Virus combined detection immunochromatography test paper, detection pen and detection product

Abstract

The application provides a virus combined detection immunochromatography test paper, a detection pen and a detection product, and relates to the technical field of immunochromatography test paper. The virus combined detection immunochromatography test paper can detect the novel coronavirus, the influenza A virus and the influenza B virus in a three-in-one manner. The sample binding pad of the immunochromatography test paper is coated with labeled antibodies, including a first influenza A virus antibody, a first influenza B virus antibody, a first novel coronavirus antibody and a first human internal reference protein antibody, and each labeled antibody is labeled with a different color of label. The immunochromatography test paper adopts a double-antibody sandwich principle, and the second influenza A virus antibody, the second influenza B virus antibody and the second novel coronavirus antibody are coated on different detection lines in the detection area. The immunochromatography test paper and the detection pen solve the problems of complex operation and difficult interpretation of the three-in-one detection product of the novel coronavirus, the influenza A virus and the influenza B virus in the prior art.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of immunochromatography test paper, in particular to a virus combined detection immunochromatography test paper, a detection pen and a detection product. BACKGROUND

[0002] Corona Virus Disease 2019 (COVID-19) is an acute respiratory infectious disease caused by infection with the 2019 novel coronavirus. The main symptoms after infection are fever, dry cough and fatigue, and some patients have nasal congestion, runny nose and other upper respiratory symptoms, which affect human daily life. Therefore, rapid detection of the new coronavirus is necessary.

[0003] Influenza, commonly known as influenza, is an acute respiratory infectious disease caused by two types of influenza viruses, A and B. Its transmission characteristics are strong infectivity, high incidence and rapid spread, mainly by respiratory inhalation, and the main symptoms after infection are high fever, fatigue and headache. Influenza A virus is prone to mutation and has caused several pandemics worldwide, which is a public health problem that the world pays attention to.

[0004] Because the new coronavirus and the influenza virus have similar modes of transmission and can spread through the respiratory tract, and the clinical symptoms after infection are very similar, even some patients have mixed infections, which are difficult to distinguish by ordinary medical means such as blood tests and medical effects. And general testing methods require professional personnel to operate in the laboratory, the detection period is long, and it is difficult to achieve rapid and accurate diagnosis, which is not conducive to timely treatment of patients. Therefore, a rapid detection product that can distinguish between the new coronavirus and influenza is essential. And for patients with similar symptoms, accurate and rapid diagnosis is beneficial for patients to receive effective treatment in a timely manner and avoid cross-infection. There are few products that can simultaneously detect the three existing antigens on one test strip.

[0005] The existing three-in-one rapid diagnostic test paper for the new coronavirus, influenza A and influenza B on the market is mostly a double-window detection card, and often requires repeated operation when adding samples. The pretreatment operation is complicated. And most of them use colloidal gold as the color developing indicator, and the color development is red in the case of co-positive. If the user misreads the T line sequence, it will cause a diagnostic error. The existing product quality control line is the quality control of the test strip itself, without sampling quality control. In actual detection, false negative or false positive results may occur due to improper sampling, leading to misdiagnosis. Most of the existing reagent kits on the market are in the form of a plate-shaped card, and after sampling is completed, the sampling head is inserted into the lysis solution, and then incubated before sampling. The operation steps are many, and the supporting parts in the detection reagent kit are many. For ordinary residents who have not touched medical devices, the operation steps are many and the operation is not professional, which may cause false negative results.

[0006] Therefore, it is necessary to provide a simple and convenient combined diagnostic detection method with easy-to-read results for the normal combined detection of the novel coronavirus, the influenza A virus and the influenza B virus.

[0007] In view of this, the present application is proposed. SUMMARY

[0008] The present application aims to provide a virus combined detection immunochromatography test strip, which realizes the three-in-one immunochromatography rapid detection of the novel coronavirus, the influenza A virus and the influenza B virus. The test strip adds an antibody specifically combined with a human internal reference protein as a sampling quality control line, which greatly avoids false negatives caused by non-professionals sampling without positioning. The present application uses different colored markers as indicators. When three detection lines are positive, the color development is different, which avoids diagnostic errors caused by user misjudgment of the T line position. The test strip can judge the results in 15 minutes, which is suitable for small-scale rapid detection and personal self-testing. Based on the above virus combined detection immunochromatography test strip, the present application further provides a detection pen mainly composed of the virus combined detection immunochromatography test strip and a shell. The present application alleviates the problems of complex operation and difficult interpretation of the three-in-one detection product of the novel coronavirus, the influenza A virus and the influenza B virus in the prior art.

[0009] To solve the above technical problems, the present application adopts the following technical solutions:

[0010] According to one aspect of the present application, the present application provides a virus combined detection immunochromatography test strip, which includes the detection of the novel coronavirus, the influenza A virus and the influenza B virus. The immunochromatography test strip includes a sample pad, a combination pad, a detection pad and a water absorption pad arranged in sequence on a fixed bottom plate along the chromatography direction of the sample to be tested.

[0011] The combination pad is coated with a marker-labeled marker antibody, and the marker antibody includes a first influenza A virus antibody, a first influenza B virus antibody, a first novel coronavirus antibody and a first human internal reference protein antibody. The first influenza A virus antibody, the first influenza B virus antibody and the first novel coronavirus antibody are respectively labeled with different colored markers.

[0012] The combination pad includes a second combination pad and a first combination pad along the chromatography direction of the sample to be tested, and the first novel coronavirus antibody is coated on the second combination pad.

[0013] The detection pad is coated with a detection antibody, and the detection antibody includes a second influenza A virus antibody, a second influenza B virus antibody, a second novel coronavirus antibody and a second human internal reference protein antibody.

[0014] The detection pad comprises a detection line T1, a detection line T2, a detection line T3 and a sample-to-site quality control line C along a chromatographic direction of a sample to be detected; the second novel coronavirus antibody is coated on the detection line T3, and the second human internal reference protein antibody is coated on the sample-to-site quality control line C.

[0015] The detection antibody and the corresponding labeled antibody can simultaneously bind to the same antigen.

[0016] According to another aspect of the present application, the present application also provides a virus combined detection pen, which comprises a virus combined detection immunochromatography test paper and a shell; the shell comprises a base and a pen body; the sample lysate is preloaded in the base;

[0017] The pen body is provided with a clamping groove for fixing the immunochromatography test paper, and an end of the pen body close to the sample pad is provided with a sampling part in contact with the sample pad; the base is provided with a buckle for fixing the pen body; after the pen body is inserted into the base, the sampling part is in contact with the sample lysate; and the pen body is provided with a visible part for observing the detection pad.

[0018] According to another aspect of the present application, the present application also provides a virus diagnostic product, which comprises the above-mentioned virus combined detection immunochromatography test paper or the above-mentioned virus combined detection pen.

[0019] Compared with the prior art, the present application has the following beneficial effects:

[0020] The virus combined detection immunochromatography test paper provided by the application can detect the novel coronavirus, the influenza A virus and the influenza B virus in three-in-one. The immunochromatography test paper includes a sample pad, a combination pad, a detection pad and a water absorption pad arranged on a fixed bottom plate in sequence along the chromatography direction of the sample to be detected. The sample combination pad is coated with a labeled antibody, and the labeled antibody includes a first influenza A virus antibody, a first influenza B virus antibody, a first novel coronavirus antibody and a first human internal reference protein antibody. The immunochromatography test paper provided by the application adopts a double-antibody sandwich principle, and therefore the second influenza A virus antibody, the second influenza B virus antibody, the second novel coronavirus antibody and the second human internal reference protein antibody capable of forming an antibody-antigen-antibody complex are further coated on the different detection lines in the detection area. The first influenza A virus antibody, the first influenza B virus antibody and the first novel coronavirus antibody in the application are respectively labeled with different color markers, so that the three detection lines have color differences when the detection lines are positive. This design avoids the situation that the diagnosis is wrong due to the judgment error of the user when reading the results because the detection lines of different viruses are of the same color. The quality control line in the immunochromatography test paper includes a sampling-in-place quality control line C, which can detect the human internal reference protein. When the sampling is not in place, the sampling-in-place quality control line C does not show a line, indicating that the sampling is not in place, and the result is invalid. When the sampling-in-place quality control line C shows a line, it indicates that the sampling is in place, the chromatography is normal, and the result is of reference value. This design avoids the false negative situation caused by the user's sampling not being in place.

[0021] According to another aspect of the application, the application further provides a virus combined detection pen composed of the above-mentioned virus combined detection immunochromatography test paper and a shell. The shell includes a base preloaded with a sample lysate. When in use, the user only needs to insert the sampled detection pen into the base. The sampling and detection are integrated, which is convenient and fast.

[0022] The immunochromatography test paper and the detection pen provided by the application combine the detection of the novel coronavirus, the influenza A virus and the influenza B virus on one test paper strip, realize one-time sampling and simultaneous detection of positive results. In addition, different color markers are used to distinguish the colors of the detection lines, which improves the readability of the results. The sampling-in-place quality control line C provided by the application can prompt the user to sample in place, thereby indicating whether the sample is valid, and avoiding the invalid sampling problem caused by the user's non-standard operation and other reasons. The design of the sampling-in-place quality control line improves the effectiveness and accuracy of the immunochromatography test paper in detecting human samples.

[0023] The immunochromatography test paper and detection pen provided by the application have the advantages of high sensitivity, and the LOD value of the detection of the new coronavirus can reach 50 pg / Test, the LOD value of the detection of the influenza A virus can reach 50 pg / Test, the LOD value of the detection of the influenza B virus can reach 1 ng / Test, the advantages of good specificity, no detection of various interference pathogens, the advantages of rapid detection, naked-eye judgment of the result within 15 minutes, meeting the daily detection needs of professionals and non-professionals, and wide application range. BRIEF DESCRIPTION OF DRAWINGS

[0024] In order to more clearly illustrate the technical solutions in the specific embodiments or prior art of the application, the drawings needed in the specific embodiments or prior art description will be briefly introduced as follows. Obviously, the drawings in the following description are some embodiments of the application, and other drawings can be obtained by those skilled in the art without creative labor on the basis of these drawings.

[0025] Figure 1 It is a schematic diagram of the virus combined detection immunochromatography test paper of Example 1.

[0026] Figure 2 It is a schematic diagram of the sampling of the virus combined detection immunochromatography test paper of Example 1 to the quality control line C.

[0027] Figure 3 It is a schematic diagram of the detection line of the virus combined detection immunochromatography test paper of Example 1.

[0028] Figure 4 It is a photo of the virus detection pen of Example 1 after detection. DETAILED DESCRIPTION

[0029] The technical solutions of the application will be described in detail below with reference to the embodiments. Obviously, the described embodiments are some of the embodiments of the application, rather than all the embodiments. Based on the embodiments in the application, all other embodiments obtained by those skilled in the art without creative labor fall within the protection scope of the application.

[0030] It should be noted that "new coronavirus", "coronavirus", "COVID-19" and "CoV" in this paper all represent new coronavirus and can be used interchangeably; "influenza A virus", "influenza A virus", "influenza A" and "FluA" in this paper all represent influenza A virus and can be used interchangeably; "influenza B virus", "influenza B virus", "influenza B" and "FluB" in this paper all represent influenza B virus and can be used interchangeably; "coating", "embedding" and "spraying point" in this paper all represent fixing antibodies on immunochromatography test paper and can be used interchangeably.

[0031] According to one aspect of the present application, the present application provides a virus combined detection immunochromatography test paper, the virus combined detection including detecting new coronavirus, influenza A virus and influenza B virus; the immunochromatography test paper includes sample pad, combination pad, detection pad and water absorption pad arranged on the fixed bottom plate in turn along the chromatography direction of the sample to be detected.

[0032] The combination pad is coated with labeled antibodies labeled with markers, and the labeled antibodies include first influenza A virus antibody, first influenza B virus antibody, first new coronavirus antibody and first human internal reference protein antibody. The first influenza A virus antibody specifically binds to influenza A virus antigen, the first influenza B virus antibody specifically binds to influenza B virus antigen, the first new coronavirus antibody specifically binds to new coronavirus antigen, and the first human internal reference protein antibody specifically binds to human internal reference protein antigen. Moreover, the first influenza A virus antibody, the first influenza B virus antibody and the first new coronavirus antibody are respectively labeled with different color markers.

[0033] Since the labeled antibodies of the present application are labeled with markers showing different colors, multiple markers need to be coated on the combination pad, therefore, the present application uses double-layer combination pad for chromatography, which can prevent the surface of the detection pad from being too colorful during the chromatography process. Along the chromatography direction of the sample to be detected, the combination pad includes a second combination pad and a first combination pad, i.e. the second combination pad is close to the sample pad, and the first combination pad is close to the detection pad. The first new coronavirus antibody is coated on the second combination pad, and the rest of the labeled antibodies are optionally coated on the first combination pad or the second combination pad. In some alternative embodiments, the first combination pad is coated with the first influenza A virus antibody and the first influenza B virus antibody, and the second combination pad is coated with the first new coronavirus antibody and the first human internal reference protein antibody.

[0034] The detection pad is coated with detection antibodies, and the detection antibodies include a second influenza A virus antibody, a second influenza B virus antibody, a second novel coronavirus antibody, and a second human internal reference protein antibody. The second influenza A virus antibody specifically binds to an influenza A virus antigen, the second influenza B virus antibody specifically binds to an influenza B virus antigen, the second novel coronavirus antibody specifically binds to a novel coronavirus antigen, and the second human internal reference protein antibody specifically binds to a human internal reference protein antigen.

[0035] The present application adopts a double-antibody sandwich principle. The detection antibodies and the corresponding labeled antibodies can simultaneously bind to the same antigen to form an antibody-antigen-antibody complex, thereby achieving detection of the virus antigen. That is, the first influenza A virus antibody and the second influenza A virus antibody can simultaneously bind to the influenza A virus antigen, the first influenza B virus antibody and the second influenza B virus antibody can simultaneously bind to the influenza B virus antigen, the first novel coronavirus antibody and the second novel coronavirus antibody can simultaneously bind to the novel coronavirus antigen, and the first human internal reference protein antibody and the second human internal reference protein antibody can simultaneously bind to the human internal reference protein. The detection antibodies and the corresponding labeled antibodies can be the same or different. If the same antibody can simultaneously bind to the same antigen, for example, to the same antigen at different positions of the repeated antigen epitope, the same antibody can simultaneously serve as a labeled antibody and a detection antibody.

[0036] The detection pad sequentially includes a detection line T3, a detection line T2, a detection line T1, and a sample-to-site quality control line C in the chromatographic direction of the sample to be detected. The second novel coronavirus antibody is coated on the detection line T3, and the second human internal reference protein antibody is coated on the sample-to-site quality control line C. The second influenza A virus antibody and the first influenza A virus antibody are coated on the detection line T2 and the detection line T1, respectively. In some alternative embodiments, the detection line T3 is coated with the second novel coronavirus antibody; the detection line T2 is coated with the second influenza A virus antibody; the detection line T1 is coated with the second influenza B virus antibody; and the sample-to-site quality control line C is coated with the second human internal reference protein antibody.

[0037] Experiments have verified that coating the second influenza A virus antibody or the second influenza B virus antibody on the detection line T3 will affect the detection effect of the detection line T1 and the detection line T2 located downstream in the sample chromatographic direction. Therefore, the present application coats the second novel coronavirus antibody on the detection line T3 closest to the combination pad. In order to increase the chromatographic distance of the novel coronavirus antigen and improve the sensitivity, the first novel coronavirus antibody is coated on the second combination pad farther from the detection pad to increase the chromatographic distance. Experiments have verified that the detection result of coating the second human internal reference protein antibody or the second influenza A virus antibody together with the second novel coronavirus antibody on the second combination pad is better than the detection result of coating the second novel coronavirus antibody on the first combination pad.

[0038] In some alternative embodiments, the first influenza A virus antibody and the second influenza A virus antibody optionally recognize any antigen of the influenza A virus, which can be a natural antigen or a recombinant antigen. In some alternative embodiments, the first influenza A virus antibody and the second influenza A virus antibody recognize the N protein (nucleocapsid protein) of the influenza A virus, which can be a recombinant N protein.

[0039] In some alternative embodiments, the first influenza B virus antibody and the second influenza B virus antibody optionally recognize any antigen of the influenza B virus, which can be a natural antigen or a recombinant antigen. In some alternative embodiments, the first influenza B virus antibody and the second influenza B virus antibody recognize the N protein of the influenza B virus, which can be a recombinant N protein.

[0040] In some alternative embodiments, the first SARS-CoV-2 antibody and the second SARS-CoV-2 antibody optionally recognize any antigen of the SARS-CoV-2, which can be a natural antigen or a recombinant antigen. In some alternative embodiments, the first SARS-CoV-2 antibody and the second SARS-CoV-2 antibody recognize the N protein of the SARS-CoV-2, which can be a recombinant N protein.

[0041] In some alternative embodiments, the target human reference protein detected by the sample-to-well quality control line C is selected from the group consisting of ribonuclease P, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), beta microglobulin, beta-tubulin, 18s RNA, beta-actin, and TATA-binding protein, preferably glyceraldehyde-3-phosphate dehydrogenase.

[0042] In some alternative embodiments, the label comprises at least one of quantum dots, colloidal gold, colored latex microspheres, fluorescent microspheres, cellulose microspheres, quantum dot particles, and immunomagnetic beads; different labeled antibodies can be selected from the same type of label or different types of labels, as long as the antibody-virus antigen-detection antibody complex formed by the specifically recognized virus-labeled antibodies exhibits different visual signals.

[0043] In some preferred embodiments, the labels are selected from colored latex particles, as long as each labeled antibody labels a colored latex particle of a different color, without limitation to the color of the colored latex particle labeled by each labeled antibody, in some alternative embodiments, the first SARS-CoV-2 antibody labels a blue latex particle, the first influenza A virus antibody labels a green latex particle, and the first influenza B virus antibody labels a red latex particle. The particle size of the colored latex particles is preferably 200-400 nm, for example, but not limited to, 200, 250, 300, 350, or 400 nm, or a particle size range between any two of the foregoing. The concentration of the colored latex particle stock solution is preferably 4-10 wt%, for example, but not limited to, 4, 5, 6, 7, 8, 9, or 10 wt%.

[0044] The coated labeled antibodies on the conjugate pad are obtained by spraying the conjugate pad with a conjugate pad coating solution containing the labeled antibodies. The mass ratio of each labeled antibody to the colored latex particles in the conjugate pad coating solution is independently 1:20-1:80, for example, but not limited to, 1:20, 1:30, 1:40, 1:50, 1:60, 1:70, or 1:80. The concentration of the labeled antibodies in the conjugate pad coating solution is preferably 2-5 mg / mL, for example, but not limited to, 2, 3, 4, or 5 mg / mL; and the spraying amount of the conjugate pad coating solution on the conjugate pad is preferably 1-2 μL / cm, for example, but not limited to, 1, 1.5, or 2 μL / cm.

[0045] The test line and the sample-to-site quality control line C of the test pad are obtained by spraying the test pad with a test pad coating solution containing the detection antibodies.

[0046] In some alternative embodiments, the concentration of the second human reference protein antibody in the test pad coating solution is 0.5-1.5 mg / mL, preferably 0.5-1 mg / mL, and more preferably 1 mg / mL.

[0047] In some alternative embodiments, the concentration of the second influenza B virus antibody in the test pad coating solution is 0.3-1.5 mg / mL, preferably 0.4-1.0 mg / mL, and more preferably 0.5 mg / mL.

[0048] In some alternative embodiments, the concentration of the second influenza A virus antibody in the test pad coating solution is 0.1-1.5 mg / mL, preferably 0.5-1.2 mg / mL, and more preferably 0.7 mg / mL.

[0049] In some alternative embodiments, the concentration of the second SARS-CoV-2 antibody in the test pad coating solution is 0.1-1.5 mg / mL, preferably 0.3-0.8 mg / mL, and more preferably 0.5 mg / mL.

[0050] In some alternative embodiments, the detection pad coating solution for each detection antibody independently contains 0.8-1.0 wt% NaCl, 0.1-0.5 wt% Na2HPO4, and 2-5 wt% sucrose. Preferably, the detection pad coating solution for each detection antibody independently contains 0.9 wt% NaCl, 0.13 wt% Na2HPO4, and 3 wt% sucrose.

[0051] In some alternative embodiments, the volume of the detection pad coating solution sprayed on the test line T1, the test line T2, the test line T3, and the sample-to- site quality control line C is independently 0.7-1.2 μL / cm.

[0052] In some alternative embodiments, the interval between the test line T3 and the test line T2 is 3.5-4.5 mm, and the interval between the test line T2 and the test line T1 is 3.5-4.5 mm.

[0053] In some alternative embodiments, the interval between the test line T1 and the sample-to-site quality control line C is 3-4 mm.

[0054] In some alternative embodiments, the sample pad is pretreated with a sample pad treatment solution, which preferably contains 0.8-1.2 wt% polyvinylpyrrolidone, 0.3-1.0 wt% S-9 (Tetronic 1307) surfactant, 0.01-0.1 wt% sodium azide, and 1-3.8 wt% borax.

[0055] The materials used to construct the various parts of the test strip of the present application can be selected from any materials acceptable in the art. The sample pad can be made of glass fiber membrane, polyester fiber membrane, or non-woven fabric, preferably glass fiber membrane; the first conjugate pad can be made of glass fiber membrane, polyester fiber membrane, or non-woven fabric, preferably polyester fiber membrane; the second conjugate pad can be made of glass fiber membrane, polyester fiber membrane, or non-woven fabric, preferably polyester fiber membrane; the detection pad can be made of nitrocellulose membrane; and the absorbent pad can be made of absorbent paper. The first conjugate pad can optionally overlap the second conjugate pad by 1-3 mm, the detection pad can optionally overlap the first conjugate pad by 1-3 mm, and the second conjugate pad can optionally overlap the sample pad by 1-3 mm. In a preferred embodiment, the absorbent pad is fixed to the base plate, and the detection pad, the first conjugate pad, the second conjugate pad, and the sample pad are arranged in this order, with each part overlapping the next by 1-3 mm.

[0056] According to another aspect of the present application, the present application also provides a virus combined detection pen, which comprises an immunochromatography test paper and a shell, and the shell comprises a base and a pen body. The sample lysate is preloaded in the base, the pen body is provided with a clamping groove for fixing the immunochromatography test paper, and the end of the pen body close to the sample pad is provided with a sampling part in contact with the sample pad; the base is provided with a buckle for fixing the pen body; after the pen body is inserted into the base, the sampling part is in contact with the sample lysate. The immunochromatography test paper can be preloaded in the shell or fixed in the clamping groove during use. After the immunochromatography test paper is fixed, the sample pad of the immunochromatography test paper is in contact with the sampling part of the pen body. After sampling, the pen body is inserted into the base and fixed by the buckle. The sampling part is in contact with the sample lysate, and the immunochromatography test paper in the pen body detects the sample to be tested through upward chromatography.

[0057] The pen body of the virus combined detection pen is provided with a visible part for observing the result. In some optional embodiments, the visible part is optionally a through hole in the area of the detection pad corresponding to the pen body, or the area of the detection pad corresponding to the pen body is made of transparent material to realize the observation of the detection result. In another optional embodiment, the pen body is made of transparent material to observe the chromatography result of the immunochromatography test paper.

[0058] The base is preloaded with a sample lysate, and the base is optionally provided with a cover to package the preloaded sample lysate. When in use, the cover is opened, and the pen body is inserted into the base to realize the contact between the sampling part and the sample lysate. The base is also optionally sealed with a sealing film. Correspondingly, the pen body is provided with a protruding end. When in use, the pen body is inserted into the base, and the protruding end breaks the integrity of the sealing film of the base, so that the sampling part is inserted into the base and fully contacts with the sample lysate, realizing the lysis and immunochromatography detection of the sample to be tested taken by the sampling part.

[0059] In some optional embodiments, the detection pen structure refers to the device described in CN114414306A or CN114869277A. The virus combined detection immunochromatography test paper provided by the present application is optionally the detection element in the collection device described in CN114414306A, or is optionally the test element in the detection device described in CN114869277A.

[0060] In some optional embodiments, the shell is the detection pen described in CN307182555S.

[0061] In some optional embodiments, the sample lysate comprises 3-5wt% NaCl, 0.2-1wt% BSA and 0.02-0.2wt% sodium azide.

[0062] In some optional embodiments, the virus combined detection pen is used according to the following steps:

[0063] S1, the virus combined detection immunochromatography test paper is loaded into the detection cavity of the pen-shaped shell with publication number CN307182555S;

[0064] S2, the pen tube tip sponge sampling head is inserted into the nasal cavity 2-3 cm, and is attached to the nasal mucosa and rotated for about 5 circles;

[0065] S3, the sampling completed sponge head is vertically inserted into the base loaded with sample lysate for lysing chromatography;

[0066] S4, after 15 min, the detection results are judged according to the color of the test strip sampling to the quality control line C and each detection line.

[0067] According to another aspect of the present application, the present application also provides a virus diagnostic product, which comprises the above-mentioned virus combined detection immunochromatography test paper or the above-mentioned virus combined detection pen. The virus diagnostic product can also optionally comprise conventional reagents or consumables for detecting viruses, for example, when the detection element of the virus diagnostic product is the above-mentioned virus combined detection immunochromatography test paper, one or more of a swab, a sample lysate, a desiccant and a sample container can be optionally included; when the virus diagnostic product takes the above-mentioned virus combined detection immunochromatography test paper or the above-mentioned virus combined detection pen as the main detection element, a colorimetric card can also be included to qualitatively or semi-quantitatively determine the results.

[0068] The technical solutions and beneficial effects of the present application will be further illustrated in combination with preferred embodiments.

[0069] Example 1

[0070] The present embodiment provides a virus combined detection pen, which is composed of a virus combined detection immunochromatography test paper and a shell. The schematic diagram of the immunochromatography test paper is shown in Figure 1 , Figure 1 , wherein 1 is a water absorption pad; 2 is a sampling to quality control line C; 3 is a detection line T1; 4 is a detection line T2; 5 is a detection line T3; 6 is a first binding pad; 7 is a second binding pad; 8 is a sample pad; 9 is a fixed base plate; and 10 is a detection pad.

[0071] The virus combined detection immunochromatography test paper includes, along the chromatography direction of the sample to be detected, a sample pad 8, a second combination pad 7, a first combination pad 6, a detection pad 10 and a water absorption pad 1 arranged on a fixed bottom plate 9 in sequence; one end of the first combination pad 6 is overlapped with the detection pad 10, and the other end is overlapped with the second combination pad 7; the other end of the second combination pad 7 is overlapped with the sample pad 8; the specific structure is that the water absorption pad 1 is located on the fixed bottom plate 9, the detection pad 10, the first combination pad 6, the second combination pad 7 and the sample pad 8 are arranged in sequence, and each part is overlapped by 1-3 mm. The material of the sample pad 8 is selected from glass fiber, the materials of the first combination pad 6 and the second combination pad 7 are selected from polyester fiber, the material of the detection pad 10 is selected from nitrocellulose membrane, and the material of the water absorption pad 1 is selected from water absorption paper.

[0072] The first combination pad 6 is coated with green latex particle labeled first influenza A virus recombinant N protein antibody and red latex particle labeled first influenza B virus recombinant N protein antibody. The second combination pad 7 is coated with blue latex particle labeled first novel coronavirus recombinant N protein antibody and black latex particle labeled GAPDH antibody. The spraying amount of the combination pad coating solution is 1-2 μL / cm.

[0073] The marking method of the multicolor latex particles in the embodiment is as follows:

[0074] First, the latex microspheres are cleaned with a MES solution with a pH value of 6.0 and a concentration of 100 mM, and after centrifugation, the supernatant is discarded; then the latex microspheres are activated with a 750 μg / mL NHS and EDC solution, and after incubation at room temperature for 1 hour, the supernatant is discarded after centrifugation; then the antibody and the latex particles are added according to the feeding ratio (mass ratio) of the antibody to the latex particles of 1:20, and the concentration of the antibody is 2-5 mg / mL, and the incubation is overnight; then 30 μL of ethanolamine is added, and after incubation for half an hour, the supernatant is removed after centrifugation, and the blocking solution is added for blocking at room temperature for 4 hours, wherein the blocking solution contains 0.02 mg / mL NaN3, 5 mg / mL PvP, 50 mM / mL Tris, 5 mg / mL T-Casein, 200 mg / mL sucrose and 50 mg / mL trehalose; after the incubation is completed, the supernatant is discarded after centrifugation, and 600 μl of the blocking solution is continuously added, and the concentration is measured after ultrasonic treatment for standby use.

[0075] The detection pad 10 is provided with, along the chromatography direction of the sample to be detected, a detection line T3 5, a detection line T2 4, a detection line T1 3 and a sample-to-site quality control line C 2 in sequence. The detection line T1 3 is coated with second influenza B virus recombinant N protein antibody, the detection line T2 4 is coated with second influenza A virus recombinant N protein antibody, and the detection line T3 5 is coated with second novel coronavirus recombinant N protein antibody. The sample-to-site quality control line C 2 is coated with human GAPDH antibody.

[0076] The first recombinant influenza A virus N protein antibody is from Hangzhou Xukang Biological Technology Co., Ltd., and the item number is R1403; the second recombinant influenza A virus N protein antibody is from Hangzhou Xukang Biological Technology Co., Ltd., and the item number is R1401; the first recombinant influenza B virus N protein antibody is from Hangzhou Xukang Biological Technology Co., Ltd., and the item number is R1404; the second recombinant influenza B virus N protein antibody is from Hangzhou Xukang Biological Technology Co., Ltd., and the item number is R1402; the first recombinant novel coronavirus N protein antibody is from Hangzhou Xukang Biological Technology Co., Ltd., and the item number is RM0032; the second recombinant novel coronavirus N protein antibody is from Hangzhou Xukang Biological Technology Co., Ltd., and the item number is RM0033. The GAPDH antibody coated on the conjugate pad and the human GAPDH antibody coated on the sample-to-control line C 2 are both from Hangzhou Huaan Biotechnology Co., Ltd., and the item number is ET1601-4.

[0077] The concentration of the antibody in the detection pad coating solution containing the second recombinant influenza B virus N protein antibody of the test line T1 3 is 0.5 mg / mL; the concentration of the antibody in the detection pad coating solution containing the second recombinant influenza A virus N protein antibody of the test line T2 4 is 0.7 mg / mL; the concentration of the antibody in the detection pad coating solution containing the second recombinant novel coronavirus N protein antibody of the test line T3 5 is 0.5 mg / mL; and the concentration of the antibody in the detection pad coating solution containing the GAPDH antibody of the sample-to-control line is 1.0 mg / mL. In addition to the antibody, the detection pad coating solution also contains 9 wt% NaCl, 0.2 wt% NaH2PO4, 3 wt% sucrose and 5 wt% BSA. The amount of the test line and the sample-to-control line is 0.7-1.2 μL / cm. When the test line and the sample-to-control line C 2 are sprayed, the interval between the test line T3 5 and the test line T2 4 and the sample-to-control line C 2 is 4 mm, the interval between the test line T2 4 and the test line T1 3 is 4 mm, and the interval between the test line T1 3 and the sample-to-control line C 2 is 3.5 mm.

[0078] The sample pad 8 is pretreated with a sample pad treatment solution containing 1.0 wt% polyvinylpyrrolidone, 0.5 wt% S-9 (Tetronic 1307) surfactant, 0.02 wt% sodium azide and 2 wt% borax.

[0079] The conjugate pad and the detection pad 10 after spraying the antibody, and the pretreated sample pad 8 are assembled: the water absorption pad 1, the detection pad 10, the first conjugate pad 6, the second conjugate pad 7 and the sample pad 8 are sequentially pasted on the fixed bottom plate 9, and then cut into strips by a strip cutting machine to obtain an immunochromatography test part.

[0080] The shell of the virus combined detection pen of the embodiment is the detection pen described in CN307182555S, and the shell includes a base and a pen body. The sample lysate is preloaded in the base, the card slot for fixing the immunochromatography test paper is arranged in the pen body, and the sampling part in contact with the sample pad 8 is arranged at one end of the pen body close to the sample pad 8, and the sampling part is a sponge sampling head. The base is provided with a buckle for fixing the pen body. After the pen body is inserted into the base, the sampling part is in contact with the sample lysate. The immunochromatography test paper is preloaded in the shell, and the sample pad of the fixed immunochromatography test paper is in contact with the sampling part of the pen body. After sampling, the pen body is inserted into the base, fixed by the buckle, the sponge sampling head is in contact with the sample lysate, and the immunochromatography test paper in the pen body realizes the detection of the sample to be detected through upward chromatography.

[0081] The use method of the virus combined detection pen provided by the embodiment includes the following steps:

[0082] 1. Collecting samples:

[0083] The sample type is a human nasal swab, and the collection method includes the following steps:

[0084] (1) Insert the sponge sampling head of the detection pen tip into the nasal cavity 2-3 cm, tightly contact the nasal mucosa, and scrape the sponge head in the nasal cavity clockwise for 5 circles. After scraping, take out and perform the same operation in the other nasal cavity. When sampling, appropriate force should be applied to ensure that the sample in the human nasal mucosa is taken.

[0085] (2) When performing drug verification in the laboratory or taking serum samples, use a pipette to evenly apply 50 μl of serum sample on the sponge sampling head.

[0086] (3) When taking samples in batches, put the sampled sponge head on the matching plastic pen cap, put it into the sample bag, seal it and mark it.

[0087] 2. Detecting samples:

[0088] (1) When detecting suspected infectious sources, the detection personnel need to wear protective clothing, medical masks, medical gloves and other protective equipment according to the protective measures for testing infectious diseases. Residents can directly sample and detect in small-scale field tests.

[0089] (2) Insert the sampling head into the base containing 950 μl of sample lysate, and let it stand for 15 minutes for chromatography. The base should be vertically placed on the desktop, and the sampling head should be inserted into the base containing the sample lysate until the “click” buckle combination sound is heard. During chromatography, the base and the detection pen are vertically to the desktop or the detection table for upward chromatography, and the pen body and the base should be avoided from shaking or being perpendicular to the detection table. Observe whether the sample is normally chromatographed. If not, the sampling head should be retracted and reinserted.

[0090] (3) After 15 min, observe the line. After the test is completed, the used test pen and the base are treated as biological medical waste.

[0091] 3. Result interpretation:

[0092] The detection line of the C quality control area is black. In order to improve the visual effect and the readability of the results, the three T lines in this embodiment are set to different colors. Based on the existing material detection line T1 3, a bright red color is set. The detection line T2 4 is set to green, and the detection line T3 5 is set to blue. The schematic diagram of the line is shown in Figure 2 and Figure 3 .

[0093] The appearance of the sampling-to-position quality control line C 2 is shown in Figure 2 , and Figure 2 the left figure in the middle is the sampling-to-position quality control line C 2, which indicates that the sampling is in place; the right figure is the sampling-to-position quality control line C 2, which indicates that the sampling is not in place.

[0094] Figure 3 In the table, A indicates that the new coronavirus, influenza A virus and influenza B virus are co-positive; B indicates that only the influenza B virus is positive, and the rest are negative; C indicates that only the new coronavirus is positive, and the rest are negative; D indicates that only the influenza A virus is positive, and the rest are negative; E indicates that the new coronavirus and the influenza B virus are positive, and the influenza A virus is negative; F indicates that the influenza A virus and the influenza B virus are positive, and the new coronavirus is negative; G indicates that the new coronavirus and the influenza A virus are positive, and the influenza B virus is negative; H indicates that the three viruses are negative and the sampling is in place. Negative indicates that the sample does not contain the corresponding virus, or the content of the virus is below the detection threshold.

[0095] In the following test examples, 3 or more parallel samples are tested, and the color development value or average value of 95% or more parallel samples is taken. The color development value of the detection line and the quality control line is referenced from a color card, which has color blocks with color development from light to dark. The color development of the color blocks is corrected by measuring the reflectivity every year according to the national metrology technical specification JJF1232-2009. The color card color development value ranges from 1 to 10, and the higher the number, the darker the color development. Among them, the colorimetric value of 3.5 and above is determined as the line visible to the naked eye. In the subsequent table, the lightness of the T line is represented in this way of reading value. Therefore, the determination standard of the line visible to the naked eye is whether the color development colorimetric value of the T line reaches the color card reading value of 3.5, and "-" indicates negative. In the following test examples, if not specified, the preparation method of the immunochromatography test paper and the test pen is the same as that of Example 1.

[0096] It should be noted that the parameters of the double window pen of the new crown-influenza of the applicant's existing product are used in the conjugate pad area and the detection line area in the present application. That is, in the conjugate pad, the spray point concentration of CoV latex particles is 0.25%; the spray point concentration of FluA latex particles is 0.3%; the spray point concentration of FluB latex particles is 0.3%; and the spray point speed is 2 μL / cm. In the detection area of the NC membrane, the coating concentration of CoV is 0.5 mg / mL; the coating concentration of FluA is 0.7 mg / mL; the coating concentration of FluB is 0.5 mg / mL; and the coating speed is 1 μL / cm.

[0097] Test Example 1

[0098] Comparison of three human reference proteins as targets of sampling-to-site quality control line C: In this test example, three reference protein antibodies, β-actin antibody, β-tubulin, and GAPDH antibody, were used as alternative sampling-to-site quality control line C materials. Thirty-five human nasal cavity samples were used for screening, and the experimental design is as follows:

[0099] Scheme One:

[0100] Three different groups of reference protein antibodies were used as targets of sampling-to-site quality control line C, and the experimental groups are as follows:

[0101] Experimental Group One: Quality control line coating: β-actin antibody 1; labeling antibody: β-actin antibody 1.

[0102] Experimental Group Two: Quality control line coating: β-tubulin antibody 1; labeling antibody: β-tubulin antibody 1.

[0103] Experimental Group Three: Quality control line coating: GAPDH antibody 1; labeling antibody: GAPDH antibody 1.

[0104] Table 1 Comparison of sampling-to-site quality control line antibodies

[0105] Experimental Group Number of C-line Out-of-line Test Cases Number of C-line No Out-of-line Test Cases Total Experimental Group One 16 19 35 Experimental Group Two 14 21 35 Experimental Group Three 29 6 35 PBS Control Group 0 10 10

[0106] As can be seen from Table 1 above, the test examples of the C line of Experimental Group Three are significantly higher than those of Experimental Group One and Experimental Group Two, indicating that using GAPDH as the target of sampling-to-site quality control line C, coating GAPDH antibody in the conjugate pad and the detection pad is superior to using β-actin and β-tubulin as the target of sampling-to-site quality control line C.

[0107] Scheme Two:

[0108] Using two GAPDH antibodies as detection line coated antibody and labeled antibody respectively, using 10 human nasal samples for verification, the two GAPDH antibodies are from Hangzhou Huawan Biotechnology Co., Ltd., GAPDH antibody 1 item number: ET1601-4; GAPDH antibody 2 item number: M1310-2. The results are shown in Table 2:

[0109] Experimental group one: detection line coating: GAPDH antibody 1; labeled antibody: GAPDH antibody 1.

[0110] Experimental group two: detection line coating: GAPDH antibody 1; labeled antibody: GAPDH antibody 2.

[0111] Experimental group three: detection line coating: GAPDH antibody 2; labeled antibody: GAPDH antibody 2.

[0112] Experimental group four: detection line coating: GAPDH antibody 2; labeled antibody: GAPDH antibody 1.

[0113] Table 2.

[0114] Experimental Group C-line Out-of-line Readings Experimental Group One 7 Experimental Group Two 3.5 Experimental Group Three 4 Experimental Group Four 6 PBS Control Group -

[0115] According to the experimental results in Table 2, it is shown that the sampling-in-place design of the sampling-in-place quality control line C of the new crown, influenza A and B and influenza B three-in-one virus combined detection immunochromatography test paper is consistent with the real sample at a rate of 100%, and the above results prove that the quality control is real and effective. Therefore, the sampling-in-place quality control line coating and the labeled antibody of the present application are preferably GAPDH antibody 1. The antigen amino acid sequence recognized by GAPDH antibody 1 is mouse GAPDH aa 94-333 / 333, which is speculated to have a repeat site that can be recognized by the monoclonal antibody in the above sequence, so that the sampling-in-place quality control line coating and the labeled antibody are both GAPDH antibody 1, which can also form an antibody-antigen-antibody complex.

[0116] Test Example 2

[0117] Detection line color and concentration exploration:

[0118] Scheme one:

[0119] Using existing parameters, different antibodies are sprayed on the detection lines T1, T2 and T3 in this test example, and the experimental groups are as follows:

[0120] Experimental group one: T1: FluA; T2: CoV; T3: FluB;

[0121] Experimental group two: T1: FluB; T2: FluA; T3: CoV;

[0122] Experimental group three: T1: CoV; T2: FluB; T3: FluA;

[0123] Control group double window pen: T1: FluB; T2: FluA; T3: CoV.

[0124] This group of experiments uses a positive quality control concentration of 2 times the double window pen LOD. The effect of T line sequence setting on sensitivity is shown in Table 3:

[0125] Table 3.

[0126] Experimental Group FluA Line Readings FluB Line Readings CoV Line Readings Experimental Group One 5 3.5 5 Experimental Group Two 5 5 7 Experimental Group Three 4 5 5 Control Group 5 6 5

[0127] As can be seen from the above table, when the H1N1 and H3N2 antibodies are T3 lines, T1 and T2 lines are interfered by T3 lines, and the detection sensitivity is significantly lower than that of the double window pen product. Therefore, the detection line T3 is coated with a new crown antibody, and the detection effect is better.

[0128] Test Example 3

[0129] The effects of different first antibodies sprayed on the binding pad on the experimental results were compared, and the experimental groups were as follows:

[0130] Experimental group one: FluA and FluB latex particles sprayed on the first binding pad; CoV and GAPDH latex particles sprayed on the second binding pad;

[0131] Experimental group two: FluB and GAPDH latex particles sprayed on the first binding pad; FluA and CoV latex particles sprayed on the second binding pad;

[0132] Experimental group three: CoV and GAPDH latex particles sprayed on the first binding pad; FluA and FluB latex particles sprayed on the second binding pad;

[0133] Control group: double window pen product T1: FluB; T2: FluA; T3: CoV;

[0134] The positive quality control concentration used in this test example is 2 times the LOD of the double window pen product, and the experimental results are shown in Table 4.

[0135] Table 4. Experimental results

[0136] Experimental Group C-line Readings T1 Line Readings T2 Line Readings T3 Line Readings Experimental Group One 8 5 5 6 Experimental Group Two 8 5 5 6 Experimental Group Three 8 5 4 5 Control Group 8 5 6 5

[0137] As shown in the above table, the T2 and T3 lines of experimental group three have significantly lower sensitivity than experimental group one and experimental group two. This shows that the chromatographic distance can affect the detection sensitivity of the T line. The above experimental results show that the detection result is better when the new crown virus antibody is sprayed on the second binding pad.

[0138] Test Example 4

[0139] Detection sensitivity evaluation

[0140] The immune detection pen provided in Example 1 was evaluated using recombinant proteins, and the minimum detection limit was:

[0141] Novel coronavirus recombinant nucleocapsid protein: 50 pg / Test;

[0142] Influenza B recombinant nucleocapsid protein: 1 ng / Test;

[0143] Influenza A recombinant nucleocapsid protein: 50 pg / Test;

[0144] Among them, the positive quality control comes from Hangzhou Xukang Biological, and the new crown recombinant nucleocapsid protein is R1320; the influenza B recombinant nucleocapsid protein is R1301, and the influenza A recombinant nucleocapsid protein is Q0015.

[0145] Scheme 1: Sensitivity verification of new crown detection.

[0146] After using new crown recombinant protein to select different concentrations of pen coating, chromatography was performed, and the sensitivity verification of new crown recombinant protein detection was performed. The experimental results are shown in Table 5, and the detection sensitivity of new crown is determined to be 50 pg / Test.

[0147] Table 5.

[0148] Detection Concentration T-line Readings 0 pg / Test - 30 pg / Test - 50 pg / Test 3.5 100 pg / Test 6 200 pg / Test 8

[0149] Scheme 2: Sensitivity verification of influenza B detection.

[0150] After using influenza B recombinant protein to select different concentrations of pen coating on the sampling head, chromatography was performed, and the sensitivity verification results of influenza B recombinant protein are shown in Table 6. The detection sensitivity of influenza B is determined to be 1 ng / Test.

[0151] Table 6.

[0152] Detection Concentration T-line Readings 200 pg / Test - 500 pg / Test - 1 ng / Test 3.5 2 ng / Test 5 10 ng / Test 8

[0153] Scheme 3: Sensitivity verification of influenza A detection

[0154] Using influenza A recombinant protein, select different concentrations of coating on the sampling head, and then perform chromatography. The sensitivity verification results of influenza A recombinant protein are shown in Table 7. The detection sensitivity of influenza A is determined to be 50 pg / Test.

[0155] Table 7

[0156] Detection Concentration T-line Readings 10 ng / Test - 20 pg / Test - 50 pg / Test 3.5 100 ng / Test 5 500 ng / Test 8

[0157] Test Example 5

[0158] Virus detection experiment:

[0159] The three viral antigens from Table 8 were detected by using the detection pen of Example 1, and the detection results are shown in Table 9 and Figure 4 As can be seen from Figure 4 Table 9, the detection pen of Example 1 can detect the three viruses in Table 8, respectively.

[0160] Table 8.

[0161]

[0162] Table 9.

[0163] Sample Type Out-of-line or Not Color Intensity Out-of-line Color Influenza B Virus Yes 8 Red Influenza A Virus Yes 6 Green SARS-CoV-2 Virus Culture Yes 7 Blue

[0164] Test Example 6

[0165] Analysis of specificity-interfering substances:

[0166] The following 38 sources of interfering substances were diluted to the concentrations to be verified in Table 10, and the detection pen of Example 1 was used to verify the interfering substances, and each interfering substance was verified 3 times. The experimental results are shown in Table 11. As can be seen from Table 11, the 38 endogenous or exogenous interfering substances in Table 10 do not affect the performance of the detection pen of Example 1.

[0167] Table 10.

[0168]

[0169]

[0170] Table 11. Verification results

[0171]

[0172]

[0173] Test Example 7

[0174] Analysis of specificity-cross reaction:

[0175] The following 48 sources of pathogens were pretreated using the information in Table 12, and the detection pen of Example 1 was used to verify the interfering substances of the pathogens in Table 12, and each pathogen was verified 3 times. The verification results are shown in Table 13. The experimental results show that the 48 pathogens in Table 12 do not interfere with the detection results of the detection pen of Example 1.

[0176] Table 12

[0177]

[0178]

[0179]

[0180]

[0181]

[0182] Table 13. Verification results

[0183]

[0184]

[0185] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present application, and are not limiting; although the present application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that they can still modify the technical solutions recorded in the foregoing embodiments, or make equivalent substitutions for part or all of the technical features; and these modifications or substitutions do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present application.

Claims

1. A virus-based immunochromatographic assay strip, characterized in that, The combined virus detection includes the detection of novel coronavirus, influenza A virus and influenza B virus; the immunochromatographic test strip includes a sample pad, a conjugate pad, a detection pad and an absorbent pad arranged sequentially on a fixed base plate along the chromatographic direction of the sample to be tested. The binding pad is coated with labeled antibodies, including antibodies against influenza A virus, influenza B virus, novel coronavirus, and human internal reference protein. The first influenza A virus antibody, the first influenza B virus antibody, and the first novel coronavirus antibody are each labeled with a different colored marker; The binding pad includes a second binding pad and a first binding pad along the chromatography direction of the sample to be tested. The first binding pad is coated with a first influenza A virus antibody and a first influenza B virus antibody. The second binding pad is coated with a first novel coronavirus antibody and a first human internal reference protein antibody. The detection pad contains the detection antibodies, which include a second influenza A virus antibody, a second influenza B virus antibody, a second novel coronavirus antibody, and a second human internal reference protein antibody. The detection pad includes detection line T3, detection line T2, detection line T1, and sampling control line C along the chromatography direction of the sample to be tested; the second human internal reference protein antibody is coated on the sampling control line C; detection line T3 is coated with a second novel coronavirus antibody; detection line T2 is coated with a second influenza A virus antibody; and detection line T1 is coated with a second influenza B virus antibody. The detection antibody and the corresponding labeled antibody can bind to the same antigen simultaneously.

2. The immunochromatographic test strip for combined virus detection according to claim 1, characterized in that, The first and second influenza A virus antibodies recognize the N protein of the influenza A virus.

3. The immunochromatographic test strip for combined virus detection according to claim 1, characterized in that, The first and second type B influenza virus antibodies recognize the N protein of the influenza B virus.

4. The immunochromatographic test strip for combined virus detection according to claim 1, characterized in that, The first and second novel coronavirus antibodies recognize the novel coronavirus N protein.

5. The immunochromatographic test strip for combined virus detection according to claim 1, characterized in that, The N proteins of influenza A virus, influenza B virus, and novel coronavirus are each independently recombinant N proteins.

6. The immunochromatographic test strip for combined virus detection according to claim 1, characterized in that, The markers include at least one of quantum dots, colloidal gold, colored latex microspheres, fluorescent microspheres, cellulose microspheres, quantum dot particles, and immunomagnetic beads.

7. The immunochromatographic test strip for combined virus detection according to claim 6, characterized in that, The markers include colored latex particles.

8. The immunochromatographic test strip for combined virus detection according to claim 7, characterized in that, The first novel coronavirus antibody was labeled with blue latex particles, the first influenza A virus antibody was labeled with green latex particles, and the first influenza B virus antibody was labeled with red latex particles.

9. The immunochromatographic test strip for combined virus detection according to claim 7, characterized in that, The first person to use internal reference protein antibody to label black latex particles.

10. The immunochromatographic test strip for combined virus detection according to claim 7, characterized in that, The first novel coronavirus antibody, the first influenza A virus antibody, the first influenza B virus antibody, and the first human internal reference protein antibody were each present in a mass ratio of 1:20 to 1:80 with colored latex particles in the conjugate pad coating solution.

11. The immunochromatographic test strip for combined virus detection according to claim 10, characterized in that, The particle size of the colored latex particles is 200~400nm.

12. The immunochromatographic test strip for combined virus detection according to claim 11, characterized in that, The concentration of the colored latex granule stock solution is 4~10wt%.

13. The immunochromatographic test strip for combined virus detection according to claim 10, characterized in that, The combined spray volume of the padding liquid is 1~2 μL / cm.

14. The immunochromatographic test strip for combined virus detection according to claim 10, characterized in that, The concentrations of each labeled antibody in the coating solution were independently 2-5 mg / mL.

15. The immunochromatographic test strip for combined virus detection according to claim 1, characterized in that, Human internal reference proteins are selected from ribonuclease P, glyceraldehyde-3-phosphate dehydrogenase, β-microglobulin, β-tubulin, 18SRNA, β-actin, or TATA-binding protein.

16. The immunochromatographic test strip for combined virus detection according to claim 15, characterized in that, The human internal reference protein is selected from glyceraldehyde-3-phosphate dehydrogenase.

17. The immunochromatographic test strip for combined virus detection according to claim 1, characterized in that, The concentration of the second human internal reference protein antibody in the detection pad coating solution was 0.5~1.5 mg / mL.

18. The immunochromatographic test strip for combined virus detection according to claim 17, characterized in that, The concentration of the second human internal reference protein antibody in the detection pad coating solution was 0.5~1 mg / mL.

19. The immunochromatographic test strip for combined virus detection according to claim 18, characterized in that, The concentration of the second human internal reference protein antibody in the detection pad coating solution was 1 mg / mL.

20. The immunochromatographic test strip for combined virus detection according to claim 1, characterized in that, The concentration of influenza B virus antibody in the coating solution of the test pad was 0.3~1.5 mg / mL.

21. The immunochromatographic test strip for combined virus detection according to claim 20, characterized in that, The concentration of influenza B virus antibody in the coating solution of the test pad was 0.4–1.0 mg / mL.

22. The immunochromatographic test strip for combined virus detection according to claim 21, characterized in that, The concentration of influenza B virus antibody in the coating solution of the test pad was 0.5 mg / mL.

23. The immunochromatographic test strip for combined virus detection according to claim 1, characterized in that, The concentration of influenza A virus antibody in the coating solution of the test pad was 0.1–1.5 mg / mL.

24. The immunochromatographic test strip for combined virus detection according to claim 23, characterized in that, The concentration of influenza A virus antibody in the coating solution of the test pad was 0.5–1.2 mg / mL.

25. The immunochromatographic test strip for combined virus detection according to claim 24, characterized in that, The concentration of influenza A virus antibody in the test pad coating solution was 0.7 mg / mL.

26. The immunochromatographic test strip for combined virus detection according to claim 1, characterized in that, The concentration of the second novel coronavirus antibody in the coating solution of the test pad was 0.1~1.5 mg / mL.

27. The immunochromatographic test strip for combined virus detection according to claim 26, characterized in that, The concentration of the second novel coronavirus antibody in the coating solution of the test pad was 0.3~0.8 mg / mL.

28. The immunochromatographic test strip for combined virus detection according to claim 27, characterized in that, The concentration of the second novel coronavirus antibody in the coating solution of the test pad was 0.5 mg / mL.

29. The immunochromatographic test strip for combined virus detection according to claim 1, characterized in that, The spray volume of the coating liquid on the test pads at test lines T1, T2, T3 and the sampling control line C is 0.7~1.2 μL / cm, respectively.

30. The immunochromatographic test strip for combined virus detection according to claim 1, characterized in that, The coating solutions for the detection pads of the second human internal reference protein antibody, the second influenza B virus antibody, the second influenza A virus antibody, and the second novel coronavirus antibody each independently comprise 0.8–1.0 wt% NaCl, 0.1–0.5 wt% Na₂HPO₄, and 2–5 wt% sucrose.

31. The immunochromatographic test strip for combined virus detection according to claim 30, characterized in that, The coating solutions for the second human internal reference protein antibody, the second influenza B virus antibody, the second influenza A virus antibody, and the second novel coronavirus antibody independently consist of 0.9 wt% NaCl, 0.13 wt% Na2HPO4, and 3 wt% sucrose.

32. The immunochromatographic test strip for combined virus detection according to claim 1, characterized in that, The interval between the detection line T1 and the sampling control line C is 3.5 ± 0.5 mm.

33. The immunochromatographic test strip for combined virus detection according to claim 1, characterized in that, The interval between detection line T3 and detection line T2 is 4±0.5mm, and the interval between detection line T2 and detection line T1 is 4±0.5mm.

34. The immunochromatographic test strip for combined virus detection according to claim 1, characterized in that, The materials of the first bonding pad and the second bonding pad are independently selected from glass fiber membrane, polyester fiber membrane or non-woven fabric.

35. The immunochromatographic test strip for combined virus detection according to claim 34, characterized in that, The materials of the first bonding pad and the second bonding pad are each independently selected from polyester fiber membranes.

36. The immunochromatographic test strip for combined virus detection according to claim 1, characterized in that, The detection pad is made of nitrocellulose membrane.

37. The immunochromatographic test strip for combined virus detection according to claim 1, characterized in that, The absorbent pad is made of absorbent paper.

38. The immunochromatographic test strip for combined virus detection according to claim 1, characterized in that, The sample pad material is selected from glass fiber membrane, polyester fiber membrane or non-woven fabric.

39. The immunochromatographic test strip for combined virus detection according to claim 38, characterized in that, The sample pad is made of glass fiber membrane.

40. The immunochromatographic test strip for combined virus detection according to claim 1, characterized in that, The first bonding pad and the second bonding pad overlap by 1~3mm.

41. The immunochromatographic test strip for combined virus detection according to claim 1, characterized in that, The detection pad and the first bonding pad overlap by 1~3mm.

42. The immunochromatographic test strip for combined virus detection according to claim 1, characterized in that, The second bonding pad overlaps the sample pad by 1-3 mm.

43. The immunochromatographic test strip for combined virus detection according to claim 1, characterized in that, The absorbent pad is located on the fixed base plate, and the detection pad, the first bonding pad, the second bonding pad and the sample pad are arranged in sequence, with each part overlapping by 1~3mm.

44. A virus combined detection pen, characterized in that, Includes the virus combined detection immunochromatographic test strip and housing as described in any one of claims 1-43; the housing includes a base and a pen body; the base is pre-filled with sample lysis buffer; The pen body has a slot for fixing the immunochromatographic test strip, and a sampling part that contacts the sample pad is provided at one end of the pen body near the sample pad; the base has a buckle for fixing the pen body; after the pen body is inserted into the base, the sampling part contacts the sample lysis solution, and the pen body has a visible part for observing the test pad.

45. The virus combined detection pen according to claim 44, characterized in that, The sample lysis buffer contains 3-5 wt% NaCl, 0.2-1 wt% BSA, and 0.02-0.2 wt% sodium azide.

46. ​​The virus combined detection pen according to claim 45, characterized in that, The sample lysis buffer contained 4 wt% NaCl, 1 wt% BSA and 0.1 wt% sodium azide.

47. The virus combined detection pen according to claim 46, characterized in that, The base is pre-filled with 500~1000μL of the sample lysis solution.

48. A virus diagnostic product, characterized in that, It includes the virus combined detection immunochromatographic test strip according to any one of claims 1-43, or the virus combined detection pen according to any one of claims 44-47.

49. The virus diagnostic product according to claim 48, characterized in that, It also includes colorimetric cards.

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