CN116836234A - Short peptide, detection kit and method for detecting novel coronavirus nucleocapsid protein antigen - Google Patents
Short peptide, detection kit and method for detecting novel coronavirus nucleocapsid protein antigen Download PDFInfo
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
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- G—PHYSICS
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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
- G01N33/569—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
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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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
- G01N33/581—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)
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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
Abstract
The invention discloses a short peptide, a detection kit and a method for detecting novel coronavirus nucleocapsid protein antigen, wherein the short peptide is polypeptide consisting of ACGTKPTKFCGGGGGS amino acid sequence, and two cysteines at C2 and C10 form disulfide bonds. The short peptide is used as a capture antibody and combined with an enzyme-linked immunosorbent assay (ELISA) to detect the novel coronavirus nucleocapsid protein antigen. The solid-phase bio-peptide probe is utilized to incubate with target protein in a sample to be detected, and then the effective detection can be realized under a catalytic chromogenic system. The method provided by the invention has the advantages of strong specificity, high sensitivity, convenience in operation and low cost in probe manufacture and purification, and provides an effective way for realizing sensitive and rapid detection of novel coronaviruses.
Description
Technical Field
The invention belongs to the technical fields of biotechnology and immunoassay, and relates to a short peptide, a detection kit and a method for detecting a novel coronavirus nucleocapsid protein antigen.
Background
The disclosure of this background section is only intended to increase the understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art already known to those of ordinary skill in the art.
The Nucleocapsid Protein (NP) of the novel coronavirus (SARS-CoV-2) is one of the major expressed structural proteins, the most abundant in the virus, and the NP antigen is one of the best early diagnostic markers for SARS-CoV-2.
The novel coronaviruses are highly transmissible and infectious and have spread rapidly worldwide. In addition, variants of SARS-CoV-2 continue to appear, ranging from Alpha (B.1.1.7) to Delta (B.1.617.2) to Omacron (B.1.1.529), with the growth rate of the variant being faster, the level in the human lungs and throat being higher, and with greater infectivity than the earlier version of the novel coronavirus. The structural proteins of coronaviruses mainly include spike proteins, envelope proteins, membrane proteins and nucleocapsid proteins NP. NPs are one of the major expressed structural proteins, the most abundant in viruses, and they mainly encapsulate the viral genome and package viral genomic RNA to form helices, playing an important role in viral replication, viral particle assembly, release, and interfering with the cell cycle process of host cells. Of the several viral proteins, NP is the best early diagnostic marker for SARS-CoV and can be detected 1 day before clinical symptoms appear.
The rapid and accurate diagnosis of suspicious cases, effective isolation of infected patients and active treatment are the most important procedures for preventing and controlling epidemics, so prevention and control are particularly important in the face of SARS-CoV-2. The precondition of prevention and control is to diagnose SARS-CoV-2 effectively, rapidly and accurately. Although nucleic acid detection is widely used for SARS-CoV-2 diagnosis, a well-equipped molecular diagnostic laboratory is required, equipped with trained staff and expensive equipment. Furthermore, nucleic acid detection can produce false negative results due to differences in viral load in different samples, and high proportion of false negative results can lead to missed diagnosis. Antibody-based tests may be used to enhance diagnostic capabilities. However, antibody reactions to pathogens do not occur at the early stages of infection. In SARS-CoV-2 infection, recent data show median serum turnover times of IgM and IgG of 18 and 20 days after exposure, respectively. Thus, antibody-based serological tests do not allow diagnosis early in the onset of infection. And the SARS-CoV-2 antibody detection has false positive, which brings difficulty to clinical detection. Therefore, it is still urgent to improve the detection sensitivity and specificity.
Disclosure of Invention
The invention aims to solve the defects of the prior art, and provides a short peptide, a detection kit and a method for detecting SARS-CoV-2 virus, which can specifically, sensitively, simply and rapidly detect SARS-CoV-2NP antigen.
In order to achieve the above purpose, the technical scheme of the invention is as follows:
in one aspect, a short peptide is provided, which is a polypeptide; the polypeptide is as follows: a polypeptide consisting of the amino acid sequence ACGTKPTKFCGGGGGS (SEQ ID NO: 1) with two cysteines at C2 and C10 forming disulfide bonds. Experiments show that the polypeptide has the advantages of high affinity, high selectivity and the like for SARS-CoV-2NP, and has the advantages of small molecular weight, good stability and the like.
In another aspect, a detection kit comprises the above-described short peptide, a monoclonal primary antibody capable of specifically binding to SARS-CoV-2NP, and a monoclonal secondary antibody conjugated to horseradish peroxidase (HRP) capable of binding to monoclonal primary antibody.
In a third aspect, the use of a short peptide or assay kit as described above for the detection of SARS-CoV-2 NP.
In a fourth aspect, a method for detecting SARS-CoV-2NP, providing the above-mentioned short peptide or detection kit, fixing the short peptide, adding a test solution containing SARS-CoV-2NP to an ELISA plate after fixing the short peptide for incubation, adding a monoclonal primary antibody for incubation, then adding a monoclonal secondary antibody conjugated with horseradish peroxidase for incubation, and measuring the optical density of each well at 452 nm using an ELISA apparatus after chromogenic reaction and termination of reaction of a method (TMB) substrate for developing color of 3,3', 5' -tetramethylbenzidine.
The beneficial effects of the invention are as follows:
(1) The short peptide of the invention has the advantages of high affinity, high selectivity and the like for SARS-CoV-2NP, and has the advantages of small molecular weight, good stability, strong specificity, lower cost, and the like, and can well replace solid phase capture antibodies in a detection system. In addition, the probe is moderately prolonged while ensuring the binding site part, so that the binding efficiency with the surface of the ELISA plate is enhanced, and the probe binding site part is more fully exposed.
(2) The short peptide of the invention has a detection limit of SARS-CoV-2NP as low as 61pg/mL, which is superior to the detection limit of most methods reported. The ELISA analysis with the advantages of strong specificity, wide linear range, high sensitivity, simple operation and the like is adopted to construct a detection system with perfect, simple, quick, good safety and low commercialization cost, the detection of SARS-CoV-2 actual virus proves that the method realizes the sensitive and specific detection of the novel coronavirus, and a promising detection method is developed in the fields of medical immunodiagnosis, biological research and the like, and has very practical significance for the detection, prevention and control of the novel coronavirus.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention.
FIG. 1 shows the result of the polypeptide probe N1 specificity test provided in example 1 of the present invention.
FIG. 2 shows the results of the test of the optimal coating concentration of the polypeptide probe N1 according to example 2 of the present invention.
FIG. 3 shows the results of the test of the optimal dilution ratio of the monoclonal antibody according to example 3 of the present invention.
FIG. 4 is a graph showing the operation of the test SARS-CoV-2NP according to example 4 of the present invention.
FIG. 5 shows the sensitivity of ELISA method for detecting SARS-CoV-2 virus as provided in example 5 of the present invention.
Detailed Description
It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the invention. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the present invention. As used herein, the singular is also intended to include the plural unless the context clearly indicates otherwise, and furthermore, it is to be understood that the terms "comprises" and/or "comprising" when used in this specification are taken to specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof.
In view of the shortcomings of complicated preparation process, low stability, high cost and the like of the existing antibody-based SARS-CoV-2NP detection, the invention provides a short peptide, a detection kit and a method for detecting SARS-CoV-2 NP.
In one exemplary embodiment of the invention, a short peptide is provided; the short peptide is as follows: a polypeptide consisting of the amino acid sequence ACGTKPTKFCGGGGGS (SEQ ID NO: 1) with two cysteines at C2 and C10 forming disulfide bonds.
Experiments show that the short peptide has the advantages of high affinity, high selectivity and the like for SARS-CoV-2NP, and has the advantages of small molecular weight, good stability and the like. The short peptide is a short-chain polypeptide. The short peptide is moderately prolonged while ensuring the binding site portion so as to enhance the binding efficiency with the polystyrene surface, and is beneficial to more sufficient exposure of the probe binding site portion.
In another embodiment of the invention, a detection kit is provided, which comprises the above short peptide, a monoclonal primary antibody, and a monoclonal secondary antibody conjugated with horseradish peroxidase, wherein the monoclonal primary antibody can be combined with SARS-CoV-2NP, and the monoclonal secondary antibody can be combined with the monoclonal primary antibody.
In one or more examples of this embodiment, the monoclonal primary antibody is a murine anti-SARS-CoV-2 NP monoclonal antibody and the monoclonal secondary antibody is a goat anti-murine immunoglobulin G monoclonal antibody.
In a third embodiment of the present invention, there is provided the use of the above-described short peptide or detection kit in SARS-CoV-2NP antigen. The use of the invention is for the diagnosis and treatment of non-diseases.
In a fourth embodiment of the present invention, there is provided a method for detecting SARS-CoV-2NP antigen, providing the above-mentioned short peptide or detection kit, fixing the short peptide, adding a test solution containing SARS-CoV-2NP antigen into the ELISA plate well after fixing the short peptide for incubation, then adding monoclonal primary antibody for incubation, then adding monoclonal secondary antibody conjugated with horseradish peroxidase for incubation, then adding TMB substrate chromogenic solution for chromogenic reaction, and adding a stop solution (50. Mu.L 2M H 2 SO 4 ) The chromogenic reaction was discontinued and the optical density of each well was measured using a microplate reader at 452, nm.
In a fifth embodiment of the present invention, there is provided a method for detecting SARS-CoV-2 virus: providing the above short peptide or detection kit, fixing the short peptide, adding a test solution containing the treated SARS-CoV-2 virus into the ELISA plate hole after fixing the short peptide, incubating, adding monoclonal primary antibody, incubating, adding monoclonal secondary antibody conjugated with horseradish peroxidase, incubating, adding TMB substrate chromogenic solution, performing chromogenic reaction, and adding stop solution (50 μL 2M H) 2 SO 4 ) The chromogenic reaction was discontinued and the optical density of each well was measured using a microplate reader at 452, nm.
In order to remove the effect of free substances, the removal of the solution is required after incubation. Preferably, the method is for the diagnosis of non-disease.
In one or more examples of this embodiment, the short peptide is immobilized by adding a short peptide solution to the elisa plate for incubation.
In this series of examples, the buffer solution used in the short peptide solution was TBS buffer. The short peptides are immobilized on wells of an elisa plate to capture the target antigen. The optimal coating buffer varies with the type of well surface and the different proteins immobilized, and to select the optimal short peptide coating buffer, the short peptides are dissolved in 50 respectivelymM PBS（pH 7.4），50 mM TBS（pH 7.5），50 mM TBS（pH 8.0），50 mM Na 2 CO 3 / NaHCO 3 (pH 8.6) and 50 mM Na 2 CO 3 / NaHCO 3 (pH 9.6) these five different buffers were used for immobilization, and the ELISA experiments showed that the short peptides were best immobilized in 50 mM TBS (pH 8.0).
In the series of embodiments, the concentration of the short peptide in the short peptide solution is 8-12 mug/mL. The concentration of the short peptide will affect the amount of short peptide immobilized on the microplate. In order to find the optimal coating amount of the short peptide, different coating amounts are respectively set: 1. mu.g/mL, 2. Mu.g/mL, 5. Mu.g/mL, 10. Mu.g/mL, 20. Mu.g/mL, 30. Mu.g/mL. As a result of ELISA experiments, the optimal amount of the short peptide was 10. Mu.g/mL.
In this series of examples, short peptide solutions were added to the elisa plate for incubation under the following conditions: 3-8 ℃ overnight. The coating time and temperature are different, so that the binding efficiency of the short peptide and the surface of the polyethylene pore plate is influenced, and therefore, the exploration of the optimal coating condition of the short peptide is imperative. Coating at 4deg.C overnight, 37deg.C for 6 hr, 37deg.C for 4 hr, 37deg.C for 2 hr respectively. Experiments prove that the conditions of 37 ℃ and 6 hours are more favorable for the immobilization and reaction of the short peptide.
In one or more examples of this embodiment, the test solution containing SARS-CoV-2NP or the treated SARS-CoV-2 virus is added to the immobilized short peptide ELISA plate for incubation under the following conditions: and the room temperature is 2.8-3.2 hours. The room temperature in the invention is the temperature of the indoor environment, and is generally 15-30 ℃. The recognition and combination of the short peptide and the antigen are critical, so that the time for full recognition and combination is ensured, the efficiency of the whole experiment is ensured, and equal amounts of SARS-CoV-2NP are respectively coated for different times at room temperature: 0.5 h, 1 h, 2 h, 3 h, 4 h. Experiments prove that the optimal capture time of the short peptide to SARS-CoV-2NP is 3 hours.
In one or more examples of this embodiment, the monoclonal primary antibody is added and incubated for 1.5 hours at room temperature, with a dilution ratio of 1:4800-5200. The monoclonal primary antibody is subjected to an optimization experiment with dilution ratio of 1:2500, 1:5000, 1:10000, 1:15000 and 1:20000, and the experimental result shows that the detection effect is optimal when the dilution ratio of the monoclonal primary antibody is 1:5000.
In one or more examples of this embodiment, the monoclonal secondary antibody conjugated to horseradish peroxidase is added and incubated for 1.5 hours at room temperature, with a dilution ratio of 1:14000-1:16000. When the dilution ratio is 1:5000, 1:10000, 1:15000, 1:20000 and 1:30000, the optimal experiment shows that the positive-negative ratio is the largest when the dilution ratio is 1:15000, which indicates that the detection effect is the best.
In order to enable those skilled in the art to more clearly understand the technical scheme of the present invention, the technical scheme of the present invention will be described in detail with reference to specific embodiments.
The short peptide (shown in SEQ ID NO: 1) used in the following example, starting from the amino terminus, was alanine-cysteine-glycine-threonine-lysine-proline-threonine-phenylalanine-glycine-serine (ACGTKPTKFCGGGGGS), wherein the second cysteine (C2) and the tenth cysteine (C10) formed disulfide bonds, and the polypeptide molecular weight was 1425.62 Da, which was designated as short peptide N1 in the following example.
Example 1
Short peptide N1 specificity test: to test whether the phage clones obtained are specific, ELISA-based methods are used to test whether the selected short peptides bind specifically to different biological macromolecules by capture experiments. In this example, SARS-COV-2NP was immobilized in the wells of the microplate at a concentration of 2. Mu.g/ml, and MERS-CoV NP, SARS-COV-2 S1, SARS-CoV-2S-RBD and BSA at a concentration of 2. Mu.g/ml were used as control experiments. Shake overnight at 4 ℃. Wash once with 0.5% PBST, block buffer with 5mg/ml BSA, block at 4℃for more than one hour. The phage monoclonal expressing the N1 short peptide was then added to each well separately, washed 6 times with 0.5% PBST, and incubated for 2 hours at room temperature with shaking. The unbound free phage were washed 6 times with 0.5% PBST. HRP-anti-M13 (1:12000) was added and incubated with shaking at room temperature1.5 hours. Wash 6 times with 0.5% pbst and add TMB color development solution. The reaction was developed by shaking at room temperature. Finally, by adding 50. Mu.L of 2M H 2 SO 4 The chromogenic reaction was interrupted and the optical density of each well was measured using an enzyme-labeled instrument at 452 and nm, as shown in FIG. 1, indicating that short peptide N1 has a strong specificity for the detection of SARS-CoV-2 NP.
Example 2
Short peptide N1 optimal coating concentration test: serial gradient dilution of specific short peptide N1, 2, 5, 10, 20 and 30 mug/mL, adding into 96-well enzyme label plate holes with 100 mug/hole, wrapping with preservative film, placing in 4 deg.C environment, shaking and incubating overnight; sucking out the short peptide N1 solution in the ELISA plate, placing the plate surface downward on a clean paper towel, drying the residue, washing with 0.05% PBST for 1 time, adding SARS-CoV-2NP, and incubating at room temperature under shaking for 3 hours. Subsequently, the mixture was washed 1 time with 0.05% PBST, and a murine anti-SARS-CoV-2 NP monoclonal antibody was added. Incubate for 1.5 hours at room temperature with shaking. Then washed 1 time with 0.05% PBST, goat anti-mouse IgG-HRP monoclonal antibody was added, and incubated for 1.5 hours at room temperature with shaking. Then, the wells were washed 1 time with 0.05% PBST, TMB color development solution was added to the wells, and the wells were subjected to color development reaction by shaking at room temperature. Finally, the chromogenic reaction was stopped by adding a stop solution and the optical density of each well was measured using a microplate reader at 452 and nm.
The detection results are shown in FIG. 2, and FIG. 2 shows that the optimal coating concentration of the specific probe is 10. Mu.g/mL.
Example 3
Determination of the optimal dilution ratio of monoclonal antibodies: specific short peptide N1 was dissolved in 50 mM TBS (pH 8.0) to a concentration of 10. Mu.g/mL, respectively, and then the prepared solution was taken in 100. Mu.L of the wells of the enzyme-labeled plate, and the sealed plate was incubated at 37℃for 6 hours; sucking out the short peptide solution in the ELISA plate, placing the plate surface downward on a clean paper towel, drying the residue, washing 1 time with 0.05% PBST, loading 100 mu L of SARS-CoV-2NP solution and 100ng/mL into the wells, simultaneously taking the ELISA plate well without SARS-CoV-2NP as a control group, and incubating the plate at room temperature under shaking for 3 hours; wash 1 time with 0.05% pbst; murine anti-SARS-CoV-2 NP monoclonal antibody was subjected to a series of gradient dilutions of 1:2500, 1:5000, 1:10000, 1:15000 and 1:20000. 100. Mu.L of each plate was incubated with shaking at room temperature for 1.5. 1.5 h, then washed 1 time with 0.05% PBST, and goat anti-mouse IgG-HRP monoclonal antibody was added thereto and incubated with shaking at room temperature for 1.5 hours. Then, the wells were washed 1 time with 0.05% PBST, TMB color development solution was added to the wells, and the wells were subjected to color development reaction by shaking at room temperature. Finally, the chromogenic reaction was stopped by adding a stop solution and the optical density of each well was measured using a microplate reader at 452 and nm.
The signal to noise ratio (P/N) was calculated as the ratio of the signal (P) from the positive group with SARS-COV-2N P to the signal (N) from the negative group without SARS-CoV-2NP, and the amounts were compared to determine the optimal monoclonal antibody dose. As shown in FIG. 3, the optimal dilution ratio of the murine anti-SARS-CoV-2 NP monoclonal antibody was 1:5000.
Example 4
Specific short peptide N1 was dissolved in 50 mM TBS (pH 8.0) to a concentration of 10. Mu.g/mL, respectively, and then the prepared solution was taken in 100. Mu.L of the wells of the enzyme-labeled plate, and the sealed plate was incubated at 37℃for 6 hours; sucking out short peptide solution in an ELISA plate, placing the plate surface downward on a clean paper towel, drying residues, washing 1 time by using 0.05% PBST, loading 100 mu L of SARS-CoV-2NP solution with different concentrations into the ELISA plate holes, incubating for 3 hours at room temperature, washing 1 time by using 0.05% PBST, diluting a mouse anti-SARS-CoV-2 NP monoclonal antibody to a concentration of 1:5000, adding 100 mu L into the holes, incubating for 1.5 hours by shaking at room temperature, and washing 1 time by using 0.05% PBST; goat anti-mouse IgG-HRP monoclonal antibody was added to dilute to a concentration of 1:15000, 100 μl was added to each well, and incubated with shaking at room temperature for 1.5 hours; then, the wells were washed 1 time with 0.05% PBST, TMB color development solution was added to the wells, and the wells were subjected to color development reaction by shaking at room temperature. Finally, the chromogenic reaction was stopped by adding a stop solution and the optical density of each well was measured using a microplate reader at 452 and nm.
The SARS-CoV-2NP working curve was plotted, as shown in FIG. 4, and the established ELISA assay was linear in the range of 0.2-200 ng/mL (y=0.00536 x+0.05796, R 2 =0.995), the detection limit was 61 pg/mL.
Example 5
Dissolving specific short peptide N1 in 50 mM TBS (pH 8.0) to a concentration of 10 mug/mL respectively, taking 100 mu L of prepared solution into an enzyme-labeled plate hole, incubating the sealed plate at 37 ℃ for 6 hours, sucking out the short peptide solution in the enzyme-labeled plate, placing the plate face down on a clean paper towel, beating the residue, washing with 0.05% PBST for 1 time, inactivating SARS-CoV-2 virus at 65 ℃ for 35 minutes, performing cleavage treatment with 15% RIPA lysate after completion, performing dilution at different concentrations, loading 100 mu L into the enzyme-labeled plate hole, incubating at room temperature for 3 hours, washing with 0.05% PBST for 1 time, diluting the mouse anti-SARS-CoV-2 NP monoclonal antibody to a concentration of 1:5000, incubating with 100 mu L of solution into the hole, shaking at room temperature for 1.5 hours, and washing with 0.05% PBST for 1 time; goat anti-mouse IgG-HRP monoclonal antibody was added to dilute to a concentration of 1:15000, 100 μl was added to each well, and incubated with shaking at room temperature for 1.5 hours; then, the wells were washed 1 time with 0.05% PBST, TMB color development solution was added to the wells, and the wells were subjected to color development reaction by shaking at room temperature. Finally, the chromogenic reaction was stopped by adding a stop solution and the optical density of each well was measured using a microplate reader at 452 and nm.
Exposure to the SARS-CoV-2 virus or clinical specimens of patients containing the SARS-CoV-2 virus can present a high risk of infection to medical personnel and researchers, and heat treatment is a simple method of inactivating the virus. In this example, SARS-COV-2 virus was inactivated by heating at 65deg.C for 35 min, and then lysed with RIPA lysate, and the treated SARS-CoV-2 virus was detected, as shown in FIG. 5, with the red dotted line as the cut-off value (average value of negative control plus three times standard deviation), although the SARS-COV-2 virus was inactivated, 50 TCID could be detected in this example 50 /ml. In view of the complete inactivation of SARS-CoV-2 virus at 65℃for a short period of 5 minutes, the detection method of this example is described as a safe diagnostic method that can be used to heat inactivate clinical specimens to avoid infection during analysis.
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Sequence listing
<110> university of Qingdao
<120> a short peptide, a detection kit and a method for detecting novel coronavirus nucleocapsid protein antigen
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 16
<212> PRT
<213> novel coronavirus (SARS-CoV-2)
<400> 1
Ala Cys Gly Thr Lys Pro Thr Lys Phe Cys Gly Gly Gly Gly Gly Ser
1 5 10 15
Claims (10)
1. A short peptide, characterized in that it is: a polypeptide consisting of the amino acid sequence ACGTKPTKFCGGGGGS, the two cysteines at C2 and C10 of which form disulfide bonds.
2. A detection kit comprising the short peptide of claim 1, a monoclonal primary antibody capable of specifically binding to a novel coronavirus nucleocapsid protein, and a monoclonal secondary antibody conjugated to horseradish peroxidase capable of binding to a monoclonal primary antibody.
3. The test kit of claim 2, wherein the monoclonal primary antibody is a murine anti-SARS-CoV-2 NP monoclonal antibody and the monoclonal secondary antibody is a goat anti-murine immunoglobulin G monoclonal antibody.
4. A novel coronavirus nucleocapsid antigen detection method, which is characterized by comprising the following steps: providing the above short peptide or detection kit, fixing the short peptide, adding a solution to be detected containing SARS-CoV-2NP antigen into the ELISA plate hole after fixing the short peptide for incubation, adding monoclonal primary antibody for incubation, then adding monoclonal secondary antibody conjugated with horseradish peroxidase for incubation, adding TMB substrate chromogenic solution for chromogenic reaction, interrupting the chromogenic reaction by adding a stop solution, and measuring the optical density of each hole at 452 nm by using an ELISA instrument.
5. A novel coronavirus detection method, comprising the steps of: providing the above short peptide or detection kit, fixing the short peptide, adding a solution to be detected of the SARS-CoV-2 virus after treatment into an ELISA plate hole after fixing the short peptide for incubation, adding a monoclonal primary antibody for incubation, then adding a monoclonal secondary antibody conjugated with horseradish peroxidase for incubation, adding TMB substrate color development liquid for color development reaction, interrupting the color development reaction by adding a stop solution, and measuring the optical density of each hole at 452 and nm by using an ELISA instrument.
6. The method according to claim 4 or 5, wherein in the step (1), the method for immobilizing a short peptide is: adding the short peptide solution to an ELISA plate for incubation;
preferably, the buffer solution adopted by the short peptide solution comprises PBS buffer solution, TBS buffer solution and Na buffer solution 2 CO 3 /NaHCO 3 Any one of the buffers;
preferably, the concentration of the short peptide in the short peptide solution is 8-12 mug/mL.
7. The method according to claim 4 or 5, wherein the short peptide solution is added to the ELISA plate under incubation conditions of: and (3) incubating at 3-8 ℃ overnight or at 37 ℃ for 2-6 hours.
8. The method according to claim 4 or 5, wherein the incubation conditions for adding the test solution containing SARS-CoV-2NP antigen or the treated SARS-CoV-2 virus are as follows: and incubating for 2.8-3.2 hours at room temperature.
9. The method of claim 4 or 5, wherein the monoclonal primary antibody is added and incubated at room temperature for 1.5 hours; incubating for 1.5 hours at room temperature after adding the monoclonal secondary antibody; the dilution ratio of the monoclonal primary antibody is 1:4800-5200; the dilution ratio of the monoclonal secondary antibody is 1:14000-1:16000.
10. The method according to claim 4 or 5, wherein the color reaction comprises 3,3', 5' -tetramethylbenzidine color reaction; the method for stopping the chromogenic reaction is to add sulfuric acid.
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