CN111562367A - Novel urine coronavirus rapid detection kit, preparation method and use method - Google Patents
Novel urine coronavirus rapid detection kit, preparation method and use method Download PDFInfo
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- CN111562367A CN111562367A CN202010540444.XA CN202010540444A CN111562367A CN 111562367 A CN111562367 A CN 111562367A CN 202010540444 A CN202010540444 A CN 202010540444A CN 111562367 A CN111562367 A CN 111562367A
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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
- G01N33/53—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
- 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
- G01N33/53—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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- 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
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- G—PHYSICS
- G01—MEASURING; TESTING
- 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
- G01N2333/165—Coronaviridae, e.g. avian infectious bronchitis virus
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2469/00—Immunoassays for the detection of microorganisms
- G01N2469/20—Detection of antibodies in sample from host which are directed against antigens from microorganisms
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Abstract
The invention discloses a novel urine coronavirus rapid detection kit, a preparation method and a use method, which comprises a detection test strip, a centrifuge tube filled with a concentrated medium and a sampling cup, wherein the detection test strip consists of a sample pad, a combination pad, a nitrocellulose membrane coating the detection line and a quality control line and absorbent paper which are fixed on a PVC plate, and the combination pad of the test strip is coated with a gold-labeled SARS-CoV-2 recombinant N antigen, SARS-CoV-2 recombinant S antigen and SA. Is used for detecting anti-SARS-CoV-2 specific IgG antibody in human urine. Collecting a urine sample by using a sampling cup, adding the urine sample into a centrifugal tube filled with a concentrated medium, and inserting the sample pad end of a test strip into the medium for detecting whether the urine sample contains a novel coronavirus (SARS-CoV-2) IgG antibody. The results can be obtained in 5-10 minutes, the application includes hospitals, epidemic prevention stations, entry and exit quarantine, on-site preliminary screening diagnosis and self-test, no wound, high speed, convenience and safety are realized, and the detection sensitivity can be obviously improved by a urine concentration technology.
Description
Technical Field
The invention relates to the technical field of novel rapid detection kits for coronaviruses, in particular to a novel rapid detection kit for coronaviruses in urine, a preparation method and a use method.
Background
The new coronavirus (SARS-CoV-2) outbreak in 12 months in 2019 has seen the pneumonia epidemic caused by the infection. Causing trillions of economic loss, seriously affecting the normal life of human beings and harming the health of human beings. The disease is taken as an acute respiratory infectious disease and is included in a B infectious disease specified in infectious disease prevention and treatment Law of the people's republic of China, and is managed according to the A infectious disease. SARS-CoV-2 belongs to coronavirus of beta genus, which has envelope, particles are round or oval, and are usually multiple in nature, and the diameter is 60-140 nm, the diagnosis of the present novel coronavirus mainly depends on molecular biology detection technology, and the detection of new coronavirus nucleic acid positive or the high homology of virus gene sequencing result and the known novel coronavirus is used as the confirmed diagnosis case.
With the gradual deepening of the knowledge of the novel coronavirus pneumonia diseases and the accumulation of prevention and control experience, researchers also develop SARS-CoV-2IgM and IgG detection kits by various methods, and the great effect is played in screening potential virus positive infectors and isolating infected people as soon as possible to block virus propagation. Therefore, it is important to develop a noninvasive, rapid, simple and convenient SARS-CoV-2 detection method, technique and product. The invention is based on a colloidal gold immunochromatographic technology platform and is used for quickly detecting the content of a novel coronavirus (SARS-CoV-2) IgG antibody in urine in vitro to preliminarily determine whether to infect SARS-CoV-2.
The content of the novel coronavirus (SARS-CoV-2) IgG antibody in the urine is about 0.1 to 1 per thousand of the content in the blood, the content of the antibody is slightly less relative to the serum, the existing products of the kit for detecting the novel coronavirus (SARS-CoV-2) antibody in the urine by the capture method are available at present, and the novel coronavirus (SARS-CoV-2) IgG antibody in the urine can be detected noninvasively. In order to solve the problem that the detection sensitivity is reduced due to the low content of a novel coronavirus (SARS-CoV-2) IgG antibody in urine, the invention adopts a mode of concentrating the urine and then detecting to improve the detection sensitivity.
Disclosure of Invention
The invention aims to overcome the defects in the prior art, provides a novel rapid detection kit for coronavirus in urine, a preparation method and a use method, concentrates and detects the urine, is noninvasive, rapid, simple and convenient, high in detection sensitivity, clear in result display and higher in detection efficiency, and can effectively solve the problems in the background technology.
In order to achieve the purpose, the invention provides the following technical scheme: a reagent kit for quickly detecting coronavirus in urine is composed of test paper strip consisting of sample pad, binding pad, nitrocellulose membrane for coating detection line and quality control line, and water-absorbing paper, centrifugal tube containing concentrated medium, and sampling cup, and the binding pad of said test paper strip is coated with the gold-labeled SARS-CoV-2 recombinant N antigen, SARS-CoV-2 recombinant S antigen and SA. Is used for detecting anti-SARS-CoV-2 specific IgG antibody in human urine.
Furthermore, the used colloidal gold particles are 40nm, the used colloidal gold concentration is four parts per million, and the labeling concentrations of the SARS-CoV-2 recombinant N antigen, the SARS-CoV-2 recombinant S antigen and the SA are all 10-20 mu g/ml.
Furthermore, the detection line of the detection test strip is coated with SPA, and the quality control line is coated with rabbit anti-SA antibody.
Further, the concentration of the SPA coated on the detection line is 1.0-2.0 mg/ml.
Further, the concentration of the anti-SA antibody coated on the quality control line is 1.0-1.5 mg/ml.
Further, the amount of the concentration medium in the centrifuge tube was 0.1 g.
Further, the ratio of the concentration medium to the urine sample is 1:6-1:8 during sample processing.
The preparation method of the novel urine coronavirus rapid detection kit comprises the following steps;
1) uniformly coating SARS-CoV-2 recombinant N antigen, SARS-CoV-2 recombinant S antigen and SA marked by colloidal gold formed by combining SARS-CoV-2 recombinant N antigen, SARS-CoV-2 recombinant S antigen and SA with colloidal gold particles on glass fiber, and drying;
2) scribing the detection line coating solution on the nitrocellulose membrane to form a detection line, scribing the quality control line coating solution on the nitrocellulose membrane to form a quality control line, and drying;
3) and sequentially overlapping the sample pad, the nitrocellulose membrane and the absorbent paper on a bottom plate to obtain the nitrocellulose filter.
A using method of the novel urine coronavirus rapid detection kit comprises the steps of putting 0.6-0.8ml of sample into a centrifugal tube filled with a concentrated medium, inserting a sample adding end of a detection test strip into the medium when reaction is carried out for 3min, and observing a result after a chromatography process is complete and 5-10 min.
Compared with the prior art, the invention has the beneficial effects that: the urine novel coronavirus rapid detection kit, the preparation method and the use method have the following advantages: the urine is concentrated and then detected, the detection is noninvasive, rapid, simple and convenient, the concentration and the detectability of a trace antibody contained in the urine can be improved through simple and rapid concentration of a concentration medium, the detection sensitivity is high, the result is clearly displayed, and the detection efficiency is higher.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The invention provides a technical scheme that: a reagent kit for quickly detecting coronavirus in urine is composed of test paper strip consisting of sample pad, binding pad, nitrocellulose membrane for coating detection line and quality control line, and water-absorbing paper, centrifugal tube containing concentrated medium, and sampling cup, and the binding pad of said test paper strip is coated with the gold-labeled SARS-CoV-2 recombinant N antigen, SARS-CoV-2 recombinant S antigen and SA. Is used for detecting anti-SARS-CoV-2 specific IgG antibody in human urine.
The used colloidal gold particles are 40nm, the used colloidal gold concentration is four parts per million, and the marking concentrations of SARS-CoV-2 recombinant N antigen, SARS-CoV-2 recombinant S antigen and SA are all 10-20 mu g/ml.
The detection line of the detection test strip is coated with SPA, and the quality control line is coated with rabbit anti-SA antibody.
The concentration of SPA coated on the detection line is 1.0-2.0 mg/ml.
The concentration of the anti-SA antibody coated on the quality control line is 1.0-1.5 mg/ml.
The amount of concentration medium in the centrifuge tube was 0.1 g.
The ratio of the concentration medium to the urine sample is 1:6-1:8 during sample treatment.
The preparation method of the novel urine coronavirus rapid detection kit comprises the following steps;
1) uniformly coating SARS-CoV-2 recombinant N antigen, SARS-CoV-2 recombinant S antigen and SA marked by colloidal gold formed by combining SARS-CoV-2 recombinant N antigen, SARS-CoV-2 recombinant S antigen and SA with colloidal gold particles on glass fiber, and drying;
2) scribing the detection line coating solution on the nitrocellulose membrane to form a detection line, scribing the quality control line coating solution on the nitrocellulose membrane to form a quality control line, and drying;
3) and sequentially overlapping the sample pad, the nitrocellulose membrane and the absorbent paper on a bottom plate to obtain the nitrocellulose filter.
A using method of the novel urine coronavirus rapid detection kit comprises the steps of putting 0.6-0.8ml of sample into a centrifugal tube filled with a concentrated medium, inserting a sample adding end of a detection test strip into the medium when reaction is carried out for 3min, and observing a result after a chromatography process is complete and 5-10 min. The urine is concentrated and then detected, and the method is noninvasive, rapid, simple and convenient.
The preparation method of the sample pad, the preparation of the gold-labeled combined pad, the preparation and packaging of the printing film and the centrifugal tube filled with the concentration medium comprises the following steps:
1. preparation of colloidal gold pad
1) Preparing a colloidal gold solution:
weighing 200ml of tetrachloroauretic acid solution, pouring the tetrachloroauretic acid solution into a clean triangular flask, stirring and heating the tetrachloroauretic acid solution to boil, adding 12ml of 1 percent trisodium citrate solution, continuously stirring the mixture until the auretic solution is in stable wine red color, and recovering the volume for later use after stirring and cooling;
2) gold-labeled SARS-CoV-2 recombinant N antigen, SARS-CoV-2 recombinant S antigen, SA:
measuring 200ml of colloidal gold solution, adjusting pH, respectively stirring, adding appropriate amount of SARS-CoV-2 recombinant N antigen, stirring for 5min, adding 50ul of 10% casein solution, and continuously stirring for 5 min;
measuring 200ml of colloidal gold solution, adjusting pH, respectively stirring, adding appropriate amount of SARS-CoV-2 recombinant S antigen, stirring for 5min, adding 50ul of 10% casein solution, and continuously stirring for 5 min;
measuring 100ml of colloidal gold solution, adjusting pH, respectively stirring, adding appropriate amount of SA, stirring for 5min, adding 50ul of 10% casein solution, and continuously stirring for 5 min;
3) and (3) purifying the gold-labeled recombinant protein:
centrifuging the marked gold-marked SARS-CoV-2 recombinant N antigen, SARS-CoV-2 recombinant S antigen and SA solution in a centrifuge at the rotating speed of 11000rpm for 50 minutes, and then removing the supernatant;
4) coating of gold-labeled recombinant protein:
the three purified gold-labeled solutions were redissolved to 50ml, and mixed together. The redissolved gold-labeled recombinant protein was uniformly coated on a glass fiber conjugate pad in an amount of 30 ml/piece of glass fiber (30 cm x 28 cm), and the conjugate pad was placed on a screen window and dried in a drying chamber and sealed for use.
2. Preparing a printing film:
1) preparing detection line membrane printing solution, namely adding a proper amount of SPA into membrane printing buffer solution to ensure that the concentration of SPA is 1.5 mg/ml;
2) preparing quality control line membrane printing solution, namely adding the anti-heavy anti-SA antibody into membrane printing buffer solution to enable the concentration to be 1.0 mg/ml;
3) preparing a printing film: and respectively coating the two prepared printing film solutions to a detection line and a quality control line of a nitrocellulose film. And (5) drying the coated printing film in a drying chamber for 4 hours, and sealing for later use.
3 a method for preparing a sample pad, comprising the steps of:
1) soaking glass fiber or non-woven fabric in 0.05M Tris-HCl pH8.0, Tween-20 thousandth solution for 1 hr;
2) taking out and putting on a screen window for natural drying, and sealing for later use.
4. The packaging method specifically comprises the following steps:
1) sequentially putting absorbent paper, a printing film, a colloidal gold pad and a sample pad on a PVC large plate;
2) and cutting into strips and packaging.
5. Concentration medium: 0.1g of the concentration medium is weighed into a centrifuge tube.
The application method of the urine novel coronavirus (SARS-CoV-2) IgG antibody rapid detection kit comprises the following steps:
1) a clean sample cup is used for taking a urine sample;
2) adding 0.8ml of urine into a centrifuge tube filled with a concentration medium;
3) when the time is 3min, the moisture in the urine is fully absorbed, and the test strip is inserted into the medium for reaction;
4) and observing the result after the chromatography is completed for 5-10 min.
Interpretation of the test results:
positive (+): the quality control line and the detection line are colored.
Negative (-): only the quality control line develops color.
And (4) invalidation: the quality control line did not develop color.
If the test strip fails due to deterioration or improper handling, retesting is recommended.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (9)
1. Novel quick detect reagent box of coronavirus in urine, its characterized in that: the detection test strip consists of a sample pad fixed on a PVC plate, a combination pad, a nitrocellulose membrane coating a detection line and a quality control line, and absorbent paper, wherein the combination pad of the test strip is coated with gold-labeled SARS-CoV-2 recombinant N antigen, SARS-CoV-2 recombinant S antigen and SA.
2. The urine novel coronavirus rapid detection kit according to claim 1, characterized in that: the used colloidal gold particles are 40nm, the used colloidal gold concentration is four parts per million, and the marking concentrations of the SARS-CoV-2 recombinant N antigen, the SARS-CoV-2 recombinant S antigen and the SA are all 10-20 mu g/ml.
3. The urine novel coronavirus rapid detection kit according to claim 1, characterized in that: the detection line of the detection test strip is coated with SPA, and the quality control line is coated with rabbit anti-SA antibody.
4. The urine novel coronavirus rapid detection kit according to claim 3, wherein the kit comprises: the concentration of the SPA coated on the detection line is 1.0-2.0 mg/ml.
5. The urine novel coronavirus rapid detection kit according to claim 3, wherein the kit comprises: the concentration of the anti-SA antibody coated on the quality control line is 1.0-1.5 mg/ml.
6. The urine novel coronavirus rapid detection kit according to claim 1, characterized in that: the amount of concentration medium in the centrifuge tube was 0.1 g.
7. The urine novel coronavirus rapid detection kit according to claim 6, which is characterized in that: the ratio of the concentration medium to the urine sample is 1:6-1:8 during sample treatment.
8. The method for preparing the urine novel coronavirus rapid detection kit as claimed in any one of claims 1 to 7, wherein the kit comprises: comprises the following steps;
1) uniformly coating SARS-CoV-2 recombinant N antigen, SARS-CoV-2 recombinant S antigen and SA marked by colloidal gold formed by combining SARS-CoV-2 recombinant N antigen, SARS-CoV-2 recombinant S antigen and SA with colloidal gold particles on glass fiber, and drying;
2) scribing the detection line coating solution on the nitrocellulose membrane to form a detection line, scribing the quality control line coating solution on the nitrocellulose membrane to form a quality control line, and drying;
3) and sequentially overlapping the sample pad, the nitrocellulose membrane and the absorbent paper on a bottom plate to obtain the nitrocellulose filter.
9. The use method of the urine novel coronavirus rapid detection kit as claimed in any one of claims 1 to 7, wherein: and (3) putting 0.6-0.8ml of sample into a centrifuge tube filled with a concentrated medium, inserting the sample adding end of the detection test strip into the medium when reacting for 3min, and observing the result after the chromatographic process is complete and 5-10 min.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2022177520A1 (en) * | 2021-02-17 | 2022-08-25 | Kurtulmus Mehmet Serhan | Urine test kit and how to use it |
| WO2022236891A1 (en) * | 2021-05-14 | 2022-11-17 | 沙滨 | Non-invasive detection kit for detecting novel coronavirus antigen, and detection method thereof |
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