CN111537727A - Test strip for combined detection of influenza and COVID-19 and application thereof - Google Patents
Test strip for combined detection of influenza and COVID-19 and application thereof Download PDFInfo
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- CN111537727A CN111537727A CN202010496609.8A CN202010496609A CN111537727A CN 111537727 A CN111537727 A CN 111537727A CN 202010496609 A CN202010496609 A CN 202010496609A CN 111537727 A CN111537727 A CN 111537727A
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Images
Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/585—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with a particulate label, e.g. coloured latex
- G01N33/587—Nanoparticles
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
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- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
- G01N2333/08—RNA viruses
- G01N2333/11—Orthomyxoviridae, e.g. influenza virus
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
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Abstract
The invention relates to a test strip for combined detection of influenza and COVID-19 and application thereof, which adopts a colloidal gold immunochromatography method and distinguishes influenza virus infection and COVID-19 virus infection through one-time detection, thereby saving the cost and improving the working efficiency, in particular to antigen detection of the three, adopts an amplification system of biotin and avidin, improves the detection sensitivity and provides important basis for clinical differential diagnosis.
Description
Technical Field
The invention belongs to the technical field of biological detection, and relates to a test strip for combined detection of influenza and COVID-19 and application thereof.
Background
New coronavirus (COVID-19) is seriously threatening the safety of life and property of mankind in china and even the world. The disease generally presents a 3-7 day latency period, and some patients have a longer latency period, and the patients have no obvious symptoms in the latency period, but have infectivity, and once the disease condition develops, the fatality rate is high, so the early screening and the rapid diagnosis are significant. At present, the accurate diagnosis can be realized only by combining nucleic acid diagnosis and CT scanning clinically, but the detection positive rate of a nucleic acid diagnosis kit is low, the requirement of nucleic acid detection on the experimental environment is higher, the requirement cannot be met by a common hospital laboratory and a basic-level health and epidemic prevention quarantine organization, the detection time is long, and the wide application of the kit is limited.
Similarly, influenza virus is an acute infectious disease, and the main pathogenic bodies of influenza virus are influenza virus type a and influenza virus type B, which cause complications in patients with underlying pathology, causing multiple pandemics in the world, and leading to death in serious cases. Because the influenza virus symptoms and the novel coronavirus symptoms have similar symptoms such as fever, cough and dyspnea clinically, pneumonia and the like, and cannot be distinguished clinically. Therefore, it is particularly important to develop a one-time test that can distinguish between influenza virus and novel coronavirus infections.
According to the current literature and patents, a kit and a technical method for combined detection of the three are not found.
Disclosure of Invention
In view of this, the present invention aims to provide a test strip for combined detection of influenza and COVID-19 and applications thereof.
In order to achieve the purpose, the invention provides the following technical scheme:
1. a test strip for joint detection of influenza virus A-type IgM antibody, influenza virus B-type IgM antibody and COVID-19IgM antibody comprises a PVC base plate, wherein a sample pad, a combination pad, a nitrocellulose membrane and a water absorption pad are sequentially attached to the PVC base plate, wherein the combination pad is sprayed with a colloidal gold-labeled anti-human IgM antibody and a colloidal gold-labeled anti-chicken IgY; the nitrocellulose membrane is sequentially coated with 3 detection lines and 1 quality control line, wherein the 3 detection lines are respectively an influenza virus A antigen, an influenza virus B antigen and a COVID-19 antigen, and the 1 quality control line is an antibody chicken IgY.
Preferably, the conjugate pad is a fiberglass membrane.
2. A kit for joint detection of influenza virus A-type IgM antibody, influenza virus B-type IgM antibody and COVID-19IgM antibody is composed of a sample diluent and a test paper card, wherein the test paper card comprises a card shell and the test paper strip, the card shell is composed of an upper cover and a lower cover, and one end of the upper cover, which is close to a sample pad, is provided with a sample adding hole.
The preparation method of the kit comprises the following specific steps:
1) preparation of sample diluent: the buffer solution adopted by the sample diluent is phosphate buffer solution PH6.0-8.0 or physiological saline, and contains 0.01-1% of surfactant Tween20 or TritonX-100.
2) Sample pad handling
The sample pad treatment solution contains 0.1-0.7% of casein sodium salt, 0.05-1% of Tween20, 2-4% of sucrose, and 0.1-0.5% of erythrocyte ascites fluid (purchased from Xiamen Bosheng Biotech Co., Ltd., for blocking erythrocytes), and contains 0.01-0.1% of mouse IgG and rabbit IgG as blocking agents, and is dissolved in a buffer solution of 20mM Tris-CL pH 8.5-9.0.
② the glass cellulose membrane is processed and soaked by a sample pad and dried for 12-24 hours at 37 ℃.
3) Preparing a colloidal gold bonding pad:
the buffer solution adopted by the combined pad processing solution is an alkaline buffer solution PH8.0-9.0, such as a borate buffer solution, a Tris-cl buffer solution and the like, and contains 0.1-2% of bovine serum albumin, 2-5% of trehalose, 0.01-1% of surfactant Tween20 or TritonX-100 and 0.1-0.5% of casein sodium salt.
② soaking the glass cellulose membrane by adopting the treatment solution of the bonding pad, and drying for 12-24 hours at 37 ℃.
Thirdly, mixing the colloidal gold-labeled conjugate according to a certain proportion (the colloidal gold-labeled anti-human IgM antibody and the colloidal gold-labeled anti-chicken IgY are mixed according to the volume ratio of 4: 1), and spraying the mixture on the treated conjugate pad.
And fourthly, drying the combined pad sprayed with the colloidal gold marker for 12 to 24 hours at 37 ℃.
4) Preparing NC membrane coating liquid: the buffer solution adopted by the sample pad treatment solution is an alkali phosphate buffer solution PH7.2-7.5, and contains 0.01-1% of bovine serum albumin and 1-5% of trehalose or sucrose.
5) Preparing an NC film: attaching the NC film on a PVC plate, and drying at 37 ℃ for 12-24 hours.
6) Cutting: and cutting the prepared sample pad, the colloidal gold combined pad and the absorbent paper, sequentially sticking the sample pad, the colloidal gold combined pad and the absorbent paper on a PVC plate with an NC film, and cutting the sample pad, the colloidal gold combined pad and the absorbent paper into test strips with the width of 3 +/-1 mm.
7) Card installation: and (4) putting the cut test paper strips into a card shell to form the test paper card.
8) Assembling: and sealing the assembled test paper card and the drying agent by using an aluminum foil bag, and storing at room temperature.
3. The test strip or the kit is applied to a combined detection reagent of an influenza virus A-type IgM antibody, an influenza virus B-type IgM antibody and a COVID-19IgM antibody.
4. The joint detection method of the influenza virus A-type IgM antibody, the influenza virus B-type IgM antibody and the COVID-19IgM antibody is realized by utilizing the kit, an indirect method is adopted, a sample to be detected is firstly dripped into a sample adding hole, a sample diluent is added to obtain a sample, the influenza virus A-type IgM antibody, the influenza virus B-type IgM antibody and the COVID-19IgM antibody in the sample are combined with an anti-human IgM antibody marked by colloidal gold on a binding pad, and are combined with an influenza virus A-type antigen, an influenza virus B-type antigen and a COVID-19 antigen on a nitrocellulose membrane under the action of chromatography, and whether the influenza virus A-type IgM antibody, the influenza virus B-type IgM antibody and the COVID-19IgM antibody are contained or not is judged according to the existence of colors.
Preferably, the sample to be tested is selected from any one of serum, plasma, whole blood or peripheral blood.
5. A test strip for combined detection of influenza virus antibodies and COVID-19 antibodies comprises a PVC base plate, wherein a sample pad, a combination pad, a nitrocellulose membrane and a water absorption pad are sequentially attached to the PVC base plate, wherein a colloidal gold labeled influenza virus fusion antigen, a COVID-19 antigen and a colloidal gold labeled anti-chicken IgY are sprayed on the combination pad; the nitrocellulose membrane is sequentially coated with 2 detection lines and 1 quality control line, wherein the 2 detection lines are respectively an influenza virus fusion antigen and a COVID-19 antigen, and the 1 quality control line is an antibody chicken IgY.
Preferably, the conjugate pad is a fiberglass membrane.
6. A kit for combined detection of influenza virus antibodies and COVID-19 antibodies comprises a sample diluent and a test paper card, wherein the test paper card comprises a card shell and the test paper strip, the card shell comprises an upper cover and a lower cover, and one end of the upper cover, which is close to a sample pad, is provided with a sample adding hole.
7. The test strip or the kit is applied to being used as a combined detection reagent of an influenza virus antibody and a COVID-19 antibody.
8. The combined detection method of the influenza virus antibody and the COVID-19 antibody is realized by utilizing the kit, a double-antigen sandwich method is adopted, a sample to be detected is firstly dripped into a sample adding hole, a sample diluent is added to obtain the sample, the influenza virus antibody and the COVID-19 antibody in the sample are combined with the influenza virus fusion antigen and the COVID-19 antigen marked by the colloidal gold on the binding pad, under the chromatography action, the influenza virus antibody and the COVID-19 antigen on the nitrocellulose membrane are combined to form a sandwich structure, and whether the influenza virus antibody and the COVID-19 antibody are contained or not is judged according to the existence of color.
Preferably, the sample to be tested is selected from any one of serum, plasma, whole blood or peripheral blood.
9. A test strip for combined detection of influenza virus A antigen, influenza virus B antigen and COVID-19 antigen comprises a PVC base plate, wherein a sample pad, a combination pad, a nitrocellulose membrane and a water absorption pad are sequentially attached to the PVC base plate, and the combination pad is sprayed with colloidal gold labeled streptavidin and colloidal gold labeled anti-chicken IgY; the nitrocellulose membrane is sequentially coated with 3 detection lines and 1 quality control line, wherein the 3 detection lines are respectively an influenza virus A monoclonal antibody, an influenza virus B monoclonal antibody and a COVID-19 monoclonal antibody, and the 1 quality control line is an antibody chicken IgY.
Preferably, the conjugate pad is a fiberglass membrane.
10. A kit for combined detection of influenza virus A antigen, influenza virus B antigen and COVID-19 antigen is composed of a sample diluent and a test paper card, wherein the test paper card comprises a card shell and the test paper strip, the card shell is composed of an upper cover and a lower cover, and one end of the upper cover, which is close to a sample pad, is provided with a sample adding hole.
The preparation method of the kit comprises the following specific steps:
1) preparation of sample diluent:
the buffer solution adopted by the sample diluent is phosphate buffer solution PH6.0-8.0, and comprises 0.01-1% of surfactant Tween20 or TritonX-100, 0.05-0.5% of casein sodium salt, 2-5% of trehalose or sucrose, 0.005-0.2% of SDS, 0.1-2% of BSA and preservative solution containing ProClin 300.
Secondly, adding a certain volume of biotinylated antibody of influenza virus A, biotinylated antibody of influenza virus B and biotinylated antibody of COVID-19 into the solution.
2) Sample pad handling
The sample pad treatment solution contains 0.05-0.3% of sodium caseinate, 0.05-1% of Tween20 and 2-4% of sucrose, and is dissolved by using a buffer solution of 20mM Tris-CL, pH 8.5-9.0.
② the glass cellulose membrane is processed and soaked by a sample pad and dried for 12-24 hours at 37 ℃.
3) Preparing a colloidal gold bonding pad:
the buffer solution adopted by the combined pad processing solution is an alkaline buffer solution PH8.0-9.0, such as a borate buffer solution, a Tris-cl buffer solution and the like, and contains 0.1-2% of bovine serum albumin, 2-5% of trehalose, 0.01-1% of surfactant Tween20 or TritonX-100 and 0.1-0.5% of casein sodium salt.
② soaking the glass cellulose membrane by adopting the treatment solution of the bonding pad, and drying for 12-24 hours at 37 ℃.
Thirdly, mixing the colloidal gold labeled conjugate according to a certain proportion (mixing the colloidal gold labeled streptavidin and the colloidal gold labeled anti-chicken IgY according to the volume ratio of 4: 1), and spraying the mixture on the treated conjugate pad.
And fourthly, drying the combined pad sprayed with the colloidal gold marker for 12 to 24 hours at 37 ℃.
4) Preparing NC membrane coating liquid: the buffer solution adopted by the sample pad treatment solution is an alkali phosphate buffer solution PH7.2-7.5, and contains 0.01-1% of bovine serum albumin and 1-5% of trehalose or sucrose.
5) Preparing an NC film: the NC film was applied to a PVC sheet, scribed in the order shown in FIG. 3, and dried at 37 ℃ for 12-24 hours.
6) Cutting: and cutting the prepared sample pad, the colloidal gold combined pad and the absorbent paper, sequentially sticking the sample pad, the colloidal gold combined pad and the absorbent paper on a PVC plate with an NC film, and cutting the sample pad, the colloidal gold combined pad and the absorbent paper into test strips with the width of 3 +/-1 mm.
7) Card installation: and (4) putting the cut test paper strips into a card shell to form the test paper card.
8) Assembling: and sealing the assembled test paper card and the drying agent by using an aluminum foil bag, and storing at room temperature.
11. The test strip or the kit is applied to being used as a combined detection reagent for influenza virus A antigen, influenza virus B antigen and COVID-19 antigen.
12. The combined detection method of the influenza virus A antigen, the influenza virus B antigen and the COVID-19 antigen is realized by utilizing the kit, a double-antibody sandwich method is adopted, a sample to be detected is uniformly mixed with a sample diluent to prepare the sample, the sample is dripped into a sample adding hole, the influenza virus A antigen, the influenza virus B antigen and the COVID-19 antigen in the sample are combined with corresponding biotinylated antibodies in the sample diluent, then the combined antibodies are combined with colloidal gold labeled streptavidin on a combining pad, and then the combined antibodies are combined with the influenza virus A monoclonal antibody, the influenza virus B monoclonal antibody and the COVID-19 monoclonal antibody on a nitrocellulose membrane under the chromatography action to form a sandwich structure, and whether the influenza virus A antigen, the influenza virus B antigen and the COVID-19 antigen are contained or not is judged according to the existence of colors.
Preferably, the sample to be tested is a nasopharyngeal swab.
The percentage of the solid is mass percentage, and the liquid is volume percentage. All antibodies and antigens related to the invention are purchased from England Biotech Limited, Shenzhen.
The invention has the beneficial effects that:
the invention adopts the colloidal gold immunochromatography, distinguishes the influenza virus infection and the COVID-19 virus infection through one-time detection, saves the cost, improves the working efficiency, particularly the antigen detection of the three, adopts an amplification system of biotin and avidin, improves the detection sensitivity, and provides an important basis for clinical differential diagnosis. The following three types of detection are specifically realized:
1. and jointly detecting the influenza virus A type IgM antibody, the influenza virus B type IgM antibody and the novel coronavirus IgM antibody.
2. The influenza virus antibody and the novel coronavirus antibody are jointly detected.
3. The combined detection of influenza A antigen, B antigen and novel coronavirus antigen.
Drawings
In order to make the object, technical scheme and beneficial effect of the invention more clear, the invention provides the following drawings for explanation:
FIG. 1 is a kit for joint detection of influenza A type IgM antibody, influenza B type IgM antibody and COVID-19IgM antibody; wherein, T1 line: influenza virus type a IgM antibody; t2 line: influenza virus type B IgM antibodies; t3 line: COVID-19IgM antibody.
FIG. 2 is a kit for the combined detection of influenza virus antibodies and COVID-19 antibodies; wherein, T1 line: an influenza virus fusion antigen; t2 line: the COVID-19 antigen.
FIG. 3 is a kit for the combined detection of influenza A antigen, influenza B antigen and COVID-19 antigen; wherein, T1 line: influenza a monoclonal antibodies; t2 line: influenza B monoclonal antibodies; t3 line: the COVID-19 monoclonal antibody.
Detailed Description
Preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings.
Example 1:
joint detection of influenza virus A-type IgM antibody, influenza virus B-type IgM antibody and COVID-19IgM antibody
Preparation of the kit:
1) preparation of sample diluent: the buffer solution adopted by the sample diluent is phosphate buffer solution PH7.2, and contains 0.1% of surfactant Tween 20.
2) Sample pad handling
The sample pad treatment solution contains 0.5% of sodium caseinate, 0.5% of Tween20, 2% of sucrose, 0.2% of erythrocyte ascites, 0.08% of mouse IgG and rabbit IgG as blocking agents, and is dissolved by a buffer solution of 20mM Tris-CL PH 8.7.
② the glass cellulose membrane is processed and soaked by a sample pad and dried for 20 hours at 37 ℃.
3) Preparing a colloidal gold bonding pad:
the buffer solution adopted by the combined pad treatment solution is an alkaline buffer solution PH8.5, such as a borate buffer solution, a Tris-cl buffer solution and the like, and comprises 1% of bovine serum albumin, 5% of trehalose, 0.5% of surfactant Tween20 and 0.3% of casein sodium salt.
② soaking the glass cellulose membrane by adopting the treatment solution of the bonding pad, and drying for 20 hours at 37 ℃.
Thirdly, mixing the colloidal gold labeled combination according to a certain proportion, and spraying the mixture on the treated combination pad.
And fourthly, drying the combined pad sprayed with the colloidal gold marker for 22 hours at 37 ℃.
4) Preparing NC membrane coating liquid: the buffer solution adopted by the sample pad treatment solution is an alkali phosphate buffer solution PH7.4, and contains 0.1% of bovine serum albumin and 3% of trehalose or sucrose.
5) Preparing an NC film: the NC film was applied to a PVC sheet, scribed in the order shown in FIG. 1, and dried at 37 ℃ for 20 hours.
6) Cutting: and cutting the prepared sample pad, the colloidal gold combined pad and the absorbent paper, sequentially sticking the sample pad, the colloidal gold combined pad and the absorbent paper on a PVC plate with an NC film, and cutting the sample pad, the colloidal gold combined pad and the absorbent paper into test strips with the width of 3 mm.
7) Card installation: and (4) putting the cut test paper strips into a card shell to form the test paper card.
8) Assembling: and sealing the assembled test paper card and the drying agent by using an aluminum foil bag, and storing at room temperature.
The detection method comprises the following steps:
1. sample type: the test samples are serum, plasma, whole blood and peripheral blood.
2. The detection method comprises the following steps:
adding 10 mu L of sample into a sample adding hole;
secondly, adding 100 mu L of sample diluent into the sample adding hole;
and thirdly, judging whether the influenza virus and the COVID-19 exist or not according to the existence of the color after reacting for 10 minutes.
And (3) performance detection:
1. and jointly detecting the influenza virus A type IgM antibody, the influenza virus B type IgM antibody and the novel coronavirus IgM antibody.
The results of clinical sample detection are shown in table 1.
TABLE 1 test results of clinical specimens in example 1
② stability detection
The kit is placed at 55 ℃ for 7 days, 14 days and 28 days for comparison, and the detection result is consistent with the above.
Third sensitivity detection
1 sample of the IgM positive influenza virus type a, 1 sample of the IgM positive influenza virus type B and 1 sample of the IgM positive COVID-19 were diluted with a sample diluent at a ratio of 1: 2. 1: 4. 1: 8. 1: 16. 1: 32. 1: 64 were diluted and the results are shown in Table 2.
TABLE 2 results of sensitivity measurement in example 1
| Sample(s) | 1：2 | 1：4 | 1：8 | 1：16 | 1：32 | 1：64 |
| Influenza type A IgM results of the reagent of the present invention | + | + | + | + | + | — |
| Commercial reagent influenza A IgM results | + | + | + | — | — | — |
| Influenza type B IgM results of the reagent of the present invention | + | + | + | + | — | — |
| Commercial reagent influenza type B IgM results | + | + | + | — | — | — |
| Reagent COVID-19IgM result of the invention | + | + | + | + | + | — |
| Commercial reagent COVID-19IgM results | + | + | + | — | — | — |
Specific detection
The detection results are negative by selecting 5 positive hepatitis B surface antigens, 5 positive hepatitis C antibodies, 5 positive syphilis antibodies, 2 samples of rheumatoid patients, 5 high cholesterol, 5 high bilirubin and 3 high hemoglobin, and are shown in Table 3. The reagent is proved to have good detection specificity.
TABLE 3 results of specific detection in example 1
According to the results, the detection sensitivity and specificity of the kit are obviously improved compared with commercial reagents, and the kit is suitable for clinical infection screening and auxiliary diagnosis.
Example 2:
combined detection of influenza virus antibodies and novel coronavirus antibodies
Referring to FIG. 2, the preparation and detection methods of the kit are the same as those of example 1.
The results of clinical sample detection are shown in Table 4.
TABLE 4 test results of clinical specimens in example 2
② stability detection
The kit is placed at 55 ℃ for 7 days, 14 days and 28 days for comparison, and the detection result is consistent with the above.
Third sensitivity detection
1 example of an influenza A virus antibody-positive sample, 1 example of an influenza B virus antibody-positive sample, 1 example of a parainfluenza virus antibody-positive sample, and 1 example of a COVID-19 antibody-positive sample were diluted with a sample diluent in a ratio of 1: 2. 1: 4. 1: 8. 1: 16. 1: 32. 1: 64 were diluted and the results are shown in Table 5.
TABLE 5 results of sensitivity measurement in example 2
| Sample(s) | 1：2 | 1：4 | 1：8 | 1：16 | 1：32 | 1：64 |
| Influenza A antibody results of the reagent of the invention | + | + | + | + | + | — |
| Commercial reagent influenza a antibody results | + | + | + | — | — | — |
| Influenza B antibody results of the inventive reagent | + | + | + | + | — | — |
| Commercial reagent influenza B antibody results | + | + | + | — | — | — |
| Reagent parainfluenza antibody results of the invention | + | + | + | + | — | — |
| Commercial reagent parainfluenza antibody results | + | + | + | — | — | — |
| Reagent of the invention, COVID-19 antibody results | + | + | + | + | + | — |
| Commercial reagent COVID-19 antibody results | + | + | + | — | — | — |
Specific detection
The detection results are negative by selecting 5 positive hepatitis B surface antigens, 5 positive hepatitis C antibodies, 5 positive syphilis antibodies, 2 samples of rheumatoid patients, 5 high cholesterol, 5 high bilirubin and 3 high hemoglobin, and are shown in Table 6. The reagent is proved to have good detection specificity.
TABLE 6 results of specific detection in example 2
According to the results, the detection sensitivity and specificity of the kit are obviously improved compared with commercial reagents, and the kit is suitable for clinical infection screening and auxiliary diagnosis.
Example 3:
combined detection of influenza virus A antigen, influenza virus B antigen and novel coronavirus antigen
1) Preparation of sample diluent:
the buffer solution adopted by the sample diluent is phosphate buffer solution PH7.2, and comprises 0.1% of surfactant Tween20, 0.2% of casein sodium salt, 4% of trehalose or sucrose, 0.1% of SDS, 1% of BSA and preservative solution containing ProClin 300.
Secondly, adding a certain volume of biotinylated antibody of influenza virus A, biotinylated antibody of influenza virus B and biotinylated antibody of COVID-19 into the solution.
2) Sample pad handling
The sample pad treatment solution contained 0.2% sodium caseinate, 0.5% Tween20, 2% sucrose, and was solubilized with 20mM Tris-CL pH8.7 buffer.
② the glass cellulose membrane is processed and soaked by a sample pad and dried for 18 hours at 37 ℃.
3) Preparing a colloidal gold bonding pad:
the buffer solution adopted by the combined pad treatment solution is an alkaline buffer solution PH8.5, such as a borate buffer solution, a Tris-cl buffer solution and the like, and comprises 1% of bovine serum albumin, 5% of trehalose, 0.1% of Tween20 and 0.2% of sodium caseinate.
② soaking the glass cellulose membrane by adopting the treatment solution of the bonding pad, and drying for 20 hours at 37 ℃.
Thirdly, mixing the colloidal gold labeled combination according to a certain proportion, and spraying the mixture on the treated combination pad.
And fourthly, drying the combined pad sprayed with the colloidal gold marker for 20 hours at 37 ℃.
4) Preparing NC membrane coating liquid: the buffer solution adopted by the sample pad treatment solution is an alkali phosphate buffer solution PH7.2, and contains 0.1% of bovine serum albumin and 3% of trehalose or sucrose.
5) Preparing an NC film: the NC film was applied to a PVC sheet, scribed in the order shown in FIG. 3, and dried at 37 ℃ for 20 hours.
6) Cutting: and cutting the prepared sample pad, the colloidal gold combined pad and the absorbent paper, sequentially sticking the sample pad, the colloidal gold combined pad and the absorbent paper on a PVC plate with an NC film, and cutting the sample pad, the colloidal gold combined pad and the absorbent paper into test strips with the width of 3 mm.
7) Card installation: and (4) putting the cut test paper strips into a card shell to form the test paper card.
8) Assembling: and sealing the assembled test paper card and the drying agent by using an aluminum foil bag, and storing at room temperature.
The detection method comprises the following steps:
1. sample type: the detection sample is nasopharyngeal swab.
2. The detection method comprises the following steps:
uniformly mixing the nasopharyngeal swab with 1000 mu L of sample diluent of 200-;
secondly, adding 100 mu L of sample diluent into the sample adding hole;
and thirdly, judging whether the influenza virus and the COVID-19 exist or not according to the existence of the color after reacting for 10 minutes.
And (3) performance detection:
the results of clinical sample detection are shown in Table 7.
TABLE 7 test results of clinical specimens in example 3
② stability detection
The kit is placed at 55 ℃ for 7 days, 14 days and 28 days for comparison, and the detection result is consistent with the above.
Third sensitivity detection
1 sample of influenza A antigen positive sample, 1 sample of influenza B antigen positive sample and 1 sample of COVID-19 antigen positive sample were diluted with a sample diluent in a ratio of 1: 2. 1: 4. 1: 8. 1: 16. 1: 32 were diluted and the results are shown in Table 8.
TABLE 8 results of sensitivity measurement in example 3
| Sample(s) | 1：2 | 1：4 | 1：8 | 1：16 | 1：32 |
| Influenza A antigen results of the reagent of the invention | + | + | + | + | — |
| Commercial reagent influenza a antigen results | + | + | — | — | — |
| Influenza B antigen results of the reagent of the invention | + | + | + | — | — |
| Commercial reagent influenza B antigen results | + | + | — | — | — |
| Reagent of the invention, COVID-19 antigen results | + | + | + | + | — |
Specific detection
The detection results are negative by selecting 5 positive hepatitis B surface antigens, 5 positive hepatitis C antibodies, 5 positive syphilis antibodies, 2 samples of rheumatoid patients, 5 high cholesterol, 5 high bilirubin and 3 high hemoglobin, and are shown in Table 9. The reagent is proved to have good detection specificity.
TABLE 9 results of specific detection in example 3
According to the results, the detection sensitivity and specificity of the influenza A type antigen and the influenza B type antigen of the kit are obviously improved compared with commercial reagents. Because COVID-19 does not have commercial antigen detection reagent at present, contrast detection is not carried out, but the test result shows good specificity and sensitivity, and the reagent is suitable for clinical infection screening and auxiliary diagnosis.
Finally, it is noted that the above-mentioned preferred embodiments illustrate rather than limit the invention, and that, although the invention has been described in detail with reference to the above-mentioned preferred embodiments, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the scope of the invention as defined by the appended claims.
Claims (9)
1. A test strip for joint detection of influenza virus A-type IgM antibody, influenza virus B-type IgM antibody and COVID-19IgM antibody comprises a PVC base plate, and a sample pad, a combination pad, a nitrocellulose membrane and a water absorption pad are sequentially attached to the PVC base plate, and is characterized in that a colloidal gold-labeled anti-human IgM antibody and a colloidal gold-labeled anti-chicken IgY are sprayed on the combination pad; the nitrocellulose membrane is sequentially coated with 3 detection lines and 1 quality control line, wherein the 3 detection lines are respectively an influenza virus A antigen, an influenza virus B antigen and a COVID-19 antigen, and the 1 quality control line is an antibody chicken IgY.
2. A kit for joint detection of influenza virus A-type IgM antibody, influenza virus B-type IgM antibody and COVID-19IgM antibody is composed of a sample diluent and a test paper card, wherein the test paper card comprises a card shell and the test paper strip of claim 1, the card shell is composed of an upper cover and a lower cover, and one end of the upper cover, which is close to a sample pad, is provided with a sample adding hole.
3. The test strip of claim 1 or the kit of claim 2, wherein the test strip is used as a combined detection reagent for influenza A IgM antibody, influenza B IgM antibody and COVID-19IgM antibody.
4. A test strip for the combined detection of influenza virus antibodies and COVID-19 antibodies comprises a PVC base plate, and a sample pad, a combination pad, a nitrocellulose membrane and a water absorption pad are sequentially stuck on the PVC base plate, and is characterized in that a colloidal gold labeled influenza virus fusion antigen, a COVID-19 antigen and a colloidal gold labeled anti-chicken IgY are sprayed on the combination pad; the nitrocellulose membrane is sequentially coated with 2 detection lines and 1 quality control line, wherein the 2 detection lines are respectively an influenza virus fusion antigen and a COVID-19 antigen, and the 1 quality control line is an antibody chicken IgY.
5. A kit for combined detection of influenza virus antibodies and COVID-19 antibodies comprises a sample diluent and a test paper card, wherein the test paper card comprises a card shell and the test paper strip of claim 4, the card shell comprises an upper cover and a lower cover, and one end of the upper cover, which is close to a sample pad, is provided with a sample adding hole.
6. The test strip or the kit is applied to being used as a combined detection reagent of an influenza virus antibody and a COVID-19 antibody.
7. A test strip for the combined detection of influenza virus A antigen, influenza virus B antigen and COVID-19 antigen comprises a PVC base plate, and a sample pad, a combination pad, a nitrocellulose membrane and a water absorption pad are sequentially stuck on the PVC base plate, and is characterized in that the combination pad is sprayed with colloidal gold labeled streptavidin and colloidal gold labeled anti-chicken IgY; the nitrocellulose membrane is sequentially coated with 3 detection lines and 1 quality control line, wherein the 3 detection lines are respectively an influenza virus A monoclonal antibody, an influenza virus B monoclonal antibody and a COVID-19 monoclonal antibody, and the 1 quality control line is an antibody chicken IgY.
8. A kit for combined detection of influenza virus A antigen, influenza virus B antigen and COVID-19 antigen comprises a sample diluent and a test paper card, wherein the test paper card comprises a card shell and the test paper strip of claim 7, the card shell comprises an upper cover and a lower cover, and one end of the upper cover, which is close to a sample pad, is provided with a sample adding hole.
9. The use of the test strip of claim 7 or the kit of claim 8 as a combined detection reagent for influenza a antigen, influenza B antigen and COVID-19 antigen.
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