CN111337668A - Novel coronavirus antigen colloidal gold rapid diagnosis kit and preparation method thereof - Google Patents
Novel coronavirus antigen colloidal gold rapid diagnosis kit and preparation method thereof Download PDFInfo
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- CN111337668A CN111337668A CN202010126261.3A CN202010126261A CN111337668A CN 111337668 A CN111337668 A CN 111337668A CN 202010126261 A CN202010126261 A CN 202010126261A CN 111337668 A CN111337668 A CN 111337668A
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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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
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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 novel coronavirus antigen colloidal gold rapid diagnosis kit and a preparation method thereof, wherein the kit comprises SARS-CoV-2 nucleocapsid protein antigen colloidal gold. The kit is simple to operate, can quickly diagnose whether a patient is infected with SARS-CoV-2, and is beneficial to the effective control of epidemic situation. The invention also discloses a preparation method of the novel coronavirus antigen colloidal gold rapid diagnosis kit.
Description
Technical Field
The invention relates to the field of disease detection, in particular to a novel coronavirus antigen colloidal gold rapid diagnosis kit and a preparation method thereof.
Background
After people are infected with coronavirus, the common signs of the person are respiratory symptoms, fever, cough, shortness of breath, dyspnea and the like. In more severe cases, the infection can lead to pneumonia, severe acute respiratory syndrome, renal failure, and even death. There is currently no specific treatment for diseases caused by the novel coronavirus. Therefore, prevention and early diagnosis of diseases are particularly important for controlling the spread of the novel coronavirus.
Disclosure of Invention
In order to improve the early diagnosis efficiency of the novel coronavirus, the invention discloses a novel coronavirus antigen colloidal gold rapid diagnosis kit which is simple to operate, can rapidly diagnose whether a patient is infected with SARS-CoV-2, and is beneficial to the effective control of epidemic situation.
The invention also discloses a preparation method of the novel coronavirus antigen colloidal gold rapid diagnosis kit.
The invention is realized by the following technical scheme:
a novel coronavirus antigen colloidal gold rapid diagnosis kit comprises SARS-CoV-2 nucleocapsid protein antigen colloidal gold.
A preparation method of a novel coronavirus antigen colloidal gold rapid diagnosis kit comprises the following steps:
(1) expressing and purifying SARS-CoV-2 nucleocapsid protein;
(2) preparing the SARS-CoV-2 nucleocapsid protein antigen colloidal gold.
The amino acid sequence of SARS-CoV-2 nucleocapsid protein is shown in SEQ ID NO 1.
Wherein, in the step (1), the expression of SARS-CoV-2 nucleocapsid protein comprises the following steps:
(11) taking a nucleotide sequence corresponding to an amino acid sequence of SARS-CoV-2 nucleocapsid protein as a template, designing a forward primer with a nucleotide sequence shown as SEQ ID NO. 2 or SEQ ID NO. 3 and a reverse primer with a nucleotide sequence shown as SEQ ID NO. 4 or SEQ ID NO. 5;
(12) cloning a nucleotide sequence corresponding to SARS-CoV-2 nucleocapsid protein into a plasmid vector by PCR amplification, double digestion PCR product and the vector;
(13) transforming the escherichia coli by using the vector obtained in the step (12), and then carrying out induction culture;
(14) and (4) centrifuging the cultured escherichia coli to obtain the SARS-CoV-2 nucleocapsid recombinant protein.
Further, in the step (12), the plasmid vector is any one of pET28a, pET22b, pET21b, pET32a, pET40, pGex-6P-1 and pMal-c2 x.
Further, in the step (13), E.coli BL21 or Rosseta strain is adopted as the Escherichia coli, and the induction culture method comprises the following steps: the transformed colibacillus is coated on a plate containing ampicillin or kanamycin, incubated at 37 ℃, then single colony is selected and inoculated in a liquid culture medium for culturing for 2-5h, and finally IPTG is added for induction and culturing for 3-18 h.
Further, in step (14), the SARS-CoV-2 nucleocapsid recombinant protein is obtained by the following steps: collecting the centrifuged colibacillus thallus, adding buffer solution to break the thallus, centrifuging to obtain supernatant, and purifying by chromatography to obtain SARS-CoV-2 nucleocapsid recombinant protein.
In step (1), after the SARS-CoV-2 nucleocapsid protein is purified, the protein is verified by using an anti-N protein antibody.
Further, in the step (2), the specific steps for preparing the SARS-CoV-2 nucleocapsid protein antigen colloidal gold are as follows:
(21) preparing a colloidal gold solution by taking a chloroauric acid solution and a trisodium citrate aqueous solution as raw materials;
(22) transferring the SARS-CoV-2 nucleocapsid recombinant protein solution into a dialysis bag, and dialyzing overnight;
(23) adding the SARS-CoV-2 nucleocapsid recombinant protein after dialysis treatment into colloidal gold solution with the same volume, standing, then adding BSA solution, fully mixing uniformly, centrifuging, removing supernatant, and redissolving the colloidal gold precipitate by using protective solution to obtain the SARS-CoV-2 nucleocapsid protein antigen colloidal gold.
In the step (22), the dialysis method comprises: diluting the SARS-CoV-2 nucleocapsid protein solution to 1mg/mL, transferring into dialysis bag, dialyzing with Tris buffer solution with concentration of 20mmol/L, pH value of 7.0 at 2-8 deg.C overnight.
Compared with the prior art, the invention has the following advantages and beneficial effects:
the invention relates to a novel coronavirus antigen colloidal gold rapid diagnosis kit, which is simple to operate, can rapidly diagnose whether a patient is infected with SARS-CoV-2, and is beneficial to effective control of epidemic situation.
Drawings
The accompanying drawings, which are included to provide a further understanding of the embodiments of the invention and are incorporated in and constitute a part of this application, illustrate embodiment(s) of the invention and together with the description serve to explain the principles of the invention. In the drawings:
FIG. 1 is a diagram showing the results of SDS-page and Western blot analysis of the N protein of the present invention: and B, the graph shows that the N antigen is diluted into different concentrations and subjected to WB verification, and negative is a normal negative serum control.
FIG. 2 is a sample test color rendering of the present invention: the left side is IgG antibody in the N antigen detection serum, and the right side is IgM antibody in the N antigen detection serum.
FIG. 3 is a graph showing the OD values of the samples according to the present invention: IgG antibody in the serum detected by the N antigen is arranged on the left side, and IgM antibody in the serum detected by the N antigen is arranged on the right side.
FIG. 4 is a statistical chart of OD values of samples in example 2 of the present invention.
FIG. 5 is a statistical chart of OD values of samples in example 3 of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail below with reference to examples and accompanying drawings, and the exemplary embodiments and descriptions thereof are only used for explaining the present invention and are not used as limitations of the present invention.
Example 1
The invention relates to a novel coronavirus antigen colloidal gold rapid diagnosis kit, which is prepared by the following steps:
1. expression and purification of SARS-CoV-2 nucleocapsid protein
Taking a nucleotide sequence corresponding to an amino acid sequence of SARS-CoV-2 nucleocapsid protein as a template, designing a forward primer with a nucleotide sequence shown as SEQ ID NO. 2 or SEQ ID NO. 3 and a reverse primer with a nucleotide sequence shown as SEQ ID NO. 4 or SEQ ID NO. 5; PCR amplification, double enzyme digestion of PCR products and vectors, cloning to pET/pGex/pMal series vectors, including but not limited to pET28a/pET22b/pET21b/pET32a/pET40/pGex-6P-1/pMal-c2 x.
Transforming a recombinant plasmid containing a nucleotide sequence of SARS-CoV-2 nucleocapsid protein into E.coli BL21 or Rosseta strains, coating the recombinant plasmid on a plate containing ampicillin or kanamycin, incubating the plate at 37 ℃ for 16 hours, selecting a single bacterial colony, inoculating the single bacterial colony into a liquid culture medium, culturing the single bacterial colony for 2 to 5 hours, and adding IPTG (isopropyl thiogalactoside) for induction; centrifuging and collecting thalli after 3-18 hours; adding buffer solution to break thallus, centrifuging to obtain supernatant, and purifying by chromatography to obtain SARS-CoV-2 nucleocapsid recombinant protein.
Wherein, the nucleotide sequence of each primer is as follows:
SEQ ID NO:2
CGCGGATCCATGTCTGATAATGGACCCCA
SEQ ID NO:3
CCGGAATTCATGTCTGATAATGGACCCCA
SEQ ID NO:4
ATAAGAATGCGGCCGCTTAGGCCTGAGTTGAGTCAGC
SEQ ID NO:5
CCGCTCGAGTTA GGCCTGAGTTGAGTCAGC
2. validation of purified N protein (SARS-CoV-2 nucleocapsid protein)
The western blot was verified with an antibody against the N protein, and the result is shown in FIG. 1, where the target protein was 49KD, which is in line with expectations.
3. Preparation of SARS-CoV-2 nucleocapsid protein antigen colloidal gold
3.1 preparation of colloidal gold solution
Heating the chloroauric acid aqueous solution with the mass concentration of 0.01% to boil, dropwise adding the trisodium citrate aqueous solution with the mass concentration of 1% while stirring, continuously boiling for 15min after the chloroauric acid aqueous solution turns from golden yellow to mauve, cooling, recovering the volume to the original volume with distilled water, and storing at the temperature of 2-8 ℃ for later use.
3.2 transferring the solution of the new coronavirus nucleocapsid recombinant protein to a dialysis bag after the concentration of the solution reaches 1mg/mL, and dialyzing the solution overnight at the temperature of 2-8 ℃ by using Tris buffer solution with the concentration of 20mmol/L, pH and the value of 7.0;
3.3 adding the SARS-CoV-2 nucleocapsid recombinant protein after dialysis treatment into colloidal gold solution with equal volume, standing at 2-8 ℃ to ensure that the new coronavirus nucleocapsid antigen is fully combined with the colloidal gold, then adding BSA solution according to the proportion of adding 2mL of BSA solution with the mass concentration of 5% into every 10mL of colloidal gold, fully and uniformly mixing, centrifuging for 0.5-1.5h, abandoning supernatant, and dissolving the colloidal gold precipitate with protective solution again to obtain the new coronavirus nucleocapsid antigen immune colloidal gold.
Example 2
Clinical sample validation for detection of IgG antibodies
17 clinically confirmed cases, 22 suspected samples (final negative exclusion of nucleic acid) and 23 normal human samples of the novel coronavirus pneumonia are collected at the early stage, and whether the purified N protein can detect anti-SARS-CoV-2 IgG antibody in serum is verified by an ELISA method, wherein the operation steps are as follows:
1. the 96-well plate was coated with N antigen at a concentration of 0.2. mu.g/well and 100. mu.l/well. 4 ℃ overnight. Negative control wells were set at coating.
2. The next day, the 96-well plate was removed, and after half an hour of rewarming at room temperature, the plate was washed with PBS 4 times, 1 minute each time.
3.1% BSA blocked 96-well plates, 100. mu.l/well, incubated for 30 min at 37 ℃.
Wash the plate 4 times with PBS for 1 minute each time.
5. Sample adding: each well was loaded with 100. mu.l after five-fold serum dilution. Incubate at 37 ℃ for 60 minutes.
Wash the plate 4 times with PBS for 1 minute each time.
7. HRP-labeled secondary antibody (anti-human IgG antibody): the stock solution is prepared according to the following steps of 1: after 2000 dilution, 100. mu.l of the buffer solution was added to each well and incubated at 37 ℃ for 30 minutes.
Wash the plate 4 times with PBS for 1 minute each time.
9. Color development: 50. mu.l of solution A was added to each well, and 50. mu.l of solution B was added to each well. The mixture was allowed to stand at room temperature for 15 minutes.
10. And (4) terminating: 100. mu.l of stop buffer was added to each well. The color is measured by a microplate reader within 10 minutes.
The results of the microplate reader are shown in FIGS. 2 (left side) and 3 (left side). OD of 17 clinically confirmed positive patients450The mean value was 1.282 (0.800-2.471), compared to the OD of the normal control group450The mean value is 0.075 (0.003-0.171), and the difference between the two values has statistical significance (t is 15.31, P<0.0001). OD of suspected specimen450The mean value is 0.092 (0.002-0.219), and the difference is significant with positive patients (t is 14.64, P<0.0001). The suspected patients had no statistical difference from normal (t 0.932, P0.36) as shown in fig. 4.
Example 3
Validation of clinical samples for detection of IgM antibodies
17 clinically confirmed cases, 22 suspected samples (final negative exclusion of nucleic acid) and 23 normal human samples of the novel coronavirus pneumonia are collected at the early stage, and whether the purified N protein can detect the anti-SARS-CoV-2 IgM antibody in serum is verified by an ELISA method, wherein the operation steps are as follows:
2. the 96-well plate was coated with N antigen at a concentration of 0.2. mu.g/well and 100. mu.l/well. 4 ℃ overnight. Negative control wells were set at coating.
3.2. The next day, the 96-well plate was removed, and after half an hour of rewarming at room temperature, the plate was washed with PBS 4 times, 1 minute each time.
4.3.1% BSA blocked 96-well plates, 100. mu.l/well, incubated for 30 min at 37 ℃.
Wash plate 4 times 1 min in PBS.
6.5. Sample adding: mu.l of serum was added to 15. mu.l of PBS, and 150. mu.l of IgG adsorbent was added. After centrifugation at 10000rpm for 10 minutes, 100. mu.l of the supernatant was assayed. Incubate at 37 ℃ for 60 minutes.
PBS wash plate 4 times, 1 min each time.
8.7. Secondary HRP-labeled antibody (anti-human IgM antibody): the stock solution is prepared according to the following steps of 1: after 2000 dilution, 100. mu.l of the buffer solution was added to each well and incubated at 37 ℃ for 30 minutes.
Wash the plate 4 times with PBS for 1 minute each time.
10. Color development: 50. mu.l of solution A was added to each well, and 50. mu.l of solution B was added to each well. The mixture was allowed to stand at room temperature for 15 minutes.
11. And (4) terminating: 100. mu.l of stop buffer was added to each well. The color is measured by a microplate reader within 10 minutes.
12. The results of the microplate reader are shown in FIGS. 2 (right side) and 3 (right side). OD of 17 clinically confirmed positive patients450Mean 0.474 (0.020-1.225), OD of normal control450The mean value is 0.076 (0.014-0.188), and the difference between the two values has statistical significance (t is 3.705, and P is 0.0009). OD of suspected specimen450The mean value was 0.117 (0.003-0.253), and was significantly different from positive patients (t: 2.850, P: 0.0084). The suspected patients had no statistical difference from normal (t 1.520, P0.1406) as shown in fig. 5.
The above-mentioned embodiments, objects, technical solutions and advantages of the present invention are further described in detail, it should be understood that the above-mentioned embodiments are only exemplary embodiments of the present invention, and are not intended to limit the scope of the present invention, and any modifications, equivalent substitutions, improvements and the like made within the spirit and principle of the present invention should be included in the scope of the present invention.
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Claims (9)
1. A novel coronavirus antigen colloidal gold rapid diagnosis kit is characterized by comprising SARS-CoV-2 nucleocapsid protein antigen colloidal gold.
2. The method for preparing the novel coronavirus antigen colloidal gold rapid diagnosis kit according to claim 1, which comprises the following steps:
(1) expressing and purifying SARS-CoV-2 nucleocapsid protein;
(2) preparing the SARS-CoV-2 nucleocapsid protein antigen colloidal gold.
3. The method for preparing the novel coronavirus antigen colloidal gold rapid diagnostic kit according to claim 2, wherein the expression of SARS-CoV-2 nucleocapsid protein in step (1) comprises the following steps:
(11) taking a nucleotide sequence corresponding to an amino acid sequence of SARS-CoV-2 nucleocapsid protein as a template, designing a forward primer with a nucleotide sequence shown as SEQ ID NO. 2 or SEQ ID NO. 3 and a reverse primer with a nucleotide sequence shown as SEQ ID NO. 4 or SEQ ID NO. 5;
(12) cloning a nucleotide sequence corresponding to SARS-CoV-2 nucleocapsid protein into a plasmid vector by PCR amplification, double digestion PCR product and the vector;
(13) transforming the escherichia coli by using the vector obtained in the step (12), and then carrying out induction culture;
(14) and (4) centrifuging the cultured escherichia coli to obtain the SARS-CoV-2 nucleocapsid recombinant protein.
4. The method for preparing a novel coronavirus antigen colloidal gold rapid diagnostic kit according to claim 3, wherein in the step (12), the plasmid vector is any one of pET28a, pET22b, pET21b, pET32a, pET40, pGex-6P-1 and pMal-c2 x.
5. The method for preparing the novel coronavirus antigen colloidal gold rapid diagnostic kit according to claim 3, wherein E.coli strain E.coli BL21 or Rosseta strain is adopted in the step (13), and the induction culture method comprises: the transformed Escherichia coli is coated on a plate containing ampicillin or kanamycin, incubated at 37 ℃, then a single colony is selected and inoculated in a liquid culture medium for culturing for 2-5h, and finally IPTG is added for induction and culturing for 3-18 h.
6. The method for preparing the novel coronavirus antigen colloidal gold rapid diagnostic kit according to claim 3, wherein the SARS-CoV-2 nucleocapsid recombinant protein is obtained by the following steps (14): and collecting the centrifuged escherichia coli thallus, adding a buffer solution to break the thallus, centrifuging to obtain a supernatant, and purifying by chromatography to obtain the SARS-CoV-2 nucleocapsid recombinant protein.
7. The method for preparing the novel coronavirus antigen colloidal gold rapid diagnostic kit according to claim 3, wherein the SARS-CoV-2 nucleocapsid protein is purified and then verified by using an anti-N protein antibody.
8. The method for preparing the novel coronavirus antigen colloidal gold rapid diagnosis kit according to claim 2, wherein the step (2) for preparing the SARS-CoV-2 nucleocapsid protein antigen colloidal gold comprises the following specific steps:
(21) preparing a colloidal gold solution by taking a chloroauric acid solution and a trisodium citrate aqueous solution as raw materials;
(22) transferring the SARS-CoV-2 nucleocapsid recombinant protein solution into a dialysis bag, and dialyzing overnight;
(23) adding the SARS-CoV-2 nucleocapsid recombinant protein after dialysis treatment into colloidal gold solution with the same volume, standing, then adding BSA solution, fully mixing uniformly, centrifuging, removing supernatant, and redissolving the colloidal gold precipitate by using protective solution to obtain the SARS-CoV-2 nucleocapsid protein antigen colloidal gold.
9. The method for preparing the novel coronavirus antigen colloidal gold rapid diagnosis kit according to claim 8, wherein in the step (22), the dialysis method comprises: diluting the SARS-CoV-2 nucleocapsid protein solution to 1mg/mL, transferring into dialysis bag, dialyzing with Tris buffer solution with concentration of 20mmol/L, pH value of 7.0 at 2-8 deg.C overnight.
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| CN111830261A (en) * | 2020-07-29 | 2020-10-27 | 安第斯抗体生物技术衡水有限公司 | Method and kit for rapidly detecting new coronavirus IgG antibody |
| WO2022027703A1 (en) * | 2020-08-06 | 2022-02-10 | 中国人民解放军陆军军医大学 | Monoclonal antibody of n antigen of sars-cov-2, detection method therefor and use thereof |
| CN111848754A (en) * | 2020-08-10 | 2020-10-30 | 四川大学华西医院 | Novel coronavirus N protein recombinant antigen and application thereof |
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