasopharynx to obtain a sufficient sample for testing. It is necessary to insert the swab and scrape it hard to obtain a sufficient sample from the mucosa, sometimes with force. In addition, when collecting a pharyngeal swab, the palatine tonsils and pharynx may be The posterior wall must be scraped thoroughly to obtain the specimen, which sometimes induces a gag reflex and can lead to vomiting. It can be difficult to collect a sufficient amount of specimen. Also, nasal aspirates require a dedicated suction device. It can only be used at certain medical institutions that have the equipment, and depending on the patient's condition, it may be that the patient only has a runny nose. It may be difficult to collect a sufficient amount of specimen. Insert a large amount into the nasal cavity and use the suction device to collect the liquid that flows out, just like a paper cup or nasal aspirate. However, putting a large amount of liquid into the nasal cavity can make the patient feel short of breath and in some cases In some cases, the inserted fluid may flow down the nose and into the throat, making it difficult to collect. Nasal aspirate can only be used for patients who are old enough to blow their nose, and Similarly, depending on the patient's condition, it may be difficult to collect a sufficient amount of specimen. For this reason, the above nasal swabs and throat swabs used in influenza virus testing The five specimen types and collection methods are: nasal aspirate, nasal wash, and nasal mucus. Overall, the burden on patients is not light, and depending on the patient's condition, the required specimen may not be available. Since the amount of sample cannot be obtained and the accuracy of the test is affected, a less invasive method is used, and a fixed amount of sample can be stably obtained. There was a need for a method of collecting samples that could be collected efficiently and for a test kit.

[0005] In addition, the five specimen types used in the above influenza virus tests and their collection The method involves introducing a large amount of biological components, which can lead to false positives, false negatives, or inaccurate results. In particular, the nasal wash fluid is prone to non-specific reactions such as dilution of the sample due to the large amount of wash fluid. This resulted in a problem of reduced 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 uses a nasal drip device to non-invasively and easily collect mucus adhering to the surface of the nasal mucosa. By collecting the sample and using it as a specimen, it is possible to quickly, easily and accurately analyze the substance to be detected in the specimen. The present invention provides a method for analyzing the [Means for solving the problem]

[0008] As a result of extensive research, the inventors of the present invention have discovered that a predetermined amount of physiological saline solution can be administered into the nasal cavity using a nasal drip device. The solution is sprayed into the nasal cavity and the adherent mucus is collected by washing the nasal mucosal surface. Furthermore, the specimen can be collected stably, and the collected specimen can be used to detect pathogens in the specimen. By establishing a measurement system that provides sufficient sensitivity when This will enable the analysis of substances to be detected in samples through simple and non-invasive sample collection. The present invention has been completed.

[0009] The aspects of the present invention are as follows. [1] A nasal lavage solution was added to the nasal cavity of a subject suspected of having an infection to wash the nasal mucosal surface. A method for detecting infectious diseases using a washing solution containing mucus adhering to the surface of the nasal mucosa as a specimen. [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] The nasal mucosal surface was washed by adding a lavage solution to the nasal cavity of a subject suspected of having an infection. A test for detecting infectious diseases using a washing containing mucus adhering to the nasal mucosal surface as a specimen A kit comprising a test reagent for detecting infectious diseases and a cleaning agent for cleaning the nasal mucosa. A test kit that includes a nasal drop device containing purified solution. [8] The nasal spray device is a single-use nasal spray device, and the device contains The test kit [7] contains 200 to 800 μL of cleaning solution to be added per use. [9] A test kit according to [7] or [8], in which the nasal irrigation solution is administered by spraying.

[10] The infectious disease is influenza virus infection, and any of the test kits [7] to [9] is used. tt.

[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 washing solution was added to the nasal cavity of a subject suspected of having an infection to wash the nasal mucosal surface. A method for collecting a sample containing a washing solution containing mucus adhering to the surface of the nasal mucosa.

[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] Those whose infectious disease is influenza virus infection,

[13] to

[15] Law. [Effects of the Invention]

[0010] According to the method of the present invention, it is possible to detect infectious diseases from a specimen containing adherent mucus obtained by washing the surface of the nasal mucosa. A diagnostic method was established.

[0011] In the method of the present invention, the adherent mucus obtained by washing the surface of the nasal mucosa is used as a specimen to detect infectious diseases. If a diagnosis is made, nasal swabs, throat swabs, nasal aspirates, nasal washes, and nasal blows Sensitivity is comparable to or better than that of nasal fluid samples used to diagnose infections This allows for the diagnosis of infectious diseases. DETAILED DESCRIPTION OF THE INVENTION

[0012] The present invention will be described in detail below. The present invention provides a nasal drip device that sprays a predetermined amount of a rinsing solution such as physiological saline solution into the nasal cavity, The mucus adhered to the mucous surface is collected as a sample by washing the mucous surface. Mucus refers to mucus that adheres to the surface of the nasal mucosa.

[0013] For example, for patients who are suspected of having an infectious disease through medical interviews, etc., A predetermined amount of the cleaning solution is sprayed or dropped into the nasal cavity using a nasal drop device. After spraying or dripping, wash the surface of the nasal mucosa with the irrigation solution and rinse the area around the entrance of the nose. The washings containing the adhered mucus are collected as a specimen. Various tests will be conducted.

[0014] The present invention is directed to spraying a nasal irrigation solution into the nasal cavity to wash the surface of the nasal mucosa with the irrigation solution, thereby The washing liquid containing the adhered mucus that flows out near the entrance of the tube can be collected as a sample.

[0015] By pouring the cleaning solution into the nasal cavity with a dropper or nozzle, the inside of the nasal cavity can be cleaned locally. Therefore, it becomes impossible to collect mucus from a wide area of ​​the nasal mucosa surface.

[0016] In contrast, spraying a nasal irrigation solution into the nasal cavity allows collection of mucus from a wide range of the nasal mucosa surface. It can be obtained.

[0017] Also, if you pour the cleaning solution into the nasal cavity using a dropper or a tool with a nozzle, the cleaning solution will flow in and The force of the swallowing causes the liquid to pass through the nasal cavity and flow into the throat, making it impossible to efficiently recover the cleaning solution.

[0018] In contrast, by spraying the nasal cavity with a cleaning solution, the solution passes through the nasal cavity and flows into the throat. This can prevent the cleaning liquid from being contaminated and allows for efficient recovery of the cleaning liquid.

[0019] In addition, by pouring the cleaning solution into the nasal cavity using a dropper or a nozzle-equipped instrument, the inside of the nasal cavity can be locally cleaned. The force of the washing solution being poured into the nasal cavity puts a strain on the nasal cavity, causing pain and discomfort to the subjects (especially children). I often feel shortness of breath.

[0020] In contrast, spraying a nasal cleanser into the nasal cavity reduces the burden on the nasal cavity, and the subjects (Especially children) become less sensitive to pain and 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 Ming nasal spray device is available as a commercially available nasal spray container, a spray device, or a medical spray device. However, it is not limited to this.

[0022] Furthermore, an example of a cleaning solution is physiological saline solution. Commercially available saline solutions and solutions prepared according to the Japanese Pharmacopoeia prescriptions can be used. The washing solution is not limited to the above. Ringer's solution, lactated Ringer's solution, glucose solution, etc. A tonic solution may also be used.

[0023] The amount of nasal irrigation is 200 to 800 μL, preferably 400 to 700 μL, and more preferably It is sufficient to spray or drip 300 to 500 μL of the rinsing solution into the nasal cavity using a nasal drip device. stomach.

[0024] There are several methods for collecting the liquid containing the adherent mucus that flows out near the entrance of the nose as a specimen. Examples of collection methods include using various collection tools, such as cotton swabs, swabs, brushes, A device with a mesh-like tip, a sponge-like device, a cloth-like absorbent material, or other material that can hold liquid. These instruments can be used to collect the liquid containing the adherent mucus. Just do that.

[0025] In the present invention, the sample collected in this manner is called a simple nasal mucosal surface adherent mucus sample. Boo.

[0026] The specimen collected with the collection device is dissolved or suspended in a liquid, and the resulting liquid is then analyzed. The specimen collected with the collection device absorbs the liquid containing the mucus attached to the collection device. Do not rub, squeeze, or rub the affected area, such as the cotton tip of a cotton swab, into the liquid. The specimen can be dissolved or suspended in a liquid by dissolving or suspending the specimen. The liquid may be a buffer solution.

[0027] In the present invention, the specimen can be used directly as a sample without treatment such as concentration or culturing. Alternatively, the sample may be mixed with a buffer solution. Examples of the buffer solution include phosphate buffer. A liquid can be used, which may contain a surfactant such as Tween 20 or serum albumin. For example, immunochromatography, a lateral flow immunoassay method using a membrane, When performing the assay, the sample collected with a cotton swab is suspended in a buffer solution and used as a specimen.

[0028] Infectious diseases detected in the present invention include viruses, bacteria, protozoa, fungi, and mycoplasma. It is an infectious disease caused by pathogens such as rickettsia, chlamydia, etc., and among these, viruses are particularly The purpose is to detect infectious diseases such as influenza virus, SARS-CoV, Coronaviruses such as MERS-CoV and SARS-CoV-2; respiratory syncytial virus; adenovirus; human methanogens; The pathogens that cause these infectious diseases are used as the detection target. Just put it out.

[0029] The methods for detecting an analyte using the present invention include immunoassay (immunological assay), gene assay, These include testing methods, isolation and culture, and identification methods.

[0030] In immunoassays, viruses in samples are analyzed using antibody-antigen reactions. The virus can be analyzed using antibodies specific to the virus antigen. Antibodies against the IgG can be obtained by known methods. immunofluorescence, enzyme immunoassay, heavy metal-labeled antibody method, and radioisotope-labeled antibody method), electrophoresis A method that combines dynamic separation with detection methods such as fluorescence, enzymes, and radioisotopes. (including Western blot and fluorescent two-dimensional electrophoresis), enzyme-linked immunosorbent assay (ELISA) ), dot blotting, latex agglutination (LA) Any method well known to those skilled in the art, such as immunometric immunoassay, immunochromatography, etc. In the present invention, "analysis" includes any of quantitative, semi-quantitative, and detection. All of these are included.

[0031] Among the above immunoassay methods, the sandwich method is preferred. For example, immunochromatography, which performs immunoassays in a lateral flow manner, is well known in certain fields. These sandwich methods can be performed by the ELISA method or the sandwich method. The method of the present invention can be carried out by the well-known sandwich method.

[0032] In immunoassays that use the sandwich method as the detection principle, the solid phase on which the antibody is immobilized is Any known technique for immobilizing an antibody can be used. For example, a technique using capillary action can be used. Any known material such as a porous thin film (membrane), particulate matter, test tube, or resin plate can be used. In addition, the substance to label the antibody can be selected from enzymes, radioisotopes, fluorescent substances, Optical substances, colored particles, colloidal particles, etc. can be used. Two or more antibodies may be used in the sandwich method, targeting different epitopes. It is preferable that the antibody recognizes the polypeptide.

[0033] Among the various immunoassay methods using the aforementioned materials, the method is particularly advantageous in terms of the simplicity and speed of clinical testing. Therefore, immunochromatography, which is a lateral flow immunoassay method using a membrane, is preferred. stomach.

[0034] The lateral flow immunoassay method of the present invention uses an antibody that captures the object to be measured (antigen). A support having a detection area where the antibody (antibody 1) is immobilized, colored polystyrene particles, gold colloid a labeled region having a mobile labeled antibody (antibody 2) labeled with an appropriate labeling substance such as a dye; The sample pad onto which the sample is dropped and the absorbent band that absorbs the developed sample liquid are all integrated into one unit. This can be carried out using an immunoassay device that is made up of a backing sheet for lamination. In this method, a lavage solution is added to the nasal cavity of a subject suspected of having an infection to irrigate the nasal mucosal surface. The sample, which is the washing solution containing the mucus adhering to the surface of the nasal mucosa obtained by washing, is dropped onto the sample pad. Then, using capillary action, colored polystyrene particles or gold particles were attached to the solid support on which antibody 1 was immobilized. The antibody 2 capable of binding to the substance to be detected (labeled reagent) is labeled with an appropriate labeling substance such as clonal antibody. As a result, the complex of the immobilized substance, the substance to be detected, and the labeled reagent is developed. The complex is formed on the solid support, and the signal of the labeling reagent (colloidal gold) emitted from the complex is detected. In this case, the part of the solid support on which the substance that can bind to the target substance is immobilized turns red. The immunoassay is carried out at a temperature of 5 to 35°C, preferably 5 to 35°C. Preferably, the treatment can be carried out at room temperature, and when pretreatment with a sample treatment solution is carried out, the treatment should be carried out at this temperature range. It can be done internally.

[0035] The number of detection regions and the type of labeled antibody contained in the labeled region are limited to one. By using antibodies corresponding to multiple target substances, two or more antigens can be measured in the same immunoassay. It can be detected with standard equipment.

[0036] The present invention provides the above-mentioned immunoassay device for analyzing an analyte in a specimen and detecting an infectious disease. A test that is an immunoassay reagent and an immunoassay kit that includes an immunoassay reagent and a nasal spray device Kits are also included.

[0037] If the number of detected substances such as viruses is small, they can be cultured and increased using isolation and identification methods. It can be analyzed from

[0038] Genetic testing methods include PCR (Polymerase Chain Reaction) and LAMP (Loop Mediated Amplification) Isothermal Amplification) method, TMA (Transcription Mediated Amplification) method, SDA ( Strand Displacement Amplification) method, ICAN (Isothermal and Chimeric Primer-initia) Examples of gene amplification methods include PCR (Reversed Amplification of Nucleic Acids) and PCR (Reversed Amplification of Nucleic Acids). A method of administering nasal washes to the nasal cavity of a subject suspected of having an infection can be suitably used. The lavage fluid containing the mucus adhering to the nasal mucosal surface obtained by washing the nasal mucosal surface is used as a sample. PCR utilizes the Taq DNA polymerase reaction. and a method for amplifying in vitro a gene region surrounded by primers having a specific sequence. The gene amplification reaction uses a pair of primers, a forward primer and a reverse primer. The three steps are a) heat denaturation of double-stranded DNA, ii) primer annealing, and iii) extension reaction. This method involves repeating the thermal cycle of steps 30 to 40 times. The method is also known.

[0039] The present invention relates to the above-mentioned gene amplification reagent for analyzing an analyte in a specimen and detecting an infectious disease. Medicines and test kits that are gene amplification kits that include gene amplification reagents and nasal spray devices The nasal drop device of the kit contains a rinsing solution to be added to the nasal cavity. The device may be a disposable type, and a single-use nasal drop device may be a single-use type. 200 to 800 μL, preferably 400 to 700 μL, of the washing solution to be added to the nasal cavity for 1 minute, The amount of the solution is preferably 300 to 500 μL. [Example]

[0040] The present invention will be described in more detail below with reference to examples, but it should be understood that the present invention is not limited to these examples. There is no.

[0041] [Example 1] 1. Preparation of nasal instillation device A 20 mL nasal drop container (Kinkei Seisakusho) was filled with 10 mL of saline (Otsuka Pharmaceuticals) and administered. The device was used. 2. Simple collection of mucus specimens adhering to the nasal mucosa Three healthy adults were administered 200μL, 300μL, 400μL, 500μL, 600μL, and 700μL doses using a nasal instillation device. 00μL, 800μL were sprayed, and the liquid that flowed out of the nasal entrance was collected by absorbing it into Ex Swab 003T. 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 attached to the nasal mucosa surface, three healthy adults were sprayed with each amount of sample. The evaluation was conducted from three perspectives: ease of intake, breathlessness, and difficulty 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, these were taken from healthy adults, so the results may differ for children and may differ depending on race. This is not a limitation as it may change.

[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, 2. was suspended in 200 μL of physiological saline. A portion of this was taken and diluted to a final concentration of 62.5 mM Tris-HCl (pH 6.5), 10 (w / v)% glycerol, Add each reagent to make 2.3 (w / v)% SDS and 0.05 (w / v)% BPB (dye), and heat-transform at 95°C for 5 minutes. The mixture was subjected to a sterilization treatment and then subjected to standard SDS-PAGE. 2. Total protein analysis using a densitometry analyzer Using a densitometry analyzer (Bio-Rad), analyze the CB of the SDS-PAGE obtained in step 1 above. Analyze the B-stained gel, measure the density of all bands in each lane, and calculate the total density. Let the value be the Density Score. 3.Comparative Consideration The results of the Density Score are shown in Table 2. From the results in Table 2, although there are individual differences, The amount of protein in the simple nasal mucus sample collected with a mist volume of 400-700 μL was high. However, this was in healthy adults, and may differ in symptomatic individuals and children. It is also possible that this may 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 nasal mucosal surface adherent mucus samples Ten nasal aspirate samples were analyzed using Mentip P1503 (manufactured by Japan Cotton Swab Co., Ltd.). The resulting solution was collected and suspended in 200 μL of physiological saline to prepare a sample for SDS-PAGE. The 400 μL nasal mucosal surface adhered mucus samples collected from three healthy subjects in 2. were mixed. Each sample was aliquoted and diluted to a final concentration of 62.5 mM Tris-HCl (pH 6.5 ), 10(w / v)% glycerol, 2.3(w / v)% SDS, 0.05(w / v)% BPB (dye) The mixture was added, heat denatured at 95°C for 5 minutes, and then subjected to standard SDS-PAGE. 2. Total protein analysis using a densitometry analyzer Using a densitometry analyzer (Bio-Rad), analyze the CB of the SDS-PAGE obtained in step 1 above. Analyze the B-stained gel and measure the total density (density score) of all bands in each lane. It was determined. 3.Comparative Consideration The results of the density score measurement are shown in Table 3. From the results in Table 3, although there were differences between samples, When comparing the mixed conditions, the nasal aspirate specimen and the simple nasal mucosal surface adhered mucus specimen Therefore, there was no significant difference in the amount of protein that could be collected by the simplified nasal mucosa of the present invention. It is believed that it is possible to test for influenza viruses using surface-adhered mucus specimens. was made.

[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 kept for a certain period of time. The spleens were removed from the mice and subjected to the method of Kohler et al. (Nature, vol. 256, p. 495-497) The resulting fused cells (hybrids) were then fused with mouse myeloma cells (P3X63) by the method of The cells (Doma) were maintained in a 37°C incubator and incubated with an anti-H. pylori extract. The antibody activity of the supernatant was confirmed by ELISA using a plate with the original material solidified. The two cell lines obtained were then cloned into pristane-treated BALB / c mice. The antibody was intraperitoneally administered to the mice, and approximately two weeks later, antibody-containing ascites was collected. The ascites obtained was subjected to affinity chromatography using a protein A column. IgG was purified from each of these samples, 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 kept for a certain period of time. The spleens were removed from the mice and subjected to the method of Kohler et al. (Nature, vol. 256, p. 495-497) The resulting fused cells (hybrids) were then fused with mouse myeloma cells (P3X63) by the method of The cells (Doma) were maintained in a 37°C incubator and incubated with an anti-H. pylori extract. The antibody activity of the supernatant was confirmed by ELISA using a plate with the original material solidified. The two cell lines obtained were then cloned into pristane-treated BALB / c mice. The antibody was intraperitoneally administered to the mice, and approximately two weeks later, antibody-containing ascites was collected. The ascites obtained was subjected to affinity chromatography using a protein A column. IgG was purified from each of these, 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 administered in 50 mM MES (2-Morpholinoethanesulfon After dialysis against a buffer solution (pH 6.0) containing ic acid, monohydrate (Dojindo Chemical Co., Ltd.), the OD280nm was 0.5. Next, 10 (W / V)% blue polystyrene lamination was prepared by diluting the solution with the same buffer solution. Tex particles (particle size 0.45 μm, surface functional groups are carboxyl groups, functional group density 65 Å / COOH group; M The mixture was mixed with EDAC (Agsphere) at a liquid ratio of 40:1 and allowed to react. (N-(3-Dimethlaminopropyl)-N'-ethylcarbodiimide hydrochloride; Sigma) at a final concentration After adding 0.1% of the solution, the mixture was allowed to react for 2 hours. After washing, the final suspension (5 mM Tris, 0. 0.4 (w / v)% BSA (bovine serum albumin), 0.4 M trehalose, 0.2 (v / v)% Triton X-100) 20 mL The latex particles were suspended in the water and dispersed in an ultrasonic disperser (Olympus).

[0050] 4. Preparation of Labeled Anti-influenza B Virus Antibodies One of the anti-type B influenza virus antibodies was administered in 50 mM MES (2-Morpholinoethanesulfon After dialysis against a buffer solution (pH 6.0) containing ic acid, monohydrate (Dojindo Chemical Co., Ltd.), the OD280nm was 0.5. Next, 10 (w / v)% blue polystyrene lamination was added to the same buffer solution to prepare 10 mL of the solution. Tex particles (particle size 0.45 μm, surface functional groups are carboxyl groups, functional group density 65 Å / COOH group; M The mixture was mixed with EDAC (Agsphere) at a liquid ratio of 40:1 and allowed to react. (N-(3-Dimethlaminopropyl)-N'-ethylcarbodiimide hydrochloride; Sigma) at a final concentration After adding 0.1% of the solution, the mixture was allowed to react for 2 hours. After washing, the final suspension (5 mM Tris, 0. 0.4 (w / v)% BSA (bovine serum albumin), 0.4 M trehalose, 0.2 (v / v)% Triton X-100) 20 mL The latex particles were suspended in the water and dispersed in an ultrasonic disperser (Olympus).

[0051] 5. Preparation of Latex Particle-labeled Antibody Dry Pads The latex particle-labeled anti-type A and type B influenza viruses obtained in 3 and 4 above The antibody was mixed with the antibody and sprayed onto the reel at a volume of 8 μL / cm using a positive pressure spray device (BioJet; BioDot). The solution was sprayed onto the entire surface of a 15 mm wide rolled cellulose nonwoven fabric. After spraying, hot air at 50°C was blown over it for 1 minute. The mixture was applied and dried to prepare a dry pad of latex particle-labeled antibody.

[0052] 6. Preparation of membrane-immobilized antibodies The purified anti-influenza A virus antibodies prepared in 1 above that were not used for labeling The filtrate was dialyzed against a solid phase solution (10 mM Tris-HCl (pH 8.0)), and after dialysis, it was filtered through a 0.22 μm filter. Prepare solid-phase anti-influenza A virus antibody by diluting with the solid-phase solution so that the nm = 3.0. Ta. The purified anti-influenza B virus antibodies prepared in 2 above that were not used for labeling The filtrate was dialyzed against a solid phase solution (10 mM Tris-HCl (pH 8.0)), and after dialysis, it was filtered through a 0.22 μm filter. Prepare solid-phase anti-influenza B virus antibody by diluting with the solid-phase solution to a concentration of 3.0 nm. Ta.

[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 nitrocellulose membrane (pore size 12 μm; A white sheet (manufactured by MAN) was used. One end of the long axis (this end is the upstream end, and the other end is the downstream end) The solid-phase anti-influenza A virus antibody was placed 6 mm away from the end of the plate, and the solid-phase anti-influenza A virus antibody was placed 8 mm away from the end of the plate. Solid-phase anti-influenza B virus antibody was applied at each position using a positive pressure sprayer at a dose of 1 μL / cm. The anti-mouse antibody was applied in a line using a BioJet (BioDot) dispenser, and placed 13 mm from one end of the long axis. The IgG antibody was diluted to OD280nm = 1.0 and sprayed at a volume of 1 μL / cm using a positive pressure sprayer (BioJet; BioDot After application, the sheet was dried by blowing hot air at 45°C for 30 minutes. Next, to fix the components and increase their strength, the antibody-coated surface of the membrane (this surface is referred to as the upper surface) was On the other side of the plate (this side is the bottom), place a plastic backing sheet (manufactured by BioDot) ) was glued. Next, cut the latex particle-labeled antibody dry pad prepared in step 5 above into a width of 15 mm and a length of 10 cm. The upstream end of the membrane was placed on the top surface of the membrane so that it overlapped by 2 mm. Furthermore, a cellulose filter paper (Whatman) measuring 23 mm wide and 10 cm long was used to dry the latex particle-labeled antibody. The sample was placed on the top surface of the pad so that it overlapped by 13 mm and attached to the pad to form a sample drop pad. Next, a cellulose filter paper (Whatman) measuring 30 mm in width and 10 cm in length was placed on top of the membrane. The pad was attached so that it overlapped the downstream end of the membrane by 5 mm to form a sample absorption pad. Next, except for the 5 mm width at the upstream end of the sample dropping pad, the entire top surface is covered with a transparent plastic laminate. The plate was coated with Nate (Adhesive Research). Finally, the membrane was cut into 5 mm sections along the longitudinal axis to prepare membrane assay devices.

[0054] 8. Influenza virus detection Influenza virus antigen kit QuickNavi (trademark)-Flu2 (manufactured by Denka Seiken Co., Ltd.) ) to collect nasal swabs and diagnose influenza virus infection. Five patients were diagnosed as influenza A virus positive (+), and five patients were diagnosed as influenza B virus positive (+). Five patients were diagnosed as virus positive (+) and five were influenza virus negative (-). The subjects were five patients diagnosed with rhinitis, and the nasal drops prepared in Example 1, paragraph 1, were administered to the nasal cavity of the subjects. The device was used to spray 400 μL of saline, and the liquid that flowed out of the nasal passage was used as an ex-swab. The mucus was collected by sucking it into 003T (manufactured by Denka Seiken), and this was used as a simple mucus specimen attached to the surface of the nasal mucosa. The cotton ball from the collected sample was placed in a sample suspension buffer (Tween 20 0.05 (w / v)% and bovine serum albumin). Immerse the tip in 0.2 mL of phosphate buffer (pH 7.4) containing 0.1% (w / v) albumin and rub the attached tissue. The mixture was mixed and extracted in a buffer solution for flotation of specimens, and this was used as a specimen sample. Apply the influenza virus detection lateral flow membrane prepared in 7. to the sample. The sample drop pad side of the assay device was immersed in the liquid. After 10 minutes, the assay device was observed and the anti- The test was considered valid if color development was observed at the point where the mouse IgG antibody was applied (control line). If color development is observed at the site where the solid-phase anti-type A influenza virus antibody was applied, Influenza type B virus positive (+), solid phase anti-influenza type B virus antibody applied If color is observed in the indicated location, the test is positive for influenza B virus (+). If no color was observed at the control line, the result was judged to be negative (-). If no color development was observed at the position, the test was deemed invalid.

[0055] 9.Comparative Consideration The assay results are shown in Table 4. From the results in Table 4, it was found that the nasal cavity swab using QuickNavi-Flu2 The results of the test and the overall findings were compared with those of the test using the specimen according to the present invention. The results were all consistent.

[0056] [Table 4]

[0057] [Example 5] 1. Sample collection for influenza virus load comparison Influenza virus antigen kit QuickNavi (trademark)-Flu2 (manufactured by Denka Seiken Co., Ltd.) ) to collect nasal swabs and diagnose influenza virus infection. The patient was diagnosed as influenza A virus positive (+) and the test results were analyzed using the method described in Example 1. Using the prepared nasal spray device, 400 μL of saline was sprayed, and the liquid that flowed out of the nasal entrance was measured. The mucus was absorbed into an Ex Swab 003T (manufactured by Denka Seiken) to collect a simple sample of mucus adhering to the surface of the nasal mucosa. This was suspended in 200 μL of saline and used as a sample for qPCR. The residual liquid from the nasal swab test of the CheckNavi-Flu2 was used as the sample. 2.Analysis by Real-time PCR Using the QIAamp Viral RNA Mini Kit (nucleic acid extraction kit, manufactured by QIAGEN), two samples from 1. Nucleic acid was extracted from the sample, and the desired PCR sample was added and analyzed using the Applied Biosystems QuantStudio™. The measurements were performed using a PCR device (Thermo Fisher Scientific). 3.Comparative Consideration The measurement results are shown in Table 5. From Table 5, it can be seen that the viral load in the simple mucus specimen attached to the nasal mucosal surface was 1. 39x10 6 copies / mL, whereas the viral load in the control nasal swab sample was 1.31x10 5 copies The results were equivalent to / mL. Compared with the conventional method of nasal swab specimens, the simplified nasal mucosa The surface-adhered mucus sample contained a higher amount of virus, suggesting that the virus was more stable than in the nasal swab. It has been revealed that it is possible to collect viruses and use them in 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 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 adhering mucus that flows out near the nasal entrance is collected, and the resulting lavage fluid containing the adhering mucus on the nasal mucosal surface is used as a sample (excluding cases where the method for detecting an infectious disease is a lateral flow assay).

2. The method according to claim 1, wherein the method for detecting an infectious disease is a sandwich method.

3. The method for detecting infections (1) an immunostaining method selected from the group consisting of a fluorescent antibody method, an enzyme antibody method, a heavy metal-labeled antibody method, and a radioisotope-labeled antibody method; (2) an electrophoresis method selected from either Western blotting or fluorescent two-dimensional electrophoresis; (3) The method according to claim 1, which is an immunoassay selected from the group consisting of enzyme-linked immunosorbent assay (ELISA), dot blotting, and latex agglutination.

4. The method according to claim 1, wherein the method for detecting an infectious disease is a genetic testing method.

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

6. A test kit for detecting an infectious disease, which comprises spraying 300 to 500 μL of a lavage solution into the nasal cavity of a human subject suspected of having an infectious disease to wash the nasal mucosal surface, collecting the lavage solution containing the adhering mucus that has flowed out near the nasal entrance, and using the resulting lavage solution containing the mucus adhering to the nasal mucosal surface as a specimen, the test reagent for detecting an infectious disease; and a single-use nasal drop device containing a cleaning solution for cleaning the nasal mucosa, wherein the device contains 300 to 500 μL of the cleaning solution to be added to the nasal cavity per use. Test kits containing the above (excluding lateral flow assay test reagents).

7. 7. The test kit according to claim 6, which is a test kit for detecting an infectious disease, and is a sandwich method.

8. Test kits to detect infectious diseases (1) An immunostaining kit selected from any one of fluorescent antibody technique, enzyme antibody technique, heavy metal-labeled antibody technique, and radioisotope-labeled antibody technique; (2) An electrophoresis kit selected from either a Western blot method or a fluorescent two-dimensional electrophoresis method; (3) An immunoassay kit selected from the enzyme-linked immunosorbent assay (ELISA), dot blotting, and latex agglutination methods; or (4) The test kit according to claim 6, which is a genetic test kit.

9. The test kit according to any one of claims 6 to 8, wherein the infectious disease is an influenza virus infection.

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