CN116223796A

CN116223796A - Virus combined detection immunochromatography test paper, detection pen and detection product

Info

Publication number: CN116223796A
Application number: CN202310156113.XA
Authority: CN
Inventors: 葛珍珍; 凌世生; 王祎凡
Original assignee: Hangzhou Anxiu Biotechnology Co ltd
Current assignee: Hangzhou Anxiu Biotechnology Co ltd
Priority date: 2023-02-20
Filing date: 2023-02-20
Publication date: 2023-06-06

Abstract

The invention 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 novel coronaviruses, influenza A viruses and influenza B viruses in a three-in-one mode. The immunochromatographic test paper sample binding pad is coated with a labeled antibody, which comprises 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 the labeled antibodies are respectively labeled with markers with different colors. The immunochromatography test paper adopts a double-antibody sandwich principle, and different detection lines in a detection area are respectively coated with a second influenza A virus antibody, a second influenza B virus antibody and a second novel coronavirus antibody. The immunochromatography test paper and the detection pen solve the problems that the novel three-in-one detection product for coronavirus, influenza A virus and influenza B virus in the prior art is complex in operation and difficult to interpret.

Description

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

Technical Field

The invention relates to the technical field of immunochromatography test paper, in particular to a virus combined detection immunochromatography test paper, a detection pen and a detection product.

Background

The novel coronavirus infection (Corona Virus Disease 2019, covd-19) is an acute respiratory infectious disease caused by 2019 novel coronavirus infection. Symptoms after infection mainly include fever, dry cough and hypodynamia, and part of patients are accompanied with symptoms of upper respiratory tract such as nasal obstruction, nasal discharge and the like, which affect the daily life of human beings. Therefore, rapid detection of new coronaviruses is highly desirable.

Influenza (influenca) is abbreviated as influenza, and human influenza is an acute respiratory infectious disease caused by influenza A virus and influenza B virus. The spread features strong infectivity, high incidence rate and quick spread, and is mainly infected by respiratory tract inhalation, and the main symptoms after infection are high fever, hypodynamia, headache and the like. Influenza a viruses are prone to mutation, have caused pandemic worldwide for many times, and are public health problems valued worldwide.

Because the new coronavirus and influenza virus are similar in transmission mode, the new coronavirus can be transmitted through respiratory tract, and clinical situation is extremely similar after infection, even mixed infection of partial patients occurs, and the new coronavirus and influenza virus are difficult to distinguish by common medical means such as blood items, medical influences and the like. And the common inspection means needs professional to operate in a laboratory, has long detection period, is difficult to realize rapid and accurate diagnosis, and is not beneficial to timely treatment of patients. Therefore, a rapid detection product that distinguishes between new coronaries and influenza is of great importance. And for patients with similar symptoms, accurate and rapid diagnosis is beneficial to the patients to be effectively treated in time, so that cross infection is avoided. The existing 3 antigens are detected on one test strip at the same time, so that the number of products is relatively small.

Three-in-one rapid diagnosis test paper for new crown, first stream and second stream existing in the market is mainly a double-window detection card, repeated operation is often needed during sample adding, and pretreatment operation is complex; in addition, most of the color development instructions are colloidal gold, the color development is red under the condition of common positive, and if a user misread the T line sequence, diagnosis errors are caused; the quality control lines of the existing products are all the quality control of the test strip, and the test strip has no sampling quality control, and in the actual detection, false negative or false positive results are generated because the sampling is not in place, so that misdiagnosis is caused; most of the detection test strips of the existing kits in the market are installed in plate-type card shells, sampling heads are inserted into lysate after sampling is completed, sampling is performed after incubation, operation steps are many, and the detection kit is internally provided with many matched parts, so that risks of false negative are caused by many operation steps and no operation professionals for ordinary residents who do not contact medical equipment.

Therefore, for the normalized combined detection of novel coronaviruses, influenza A viruses and influenza B viruses, the invention is very necessary to provide a combined diagnosis detection means which is simple and convenient to operate and easy to read.

In view of this, the present invention has been proposed.

Disclosure of Invention

The invention aims to provide a virus combined detection immunochromatography test paper for realizing three-in-one immunochromatography rapid detection of novel coronaviruses, influenza A viruses and influenza B viruses, and the immunochromatography test paper is added with an antibody specifically combined with human internal reference protein as a sampling in-place quality control line, so that false negative caused by non-professional self-sampling failure is avoided greatly; the invention uses markers with different colors as indication, and the three detection lines are different in color development when positive, so that the diagnosis error caused by misjudgment of the T line position of a user is avoided. The test strip can read the result within 15 minutes, and is suitable for small-sized on-site rapid detection and personal self-test. Based on the virus combined detection immunochromatography test paper, the invention also provides a detection pen mainly composed of the virus combined detection immunochromatography test paper and a shell. The invention relieves the problems of complex operation and difficult interpretation of the novel three-in-one detection product of coronavirus, influenza A virus and influenza B virus in the prior art.

In order to solve the technical problems, the invention adopts the following technical scheme:

according to one aspect of the present invention, there is provided a virus joint detection immunochromatographic test strip, the virus joint detection comprising detecting novel coronaviruses, influenza a viruses and influenza b viruses; the immunochromatographic test paper comprises a sample pad, a combination pad, a detection pad and a water absorption pad which are sequentially arranged on a fixed bottom plate along the chromatographic direction of a sample to be tested;

The binding pad is coated with a labeled antibody labeled by a marker, wherein the labeled antibody comprises 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 markers of different colors;

the binding pad comprises a second binding pad and a first binding pad along the chromatographic direction of a sample to be tested, and the first novel coronavirus antibody is coated on the second binding pad;

the detection pad is coated with detection antibodies, and the detection antibodies comprise 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 comprises a detection line T1, a detection line T2, a detection line T3 and a sampling position quality control line C along the chromatographic direction of a sample to be detected; the second novel coronavirus antibody is coated on a detection line T3, and the second human internal reference protein antibody is coated on a sampling in-place quality control line C;

the detection antibody and the corresponding labeled antibody are capable of binding to the same antigen simultaneously.

According to another aspect of the invention, the invention further provides a virus combined detection pen, wherein the virus combined detection immunochromatography test paper and the shell; the shell comprises a base and a pen body; the base is preloaded with sample lysate;

a clamping groove for fixing immunochromatographic test paper is formed in the pen body, and a sampling part which is contacted with the sample pad is arranged at one end of the pen body, which is close to 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 contacted with the sample pyrolysis liquid, and the pen body is provided with a visible part for observing and detecting the pad.

According to another aspect of the present invention, there is also provided a virus diagnostic product comprising the above-described virus combined detection immunochromatographic test strip, or the above-described virus combined detection pen.

Compared with the prior art, the invention has the following beneficial effects:

the virus combined detection immunochromatography test paper provided by the invention can detect novel coronaviruses, influenza A viruses and influenza B viruses in a three-in-one way. The immunochromatographic test paper comprises a sample pad, a combination pad, a detection pad and a water absorption pad which are sequentially arranged on a fixed bottom plate along the chromatographic direction of a sample to be tested. The sample binding pad is coated with a labeled antibody 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. The immunochromatographic test paper provided by the invention adopts a double-antibody sandwich principle, so that different detection lines in a detection area are respectively coated with 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 which can form an antibody-antigen-antibody complex. The first influenza A virus antibody, the first influenza B virus antibody and the first novel coronavirus antibody are respectively marked with the markers with different colors, so that the positive outgoing lines of 3 detection lines are different in color, and the design avoids the occurrence of diagnosis errors caused by the fact that the detection lines representing the positive of different viruses are same in color and the judgment errors of the outgoing line positions of the detection lines are wrong when a user reads the results. The quality control line in the immunochromatographic test paper comprises a sampling in-place quality control line C which can detect the human internal reference protein. When the sample is not taken or the sample is not in place, the sample in-place quality control line C is not out of line, the sample is not in place, and the result is invalid; and when the sampling in-place quality control line C is out, the sampling in-place is indicated, the chromatography is normal, and the result has the referential property. This design avoids false negative conditions due to the user not sampling in place.

According to another aspect of the invention, the invention also provides a virus combined detection pen, which consists of the virus combined detection immunochromatographic test paper and a shell, wherein the shell comprises a base preloaded with sample lysate, and the detection pen after sampling is inserted into the base when in use, so that the sampling and loading are simple and convenient, the operation steps are few, the number of parts of the product is few, the feasibility is high, and the result is concise and clear. The detection pen provided by the invention can detect three antigens simultaneously, and is combined with sampling detection into a whole, thereby being convenient and rapid.

The immunochromatography test paper and the detection pen provided by the invention combine the detection of the novel coronavirus, the influenza A virus and the influenza B virus on one test paper strip, so that the simultaneous detection of common positive and single sample addition is realized. Meanwhile, the common positive result is considered, the colors of the outgoing lines of each detection line are distinguished by using different color markers, and the legibility of the result is improved. The invention sets the sampling in-place quality control line C to prompt the user whether the sampling is in place or not, thereby indicating whether the sample is effective or not, and avoiding the problem of invalid sampling caused by the fact that the operation method of the user is not standard and other reasons. The design of adding the sampling in-place quality control line improves the effectiveness and accuracy of the immunochromatography test paper products in taking human body samples for detection.

The immunochromatography Test paper and the detection pen provided by the invention have the advantage of high sensitivity, and the LOD value for detecting the novel coronavirus can reach 50pg/Test; the LOD value for detecting influenza A virus can reach 50pg/Test; the LOD value for detecting the influenza B virus can reach 1ng/Test; the kit also has the advantage of good specificity, and does not detect various interference pathogens; the method has the advantage of rapid detection, can judge the result by naked eyes within 15 minutes, meets the daily detection requirements of professional and non-professional staff, and has wide application range.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.

FIG. 1 is a schematic diagram of a combined virus detection immunochromatographic test strip of example 1;

FIG. 2 is a schematic diagram showing the condition of sampling the in-place quality control line C by using the combined detection immunochromatographic test paper for viruses of example 1;

FIG. 3 is a schematic diagram of the detection line outlet of the combined detection immunochromatographic test paper of example 1;

FIG. 4 is a photograph of the virus combination test pen of example 1 after virus detection.

Detailed Description

The technical solutions of the present invention will be clearly and completely described in connection with the embodiments, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

It should be noted that, herein, "novel coronavirus", "covd-19" and "CoV" each represent a novel coronavirus, and may be used interchangeably; herein, "influenza a virus", "a stream" and "FluA" all refer to influenza a virus, and may be used interchangeably; herein, "influenza b virus", "b virus" and "FluB" all refer to influenza b virus, and may be used interchangeably; herein, a method for manufacturing a semiconductor device is provided. The terms "coating", "embedding" and "spot" are used interchangeably to denote the immobilization of antibodies to immunochromatographic test strips.

According to one aspect of the present invention, there is provided a virus joint detection immunochromatographic test strip, the virus joint detection comprising detecting novel coronaviruses, influenza a viruses and influenza b viruses; the immunochromatographic test paper comprises a sample pad, a combination pad, a detection pad and a water absorption pad which are sequentially arranged on a fixed bottom plate along the chromatographic direction of a sample to be tested.

The binding pad is coated with a labeled antibody labeled with a label, the labeled antibody 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. The first influenza a virus antibody specifically binds to an influenza a virus antigen, the first influenza b virus antibody specifically binds to an influenza b virus antigen, the first novel coronavirus antibody specifically binds to a novel coronavirus antigen, and the first human internal reference protein antibody specifically binds to a human internal reference protein antigen. And, the first influenza a virus antibody, the first influenza b virus antibody and the first novel coronavirus antibody label the different colored labels, respectively.

Because the labeled antibody of the invention is labeled to show different colors of markers, and the binding pad is required to be coated with a plurality of markers, the invention uses the double-layer binding pad for chromatography, so that the surface of the detection pad can not be too stained in the chromatography process. Along the chromatographic direction of the sample to be tested, the binding pad comprises a second binding pad and a first binding pad, wherein the second binding pad is close to the sample pad, and the first binding pad is close to the detection pad. The first novel coronavirus antibody is coated on the second binding pad, and the rest of the labeled antibody is optionally coated on the first binding pad or the second binding pad. In some alternative embodiments, the first conjugate pad is coated with a first influenza a virus antibody and a first influenza b virus antibody and the second conjugate pad is coated with a first novel coronavirus antibody and a first human internal reference protein antibody.

The detection pad is coated with detection antibodies, and the detection antibodies comprise 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.

The invention adopts the double-antibody sandwich principle, and the detection antibody and the corresponding labeled antibody can be combined with the same antigen at the same time to form an antibody-antigen-antibody complex, thereby realizing the detection of virus antigen. That is, the first influenza a virus antibody and the second influenza a virus antibody can bind to the influenza a virus antigen simultaneously, the first influenza b virus antibody and the second influenza b virus antibody can bind to the influenza b virus antigen simultaneously, the first novel coronavirus antibody and the second novel coronavirus antibody can bind to the novel coronavirus antigen simultaneously, and the first human internal protein antibody and the second human internal protein antibody can bind to the human internal protein simultaneously. The detection antibody and the corresponding labeled antibody may be the same or different, and if the same antibody can bind to the same antigen at the same time, for example, to repetitive epitopes at different positions on the same antigen, the same antibody can be used as both the labeled antibody and the detection antibody.

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

Experiments prove that the detection effect of the detection lines T1 and T2 positioned at the downstream of the sample chromatographic direction can be influenced by coating the detection line T3 with the second influenza A virus antibody or the second influenza B virus antibody, so that the detection line T3 closest to the detection pad is coated with the second novel coronavirus antibody, and the second detection pad farther from the detection pad is coated with the first novel coronavirus antibody to increase the chromatographic distance in order to increase the chromatographic distance of the novel coronavirus antigen and the sensitivity. Experiments prove that the detection result of coating the second human internal reference protein antibody or the second influenza A virus antibody and the second novel coronavirus antibody on the second binding pad is better than the detection result of coating the second novel coronavirus antibody on the first binding pad.

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, either a natural antigen or a recombinant antigen, and in some alternative embodiments, the first influenza a virus antibody and the second influenza a virus antibody recognize influenza a virus N protein (nucleocapsid protein), optionally recombinant N protein.

In some alternative embodiments, the first and second influenza b antibodies optionally recognize any antigen of influenza b virus, which may be a natural antigen or a recombinant antigen, and in some alternative embodiments, the first and second influenza b antibodies recognize influenza b N proteins, optionally recombinant N proteins.

In some alternative embodiments, the first and second novel coronavirus antibodies optionally recognize any antigen of the novel coronavirus, either a natural antigen or a recombinant antigen, and in some alternative embodiments, the first and second novel coronavirus antibodies recognize novel coronavirus N proteins, optionally recombinant N proteins.

In some alternative embodiments, the target human internal reference protein that is sampled for detection by the in-situ quality control line C is selected from ribonuclease P, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), beta microglobulin, beta tubulin (beta-tubulin), 18sRNA, beta-actin (beta-actin), or TATA binding protein, preferably glyceraldehyde-3-phosphate dehydrogenase.

In some alternative embodiments, the label comprises at least one of a quantum dot, colloidal gold, colored latex microsphere, fluorescent microsphere, cellulose microsphere, quantum dot particle, and immunomagnetic bead; the different labeled antibodies may be selected from the same type of label or different types of label, so long as the antibody-virus antigen-detection antibody complex formed by the labeled antibody specifically recognizing the virus exhibits different visual signals.

In some preferred embodiments, the label is selected from the group consisting of colored latex particles, provided that each labeled antibody labels a colored latex particle of a different color, the color of the colored latex particle specifically labeled by each labeled antibody is not limited, and in some alternative embodiments, the first novel coronavirus 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 to 400nm, and may be, for example, but not limited to, 200, 250, 300, 350 or 400nm, or a range of particle sizes between any two of the above. The concentration of the color latex particle stock solution is preferably 4 to 10wt%, and may be, for example, but not limited to, 4, 5, 6, 7, 8, 9, or 10wt%.

The labeled antibody coated on the binding pad is obtained by spraying a binding pad coating liquid containing the labeled antibody on the binding pad, wherein the mass ratio of each labeled antibody in the binding pad coating liquid to the color latex particle is respectively and independently 1:20-1:80, and can be, for example, but not limited to, 1:20, 1:30, 1:40, 1:50, 1:60, 1:70 or 1:80. The concentration of labeled antibody in the conjugate pad coating solution is preferably 2 to 5mg/mL, and may be, for example, but not limited to, 2, 3, 4 or 5mg/mL; the spraying amount of the coating liquid of the bonding pad on the bonding pad is preferably 1-2 mu L/cm, and can be, for example, but not limited to, 1, 1.5 or 2 mu L/cm.

The detection line and the sampling in-place quality control line C of the detection pad are obtained by spraying a detection pad coating liquid containing detection antibodies on the detection pad.

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

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

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

In some alternative embodiments, the concentration of the second novel coronavirus antibody in the test pad coating solution is 0.1-1.5 mg/mL, preferably 0.3-0.8 mg/mL, more preferably 0.5mg/mL.

In some alternative embodiments, the detection pad coating liquid of each detection antibody contains 0.8-1.0wt% NaCl and 0.1-0.5wt% Na independently ₂ HPO ₄ And 2 to 5wt% sucrose. Preferably, detection of each detection antibodyThe pad coating liquid independently contains 0.9wt% NaCl and 0.13wt% Na ₂ HPO ₄ And 3wt% sucrose.

In some alternative embodiments, the spraying amount of the detection pad coating liquid on the detection line T1, the detection line T2, the detection line T3 and the sampling position quality control line C is respectively and independently 0.7-1.2 mu L/cm.

In some alternative embodiments, the separation of the test line T3 and the test line T2 is 3.5-4.5 mm, and the separation of the test line T2 and the test line T1 is 3.5-4.5 mm.

In some alternative embodiments, detection line T1 is 3-4 mm apart from sample-to-site quality control line C.

In some alternative embodiments, the sample pad is pre-treated with a sample pad treatment solution, preferably containing 0.8 to 1.2wt% polyvinylpyrrolidone, 0.3 to 1.0wt% S-9 (Tetronic 1307) surfactant, 0.01 to 0.1wt% sodium azide, and 1 to 3.8wt% borax.

The materials comprising the various portions of the combined test immunochromatographic strip of the present invention may be selected from materials acceptable in the art and well known. The sample pad material is selected from glass fiber film, polyester fiber film or non-woven fabric, preferably glass fiber film; the material of the first bonding pad is selected from glass fiber film, polyester fiber film or non-woven fabric, preferably polyester fiber film; the material of the second bonding pad is selected from glass fiber film, polyester fiber film or non-woven fabric, preferably polyester fiber film; the detection pad material is preferably a nitrocellulose membrane; the absorbent pad is preferably selected from absorbent paper. The first conjugate pad optionally overlaps the second conjugate pad by 1-3 mm, the detection pad optionally overlaps the first conjugate pad by 1-3 mm, and the second conjugate pad optionally overlaps the sample pad by 1-3 mm. The preferred embodiment is that the water absorption pad is positioned on the fixed bottom plate, and the detection pad, the first bonding pad, the second bonding pad and the sample pad are sequentially arranged, and each part is overlapped by 1-3 mm.

According to another aspect of the invention, the invention further provides a virus combined detection pen, which comprises an immunochromatographic test paper and a shell, wherein the shell comprises a base and a pen body. The base is preloaded with sample lysate, the pen body is internally provided with a clamping groove for fixing immunochromatographic test paper, and one end of the pen body, which is close to the sample pad, is provided with a sampling part which is contacted 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 contacted with the sample lysate. The immunochromatographic test paper can be preloaded in the shell, or is fixed in the clamping groove when in use, the sample pad of the immunochromatographic test paper after being fixed is contacted with the sampling part of the pen body, the pen body is inserted into the base after being sampled, the sampling part is contacted with the sample lysate through fastening fixation, and the immunochromatographic test paper in the pen body is used for detecting a sample to be detected through the upward chromatographic effect.

The pen body of the virus combined detection pen is provided with a visible part for observing results, and in some optional embodiments, the visible part is optionally a through hole in a region of the detection pad corresponding to the pen body, or is optionally made of transparent materials, so that the detection result is observed. In other alternative embodiments, the pen body is made of transparent material so as to observe the chromatographic result of the immunochromatographic test paper.

The sample pyrolysis liquid is preloaded in the base, the base optionally sets up the lid to realize encapsulating the sample pyrolysis liquid of preassembling, punch the card the lid when using, insert the pen body in the base, realize the contact of sample portion and sample schizolysis. The base is also optionally sealed by adopting a sealing film, and correspondingly, the pen body is provided with a protruding end, when the pen body is inserted into the base in use, the integrity of the sealing film of the base is damaged along with the protruding end, so that the sampling part is inserted into the base and fully contacted with sample pyrolysis liquid, and the pyrolysis and immunochromatography detection of a sample to be detected, which is taken by the sampling part, are realized.

In some alternative embodiments, the test pen structure refers to the device described in CN114414306a or CN114869277 a. The combined detection immunochromatographic test paper for viruses provided by the invention is optionally a detection element in a collection device described in CN114414306A or is optionally a detection element in a detection device described in CN 114869277A.

In some alternative embodiments, the housing is a test pen as described in CN 307182555S.

In some alternative embodiments, the sample lysate includes 3-5 wt% NaCl, 0.2-1 wt% BSA, and 0.02-0.2 wt% sodium azide.

In some alternative embodiments, the virus joint detection pen is used as follows:

s1, loading virus combined detection immunochromatography test paper into a detection cavity of a pen-shaped shell with the publication number of CN 307182555S;

s2, inserting a pen tube tip sponge sampling head into a nasal cavity at a position of 2-3 cm, and rotating for about 5 circles by sticking to a nasal mucosa;

s3, vertically inserting the sponge head after sampling into a base filled with sample lysate for lysis chromatography;

s4, after 15min, sampling the test strip to the quality control line C and judging the detection result according to the line outlet color of each detection line.

According to another aspect of the present invention, there is also provided a virus diagnostic product comprising the above virus combined detection immunochromatographic test strip, or the above virus combined detection pen. The virus diagnostic product may optionally further comprise conventional reagents or consumables for detecting viruses, for example, when the detection element of the virus diagnostic product is a virus combined detection immunochromatographic strip as described above, and may further optionally comprise one or more of a swab, a sample lysate, a desiccant and a sample-placing container; when the virus diagnosis product uses the virus combined detection immunochromatography test paper or the virus combined detection pen as a main detection element, a colorimetric card can be further included to perform qualitative or semi-quantitative determination on the result.

The technical solution and advantageous effects of the present invention are further described below in connection with preferred embodiments.

Example 1

The embodiment provides a virus joint detection pen, which consists of virus joint detection immunochromatography test paper and a shell. The immunochromatographic test paper is schematically shown in FIG. 1, wherein 1 is a water absorption pad in FIG. 1; 2 is a sampling in-place 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 bond pad; 7 is a second bond pad; 8 is a sample pad; 9 is a fixed bottom plate; 10 is a test pad.

The virus combined detection immunochromatographic test paper comprises a sample pad 8, a second binding pad 7, a first binding pad 6, a detection pad 10 and a water absorption pad 1 which are sequentially arranged on a fixed bottom plate 9 along the chromatographic direction of a sample to be detected; one end of the first bonding pad 6 is lapped with the detection pad 10, the other end is lapped with the second bonding pad 7, and the other end of the second bonding pad 7 is lapped with the sample pad 8, and the specific structure is as follows: the water absorption pad 1 is positioned on the fixed bottom plate 9, and the detection pad 10, the first bonding pad 6, the second bonding pad 7 and the sample pad 8 are sequentially arranged, and each part is overlapped by 1-3 mm. The sample pad 8 is made of glass fiber, the first bonding pad 6 and the second bonding pad 7 are made of polyester fiber, the detection pad 10 is made of nitrocellulose membrane, and the water absorbing pad 1 is made of water absorbing paper.

The first binding pad 6 is coated with a first influenza A virus recombinant N protein antibody marked by green latex particles and a first influenza B virus recombinant N protein antibody marked by red latex particles. The second binding pad 7 is coated with a first novel coronavirus recombinant N protein antibody marked by blue latex particles and a GAPDH antibody marked by black latex particles. The spraying amount of the binding pad coating liquid is 1-2 mu L/cm.

The method for marking the multicolor latex particles in this example is as follows:

firstly, washing latex microspheres by using MES solution with the pH value of 6.0 and the concentration of 100mM, centrifuging, discarding supernatant, activating the latex microspheres by using 750 mug/mL NHS and EDC solution, incubating for 1 hour at normal temperature, centrifuging, discarding supernatant, adding the MES solution, and adding the MES solution according to the feeding ratio (mass ratio) of the antibody to the latex particles of 1:20 adding antibody and latex particles, and making antibody concentration be 2-5 mg/mL, incubating overnight, then adding 30 mu L of ethanolamine, after half an hour of incubation, centrifuging to remove supernatant, adding a blocking liquid to make normal temperature blocking for 4 hours, wherein the blocking liquid contains 0.02mg/mL NaN ₃ 5mg/mL of PvP, 50mM/mL of Tris, 5mg/mL of T-Casein, 200mg/mL of sucrose and 50mg/mL of trehalose, centrifuging after incubation, discarding the supernatant, continuously adding 600 μl of blocking solution, and measuring the concentration for later use after ultrasound.

The detection pad 10 is sequentially provided with a detection line T3 5, a detection line T2 4, a detection line T1 3 and a sampling in-place quality control line C2 along the chromatographic direction of a sample to be detected. The detection line T1 3 is coated with a second influenza B virus recombinant N protein antibody, the detection line T2 4 is coated with a second influenza A virus recombinant N protein antibody, and the detection line T3 5 is coated with a second novel coronavirus recombinant N protein antibody. The sample was taken to the site and the control line C2 was coated with human GAPDH antibody.

The first influenza a virus recombinant N protein antibody is from the organism of the genus hangzhou, with the accession number R1403; the second influenza a virus recombinant N protein antibody is from the organism of the genus chuanxiiaceae, cat No. R1401; the first influenza b virus recombinant N protein antibody is from the singultus of the hangzhou family, cat No. R1404; the second influenza b virus recombinant N protein antibody is from the singultus of the hangzhou family, accession No. R1402; the first novel coronavirus recombinant N protein antibody is from Xuezu Hangzhou, product number RM0032; the second novel coronavirus recombinant N protein antibody was from Xuezu Hangzhou, cat# RM0033. Both the GAPDH antibody coated on the binding pad and the human GAPDH antibody coated on the sample in-place control line C2 were GAPDH antibodies (cat# ET 1601-4) from Hangzhou Hua' an Biotechnology Co., ltd.

The concentration of the antibody in the detection pad coating liquid containing the second influenza B virus recombinant N protein antibody of the spray point detection line T1 3 is 0.5mg/mL; the concentration of the antibody in the detection pad coating liquid containing the second influenza A virus recombinant N protein antibody of the spray point detection line T2 4 is 0.7mg/mL; the concentration of the antibody in the detection pad coating liquid containing the second novel coronavirus recombinant N protein antibody of the spray point detection line T3 5 is 0.5mg/mL; the concentration of the antibody in the detection pad coating liquid containing the GAPDH antibody of the spray point sampling in-place quality control line is 1.0mg/mL. The detection pad coating liquid contains 9wt% NaCl and 0.2wt% NaH except the antibody ₂ PO ₄ 3wt% sucrose and 5wt% BSA. The amount of the spray points of the detection line and the sampling-in-place quality control line is 0.7-1.2 mu L/cm. When the spray point detection line and the sampling in-place quality control line C2 are adopted, the interval between the detection line T3 5 and the detection line T2 4 is 4mm, the interval between the detection line T2 4 and the detection line T1 3 is 4mm, and the interval between the detection line T1 3 and the sampling in-place quality control line C2 is 3.5mm.

Sample pad 8 was pretreated with a sample pad treatment solution containing 1.0wt% polyvinylpyrrolidone, 0.5wt% s-9 (Tetronic 1307) surfactant, 0.02wt% sodium azide, and 2wt% borax.

Conjugate pad after spotting antibody and detection pad 10, sample pad 8 after pretreatment is assembled: the water absorption pad 1, the detection pad 10, the first bonding pad 6, the second bonding pad 7 and the sample pad 8 are sequentially adhered to the fixed bottom plate 9, and cut by a slitter to obtain an immunochromatography test part.

The shell of the virus combined detection pen in this embodiment is the detection pen described in CN307182555S, and the shell includes a base and a pen body. The base is preloaded with sample lysate, the pen body is internally provided with a clamping groove for fixing immunochromatographic test paper, one end of the pen body, which is close to the sample pad 8, is provided with a sampling part which is in contact with 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 contacted with the sample lysate. The immunochromatographic test paper is preloaded in the shell, a sample pad of the fixed immunochromatographic test paper is contacted with a sampling part of the pen body, the pen body is inserted into the base after sampling, the sponge sampling head is fixed through a buckle and contacted with sample lysate, and the immunochromatographic test paper in the pen body is used for detecting a sample to be detected through an upward chromatographic effect.

The application method of the virus joint detection pen provided by the embodiment comprises the following steps:

1. collecting a sample:

the sample type is a human nasal swab, and the collection method comprises the following steps:

(1) The sponge sampling head of the pen point of the detection pen is plugged into the nasal cavity at a position of 2-3 cm, the sponge head is scraped clockwise for 5 circles in the nasal cavity by clinging to the nasal mucosa, and the sponge head is taken out after scraping is finished, and the same operation is carried out in the nasal cavity at the other side. When sampling, proper force is applied to ensure that the sample in the nasal mucosa of the human body is taken.

(2) At the time of drug validation in the laboratory, or serum sampling, 50 μl of serum sample was uniformly spread on the sponge sampling head using a pipette.

(3) And (3) in batch sampling, putting a plastic pen cap matched with the sponge head cover after sampling into a sample bag, sealing and marking.

2. Detecting a sample:

(1) When screening the suspected infectious agents, the inspector needs to wear epidemic prevention clothing and protective equipment such as medical masks, medical surgical gloves and the like according to the protective measures for inspecting infectious diseases. The resident self-test and other small-sized on-site direct sampling and detection can be carried out.

(2) The sampled sampling head was inserted into a base containing 950. Mu.l of sample lysate and allowed to stand for 15 minutes for chromatography. The base should be placed vertically on the desktop, and the sample head should be based on hearing the "click" snap-fit sound when inserting the base that is equipped with sample lysate. When in chromatography, the base and the detection pen are vertical to the desktop or the detection table for upward chromatography, and the pen body and the base are prevented from shaking or being vertical to the detection table as much as possible. And observing whether the sample is subjected to normal chromatography, and if the sample is not subjected to normal chromatography, re-inserting the sample head.

(3) After 15min, the outgoing line condition was observed. After the test is finished, the used detection pen and the base are treated according to biomedical waste.

3. Interpretation of the results:

the C-line quality control area detection line is set to black, and in order to improve the look and feel and result legibility, 3T lines in this embodiment are set to different colors, bright red is set based on the existing material detection line T1 3, the detection line T2 4 is set to green, and the detection line T3 5 is set to blue. Schematic diagrams of the outgoing line situation are shown in fig. 2 and 3.

The schematic diagram of the appearance of the sampling in-place quality control line C2 is shown in FIG. 2, and the left diagram in FIG. 2 is the outgoing line of the sampling in-place quality control line C2, which illustrates sampling in place; the right graph shows that the sampling in-place quality control line C2 is not out of line, indicating that the sampling is not in place.

In fig. 3, a represents co-positivity of a new coronavirus, an influenza a virus and an influenza b virus; b represents that only influenza B virus is positive, and the rest are negative; c represents that only the new coronavirus is positive, and the rest are negative; d represents that only influenza A virus is positive, and the rest are negative; e represents that the novel coronavirus and the influenza B virus are positive and the influenza A virus is negative; f represents influenza a virus and influenza b virus positive, new coronavirus negative; g represents that the novel coronavirus and the influenza A virus are positive and the influenza B virus is negative; h indicates that all three viruses were negative and sampled in place. Negative indicates that the sample does not contain the corresponding virus, or that the virus content is below the detection threshold.

In the following test examples, 3 or more parallel samples were used for the test, and the color development value or average value of 95% or more parallel samples was obtained. The color development values of the detection line and the quality control line are referenced from a color comparison card, color blocks with light color development and dark color development are printed on the color comparison card, and the color development of the color blocks carries out measurement reflectivity correction according to the national metering technical specification JJF1232-2009 every year. The color chart has a color rendering value ranging from 1 to 10, with higher numbers indicating darker color rendering. Wherein chromaticity of 3.5 or above is judged as a visible outgoing line. The occurrence depth of the T line in the subsequent table is expressed in the read value mode. Therefore, the macroscopic line is judged as whether the color development chromaticity of the T line reaches the color card reading value of 3.5, wherein "-" indicates negative. Unless otherwise specified in the following test examples, the immunochromatographic test strip and the test pen were prepared in the same manner as in example 1.

It should be noted that the parameters of the dual window pen of the applicant's existing product new coronal influenza are used in the conjugate pad area and the detection line area in the present invention. I.e., the spray point concentration of CoV latex particles in the conjugate pad was 0.25%; the latex particle spot concentration of FluA was 0.3%; the latex particle spot concentration of FluB was 0.3%; the spray point speeds were all 2. Mu.L/cm. In the detection zone of NC membrane, the coating concentration of CoV is 0.5mg/mL; the coating concentration of FluA was 0.7mg/mL; the coating concentration of FluB is 0.5mg/mL; the coating speed was 1. Mu.L/cm.

Test example 1

Three human internal proteins were compared as targets for sampling into place quality control line C: in the test example, three reference protein antibodies, namely a beta-actin antibody, a beta-tubulin antibody and a GAPDH antibody, are adopted as alternative sampling target quality control line C materials, 35 human nasal cavity samples are used for screening, and the design experimental scheme is as follows:

scheme one:

three different sets of reference protein antibodies were used as targets for sampling into the site quality control line C, the experimental groupings were as follows:

experiment group one: coating a quality control line: beta-actin antibody 1; labeling the antibody: beta-actin antibody 1.

Experimental group two: coating a quality control line: beta-tubulin antibody 1; labeling the antibody: beta-tubulin antibody 1.

Experimental group three: coating a quality control line: GAPDH antibody 1; labeling the antibody: GAPDH antibody 1.

TABLE 1 sample in place quality control line antibody comparison results

Experimental group	Number of C-line outgoing test cases	Number of C-line non-outgoing test cases	Totals to
				Experiment group one	16	19	35
Experiment group II	14	21	35
				Experiment group III	29	6	35
PBS controlGroup of	0	10	10

As can be seen from table 1, the test cases of the C-line outgoing line of the third test group are significantly higher than those of the first test group and the second test group, which indicates that the GAPDH is used as the target of the sampling-in-place quality control line C, and the GAPDH antibodies are coated on the binding pad and the detection pad, which is superior to the targets of the sampling-in-place quality control line C using β -actin and β -tubulin.

Scheme II:

10 human nasal samples were used to verify using two GAPDH antibodies, from han hua bio-technology limited, GAPDH antibody 1, as detection line coated and labeled antibodies, respectively: ET1601-4; GAPDH antibody 2 accession number: m1310-2. The results are shown in Table 2:

experiment group one: and (3) coating a detection line: GAPDH antibody 1; labeling the antibody: GAPDH antibody 1.

Experimental group two: and (3) coating a detection line: GAPDH antibody 1; labeling the antibody: GAPDH antibody 2.

Experimental group three: and (3) coating a detection line: GAPDH antibody 2; labeling the antibody: GAPDH antibody 2.

Experimental group four: and (3) coating a detection line: GAPDH antibody 2; labeling the antibody: GAPDH antibody 1.

Table 2.

Experimental group	C-line outgoing line reading value
		Experiment group one	7
Experiment group II	3.5
		Experiment group III	4
Experiment group four	6
		PBS control group	-

According to the experimental results of table 2, the sampling in-place design and the real sample consistency ratio of the novel combined detection immunochromatography test paper for detecting the coronal virus, the first stream virus and the second stream virus of the in-place quality control line C are 100%, and the results prove that the quality control is real and effective. Thus, both the sample to site control line coating and the labeled antibody of the present invention are preferably GAPDH antibody 1. The amino acid sequence of the antigen recognized by the GAPDH antibody 1 is mouse GAPDH aa 94-333/333, and presumably the repeated site recognized by the monoclonal antibody exists in the sequence, so that the GAPDH antibody 1 can be used as both the coating of the position control line and the labeled antibody, and an antibody-antigen-antibody complex can be formed.

Test example 2

Detection line color and concentration exploration:

scheme one:

different antibodies are detected on the T1, T2 and T3 pad spray points by adopting the existing parameters in the test example, and the experiment is divided into the following groups:

experiment group one: t1: fluA; t2: coV; t3: fluB;

experimental group two: t1: fluB; t2: fluA; t3: coV;

experimental group three: t1: coV; t2: fluB; t3: fluA;

control group double window pen: t1: fluB; t2: fluA; t3: coV.

This set of experiments used a positive quality control concentration of 2-fold double window pen LOD. The effect of the T-line sequence setting on sensitivity is shown in table 3:

table 3.

Experimental group	FluA line reads	FluB line read values	CoV line read value
				Experiment group one	5	3.5	5
Experiment group II	5	5	7
				Experiment group III	4	5	5
Control group	5	6	5

As can be seen from the comparison of the table, when the antibody of the first stream and the second stream is a T3 line, the T1 line and the T2 line are interfered by the T3 line, and the detection sensitivity is obviously reduced compared with a double window pen product, so that the detection line T3 is coated with the novel crown antibody, and the detection effect is better.

Test example 3

The effect of different primary antibodies on the assay results was compared with the binding pad spray points, and the assay groupings were as follows:

experiment group one: first conjugate pad spray points FluA and FluB latex particles; second bond pad spray points CoV and GAPDH latex particles;

experimental group two: first conjugate pad spray points FluB and GAPDH latex particles; second conjugate pad spray points FluA and CoV latex particles;

Experimental group three: first bond pad spray points CoV and GAPDH latex particles; second conjugate pad spray points FluA and FluB latex particles;

control group: double window pen product T1: fluB; t2: fluA; t3: coV;

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

TABLE 4 experimental results

Experimental group	C line reading	T1 line read value	T2 line read value	T3 line read value
					Experiment group one	8	5	5	6
Experiment group II	8	5	5	6
					Experiment group III	8	5	4	5
Control group	8	5	6	5

As shown in the table above, the T2 and T3 line sensitivities of experimental group three were significantly lower than those of experimental group one and experimental group two. It is explained that the chromatographic distance affects the detection sensitivity of the T line. The above experimental results demonstrate that the detection results of the novel coronavirus antibodies are better when spotted on the second conjugate pad.

Test example 4

Evaluation of detection sensitivity

The immunodetection pen provided in example 1 was evaluated using recombinant protein, resulting in a minimum detection limit of:

novel coronavirus recombinant nucleocapsid protein: 50pg/Test;

influenza b recombinant nucleocapsid protein: 1ng/Test;

influenza a recombinant nucleocapsid protein: 50pg/Test;

wherein the positive quality control is derived from Xuezuke, hangzhou, wherein the new crown recombinant nucleocapsid protein accession number is R1320; the influenza B recombinant nucleocapsid protein has the code of R1301 and the influenza A recombinant nucleocapsid protein has the code of Q0015.

Scheme one: and (5) verifying the detection sensitivity of the new crown.

And (3) performing chromatography after using the recombinant protein of the new crown to select different concentrations for painting, verifying the detection sensitivity of the recombinant protein of the new crown, wherein the experimental result is shown in table 5, and determining that the detection sensitivity of the new crown is 50pg/Test.

Table 5.

Detecting concentration	T line reading
		0pg/Test	-
30pg/Test	-
		50pg/Test	3.5
100pg/Test	6
		200pg/Test	8

Scheme II: and (5) verifying the influenza B detection sensitivity.

And (3) selecting different concentrations of the recombinant protein of the influenza B, coating the recombinant protein of the influenza B on a pen point sampling head, and then carrying out chromatography, wherein the sensitivity verification result of the recombinant protein of the influenza B is shown in a table 6, and the detection sensitivity of the influenza B is determined to be 1ng/Test.

Table 6.

Detecting concentration	T line reading
		200pg/Test	-
500pg/Test	-
		1ng/Test	3.5
2ng/Test	5
		10ng/Test	8

Scheme III: influenza a detection sensitivity verification

The influenza A recombinant protein is used, the continuous concentration is selected and coated on a pen point sampling head for chromatography, the sensitivity verification result of the influenza A recombinant protein is shown in table 7, and the detection sensitivity of the influenza A is determined to be 50pg/Test.

TABLE 7

Detecting concentration	T line reading
		10ng/Test	-
20pg/Test	-
		50pg/Test	3.5
100ng/Test	5
		500ng/Test	8

Test example 5

Virus detection experiment:

the test pen of example 1 was used to load 3 virus antigens derived from table 8, and the test results are shown in table 9 and fig. 4. It can be seen from fig. 4 that the test pen of example 1 can detect three viruses of table 8.

Table 8.

Table 9.

Sample type	Whether or not to be led out	Color development intensity	Color of outgoing line
				Influenza b virus	Is that	8	Red color
Influenza A virus	Is that	6	Green colour
				SARS-CoV-2 virus culture	Is that	7	Blue color

Test example 6

Analytical specificity-evaluation of interfering substances:

the following 38 kinds of interfering substances were diluted to the concentrations to be verified in table 10, and the interfering substances in table 10 were used to verify the interfering substances in the test pen of example 1, the number of verification times of each interfering substance was 3, and the experimental results are shown in table 11. It can be seen from table 11 that the 38 kinds of endogenous or exogenous interfering substances in table 10 do not affect the performance of the test pen of example 1.

Table 10.

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TABLE 11 verification results

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Test example 7

Analytical specificity-cross-reaction assessment:

the following 48 pathogens were pre-treated with the information of table 12, and the test pens provided in example 1 were tested for interfering substances using the pathogens of table 12, the test pens were tested 3 times for each pathogen, and the test results are shown in table 13, and the test results indicate that the 48 pathogens in table 12 do not interfere with the test results of the test pens of example 1.

Table 12

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TABLE 13 verification results

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Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.

Claims

1. The combined detection immunochromatographic test paper for viruses is characterized in that the combined detection for viruses comprises detection of novel coronaviruses, influenza A viruses and influenza B viruses; the immunochromatographic test paper comprises a sample pad, a combination pad, a detection pad and a water absorption pad which are sequentially arranged on a fixed bottom plate along the chromatographic direction of a sample to be tested;

2. The virus joint detection immunochromatographic test strip according to claim 1, wherein 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;

preferably, the detection line T3 is coated with a second novel coronavirus antibody; the detection line T2 is coated with a second influenza A virus antibody; and the detection line T1 is coated with a second influenza B virus antibody.

3. The virus joint detection immunochromatographic test strip of claim 1, wherein the first influenza a virus antibody and the second influenza a virus antibody recognize influenza a virus N protein;

preferably, the first influenza b antibody and the second influenza b antibody recognize influenza b N protein;

preferably, the first and second novel coronavirus antibodies recognize novel coronavirus N proteins;

preferably, the N proteins of influenza a virus, influenza b virus and novel coronavirus are each independently recombinant N proteins.

4. The virus joint detection immunochromatographic test strip according to claim 1, wherein the marker comprises at least one of a quantum dot, a colloidal gold, a color latex microsphere, a fluorescent microsphere, a cellulose microsphere, a quantum dot particle and an immunomagnetic bead;

preferably, the marker comprises coloured latex particles;

preferably, the first novel coronavirus antibody is labeled with blue latex particles, the first influenza a virus antibody is labeled with green latex particles, and the first influenza b virus antibody is labeled with red latex particles;

preferably, the first human internal reference protein antibody marks a black latex particle;

preferably, the mass ratio of the first novel coronavirus antibody standard, the first influenza A virus antibody, the first influenza B virus antibody and the first human internal reference protein antibody to the color latex particles in the binding pad coating liquid is 1:20-1:80;

preferably, the particle size of the colored latex particles is 200-400 nm;

preferably, the concentration of the color latex particle stock solution is 4-10wt%;

preferably, the spraying amount of the bonding pad coating liquid is 1-2 mu L/cm;

preferably, the concentration of each labeled antibody in the binding pad coating solution is 2-5 mg/mL independently.

5. The virus joint detection immunochromatographic test strip according to claim 1, wherein the human internal reference protein is selected from ribonuclease P, glyceraldehyde-3-phosphate dehydrogenase, beta microglobulin, beta tubulin, 18sRNA, beta-actin or TATA binding protein;

preferably, the reference protein is selected from glyceraldehyde-3-phosphate dehydrogenase.

6. The virus combined detection immunochromatographic test strip according to claim 1, characterized in that the concentration of the second human internal reference protein antibody in the detection pad coating liquid is 0.5 to 1.5mg/mL, preferably 0.5 to 1mg/mL, more preferably 1mg/mL;

preferably, the concentration of the second influenza B virus antibody in the detection pad coating liquid is 0.3-1.5 mg/mL, preferably 0.4-1.0 mg/mL, more preferably 0.5mg/mL;

preferably, the concentration of the second influenza A virus antibody in the detection pad coating liquid is 0.1-1.5 mg/mL, preferably 0.5-1.2 mg/mL, more preferably 0.7mg/mL;

preferably, the concentration of the second novel coronavirus antibody in the detection pad coating solution is 0.1-1.5 mg/mL, preferably 0.3-0.8 mg/mL, more preferably 0.5mg/mL;

preferably, the spraying amount of the detection pad coating liquid on the detection line T1, the detection line T2, the detection line T3 and the sampling in-place quality control line C is respectively and independently 0.7-1.2 mu L/cm;

Preferably, the detection pad coating liquid 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 respectively and independently comprises 0.8 to 1.0 weight percent of NaCl and 0.1 to 0.5 weight percent of Na ₂ HPO ₄ And 2 to 5wt% sucrose;

preferably, the detection pad coating liquid 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 respectively and independently comprises 0.9wt% NaCl and 0.13wt% Na ₂ HPO ₄ And 3wt% sucrose;

preferably, the interval between the detection line T1 and the sampling in-place quality control line C is 3.5+/-0.5 mm;

preferably, the interval between the detection line T3 and the detection line T2 is 4+/-0.5 mm, and the interval between the detection line T2 and the detection line T1 is 4+/-0.5 mm.

7. The virus joint detection immunochromatographic test strip according to claim 1, wherein the material of the first binding pad and the material of the second binding pad are respectively and independently selected from a glass fiber film, a polyester fiber film or a non-woven fabric; more preferably from a polyester fiber film;

preferably, the detection pad material is selected from nitrocellulose membranes;

preferably, the absorbent pad material is selected from absorbent paper;

Preferably, the sample pad material is selected from glass fiber film, polyester fiber film or non-woven fabric; more preferably a glass fiber film;

preferably, the first bonding pad and the second bonding pad are overlapped in a staggered way by 1-3 mm;

preferably, the detection pad and the first bonding pad are overlapped in a staggered way by 1-3 mm;

preferably, the second bonding pad and the sample pad are overlapped in a staggered way by 1-3 mm;

preferably, the water absorption pad is positioned on the fixed bottom plate, and the detection pad, the first bonding pad, the second bonding pad and the sample pad are sequentially arranged, and each part is overlapped by 1-3 mm.

8. A virus combined detection pen, which is characterized by comprising the virus combined detection immunochromatographic test paper and a shell according to any one of claims 1 to 7; the shell comprises a base and a pen body; the base is preloaded with sample lysate;

9. The virus combination detection pen according to claim 8, wherein the sample lysate comprises 3-5 wt% nacl, 0.2-1 wt% bsa, and 0.02-0.2 wt% sodium azide;

preferably, the sample lysate comprises 4wt% nacl, 1wt% bsa, and 0.1wt% sodium azide;

preferably, 500-1000 mu L of the sample lysate is preloaded in the base.

10. A virus diagnostic product comprising the virus joint detection immunochromatographic strip of any one of claims 1 to 7, or the virus joint detection pen of claim 8 or 9;

preferably, a color chart is also included.