CN111426824A - Colloidal gold test paper and preparation method and application thereof - Google Patents

Colloidal gold test paper and preparation method and application thereof Download PDF

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Publication number
CN111426824A
CN111426824A CN202010246231.6A CN202010246231A CN111426824A CN 111426824 A CN111426824 A CN 111426824A CN 202010246231 A CN202010246231 A CN 202010246231A CN 111426824 A CN111426824 A CN 111426824A
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colloidal gold
test paper
antigen
gold
rbd
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蔡祥胜
项磊
马兴标
杨小蓉
陈兴露
陈三三
卢汉威
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First Affiliated Hospital of Guangdong Pharmaceutical University
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First Affiliated Hospital of Guangdong Pharmaceutical University
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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
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • 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 provides colloidal gold test paper and a preparation method and application thereof, and relates to the field of medical detection instruments. The preparation method comprises the following steps: preparing an antigen: adopting 293T cells to express a recombinant antigen RBD, wherein the amino acid sequence of the recombinant antigen RBD is shown as SEQ ID No. 1; preparing an antigen membrane: coating the antigen with a coating buffer solution, soaking in a sealing solution, and drying; preparing a gold-labeled antibody: preparing chloroauric acid solution, adding trisodium citrate solution, heating until the liquid is bright red, adjusting pH to 7.3-7.8, adding antibody, mixing, centrifuging, cleaning precipitate, and dissolving with gold-labeled antibody preservative solution; freeze-drying: spreading the gold-labeled antibody on a glass fiber membrane, and freeze-drying; preparing a test paper board: and (3) attaching the antigen membrane, the absorbent paper and the freeze-dried gold-labeled antibody glass fiber membrane to a plastic bottom plate to obtain the colloidal gold test paper. The invention aims at the specific fragment of the 2019-nCoV novel coronavirus to obtain the recombinant antigen RBD, and the recombinant antigen RBD is applied to colloidal gold test paper, so that the detection false positive can be reduced, and the detection accuracy and efficiency can be improved.

Description

Colloidal gold test paper and preparation method and application thereof
Technical Field
The invention relates to the field of medical detection instruments, in particular to colloidal gold test paper and a preparation method and application thereof.
Background
On 11/2/2020, the International Committee for the classification of Viruses (International Committee on Taxonomyof Viruses, ICTV) announced the formal name of the new coronavirus: severe acute respiratory syndrome coronavirus 2 (lung acute respiratory syndrome coronavirus 2, 2019-nCoV). On the day, the World Health Organization (WHO) was always on the spot in russell, pneumonia infected with the novel coronavirus would be formally named "COVID-19".
Currently there are still no specific drugs and vaccines for new coronaviruses, the most effective method is isolation and monitoring. However, due to the difference between the sampling site and the operation technique, missed detection often occurs, and other effective detection methods are urgently needed clinically as auxiliary detection methods. Furthermore, for some ambulatory populations, it is not practical to use nucleic acid detection, a method that requires reliance on large machinery. IgM and IgG detection in patient sera against novel coronavirus antigens is a reliable method. The colloidal gold labeling method is simple, convenient and quick, does not need machines, is suitable for large-scale screening, and is a powerful tool for detecting IgM and IgG generated by the induction of new coronavirus. At present, the domestic new coronavirus colloidal gold is not yet mature products to be marketed, and the early-stage products have high false positive, one reason of the high false positive is that the selection of antigens is different, and people easily think that the use of full-length antigens is more accurate, but neglect that the full-length antigens are easy to generate cross reaction.
2019-nCoV has a plurality of structural proteins such as spinous process protein S, matrix protein M, envelope protein E and nucleoprotein N. Currently, S antigen is commonly used as a detection reagent, however, the use of full-length S antigen is reflected in the industry as being prone to false positives. Therefore, screening more suitable antigens to prepare the colloidal gold detection card is an important problem to be solved by the current colloidal gold detection card.
Disclosure of Invention
Therefore, it is necessary to provide a method for preparing a colloidal gold test strip, which is used to obtain an RBD fragment of S protein for a specific fragment of a novel coronavirus, and is beneficial to reducing detection false positives and improving detection accuracy and efficiency.
A preparation method of the colloidal gold test paper comprises the following steps:
preparing an antigen: adopting 293T cells to express RBD antigen fragments on the S protein of 2019-nCoV, separating and purifying to obtain recombinant RBD antigen, wherein the amino acid sequence of the recombinant RBD antigen is shown as SEQ ID No. 1;
preparing an antigen membrane: coating the antigen with a coating buffer solution, soaking in a sealing solution, drying, and taking out for later use;
preparing a gold-labeled antibody: preparing a chloroauric acid solution, heating, adding a trisodium citrate solution, continuously heating until the liquid is bright red, stopping heating to obtain colloidal gold, adjusting the pH value of the colloidal gold to 7.3-7.8, adding a 2019-nCoV antibody, uniformly mixing, centrifuging, discarding supernatant, cleaning precipitate, and dissolving by using a gold-labeled antibody preservation solution for later use;
freeze-drying: spreading the gold-labeled antibody on a glass fiber membrane, and freeze-drying to obtain a freeze-dried gold-labeled antibody glass fiber membrane;
preparing a test paper board: cutting the gold-labeled antibody glass fiber membrane, and sticking the antigen membrane, the absorbent paper and the freeze-dried gold-labeled antibody glass fiber membrane on the plastic bottom plate to obtain the colloidal gold test paper.
SEQ ID No.1：
RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFK CYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSN NLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTN GVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFADDDDKAVPRDSGCKPCICTV PEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFN STFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMA KDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK
According to the preparation method, aiming at the specific fragment of the 2019-nCoV novel coronavirus, the selected fragment is a receptor binding domain protein (RBD) of a novel coronavirus S protein, namely the place where the S protein is connected with ACE2 and is an important fragment for determining the infection capacity of the novel coronavirus, research results show that the novel coronavirus S protein exists in a trimer form, each monomer contains more than 1300 amino acids, wherein more than 300 amino acids form the Receptor Binding Domain (RBD), and the spatial structure is also an important index for determining the antigen specificity.
In one embodiment, the antigen preparation step is specifically: screening RBD fragment genes of the S antigen of 2019-nCoV, connecting the RBD fragment genes with a vector, introducing the RBD fragment genes into 293T cells, culturing the 293T cells, expressing the recombinant antigen RBD, collecting culture supernatant, separating and purifying to obtain the recombinant antigen RBD.
It is understood that the nucleotide sequence of the RBD fragment of the S protein or a fragment thereof can be generally obtained by PCR amplification, recombinant methods, or synthetic methods. Once the target sequence is obtained, the sequence of interest can be obtained in large quantities by recombinant methods, i.e., cloning into a vector, transferring into a host cell, and isolating the sequence of interest from the propagated host cell by conventional methods.
In one embodiment, the vector is selected from a plasmid or a viral particle. The DNA sequence encoding the protein of the invention (or a fragment or derivative thereof) may be obtained entirely by chemical synthesis and may then be introduced into known vectors and cells. The polynucleotide sequence in the expression vector may be operably linked to an expression control sequence, and further comprises a ribosome binding site for translation initiation and transcription termination, and preferably one or more selectable markers.
The vector may enter the host cell by transformation, transduction, transfection or the like. The host cells are cultured for a period of time by methods well known in the art, and the supernatant is collected and the antigenic protein is purified by conventional methods such as physical or chemical methods.
The invention also provides the colloidal gold test paper prepared by the method. The test paper can detect 2019-nCoV efficiently, quickly and accurately.
The invention also provides a novel coronavirus colloidal gold test paper detection card, which comprises the following components in part by weight:
the base is provided with an installation groove, and the length direction of the installation groove extends along the length direction of the base;
the cover body covers the base and comprises a transparent observation window, a sample adding hole and a sample adding cover, and the sample adding cover is connected to the cover body and is embedded into the sample adding hole to seal the sample adding hole;
above-mentioned colloidal gold test paper is located in the mounting groove and the below of observation window, the detection zone of colloidal gold test paper is located the below of application of sample hole, at the serum warp the application of sample hole is instiled into behind the detection zone, through the observation window is observed the testing result of colloidal gold test paper.
Above-mentioned novel coronavirus's colloidal gold test paper detects card, adopt above-mentioned colloidal gold test paper on the one hand, be favorable to improving detection efficiency and accuracy, on the other hand, be in airtight state with the colloidal gold test paper setting in the lid, instil into serum through the application of sample hole when the test for the serum can be detected under inclosed environment, observe the testing result through the observation window, thereby can reduce aerosol transmission capacity, reduce the infection risk of new corona virus when detecting.
In one embodiment, the base further includes a plurality of clamping seats, and the clamping seats are arranged near the mounting groove and used for clamping the colloidal gold test paper.
In one embodiment, the base further includes a guide part disposed at an end of the mounting groove and used for guiding the colloidal gold test paper to move into the mounting groove.
In one embodiment, the cover further includes a stopper corresponding to the mounting groove, the stopper is hinged to the cover, and the stopper returns to the original position to seal the cover after the colloidal gold test paper rotates the stopper into the mounting groove.
In one embodiment, the area of the cross section of the sampling hole decreases in a direction away from the colloidal gold test paper.
In one embodiment, the area of the cross section of the sampling hole is conical with the decreasing area along the direction far away from the colloidal gold test paper.
In one embodiment, the sample adding device further comprises a connecting belt, one end of the connecting belt is connected to the sample adding cover, and the other end of the connecting belt is connected to the cover body.
In one embodiment, the cover is made of a transparent material.
Compared with the prior art, the invention has the following beneficial effects:
the preparation method of the colloidal gold test paper obtains RBD antigen genes through gene cloning, successfully expresses and purifies in 293T mammalian cells, and the colloidal gold test paper prepared by the recombinant antigen can reduce false positive detection and improve detection accuracy and efficiency.
According to the colloidal gold test paper detection card, on one hand, the colloidal gold test paper is adopted, so that the detection efficiency and the detection accuracy are improved, on the other hand, the colloidal gold test paper is arranged in the cover body and is in a closed state, serum is dripped through the sample adding hole during the test, the serum can be detected in a closed environment, the detection result is observed through the observation window, the aerosol transmission capacity can be reduced, and the infection risk of the new coronavirus during the detection is reduced.
Drawings
FIG. 1 is a schematic diagram showing the structure of a colloidal gold test paper detection card for the novel coronavirus in the example;
FIG. 2 is a schematic cross-sectional view taken along line A-A of FIG. 1;
FIG. 3 is a schematic diagram showing the structure of the base and the colloidal gold test paper in the example;
FIG. 4 is the results of a protein purification test;
FIG. 5 shows the results of the colloidal gold test card test;
FIG. 6 shows the results of IgM testing in example 1 and comparative example 1.
In the figure:
10-a cover body; 11-a viewing window; 12-a well; 13-a sample application cover; 131-a connecting band; 14-a shutter; 20-a base; 21-mounting a groove; 22-a card holder; 23-a guide.
Detailed Description
To facilitate an understanding of the invention, a more complete description of the invention will be given below in terms of preferred embodiments. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
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. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.
Example 1
The colloidal gold test paper is prepared by the following method:
1) preparing an antigen: expressing the 2019-nCoV gene recombinant antigen RBD by adopting 293T cells, collecting cell supernatant, separating and purifying to obtain the recombinant antigen RBD, wherein the amino acid sequence of the antigen RBD is shown as SEQ ID No. 1;
2) preparing an antigen membrane: coating the antigen with a coating buffer solution, airing at room temperature for 20min, soaking in a sealing solution at 37 ℃ for 60min, drying at 37 ℃ for 2h, and taking out for later use; the coating liquid is as follows: 0.05M pH9.6 sulfate buffer solution, 0.22 μ M membrane filtration, 4 deg.C storage, within a week; the closed working solution is: 2% BSA, 2% skim milk, 0.01M PBS (pH7.0), 0.22 μ M membrane filtration, 4 deg.C storage, within a week;
3) preparing a gold-labeled antibody, namely preparing a chloroauric acid solution with the mass fraction of 0.01%, heating, adding a trisodium citrate solution with the mass fraction of 1%, adding a trisodium citrate solution with the mass fraction of 4m L per 100m L chloroauric acid solution, continuously heating until the liquid is bright red, stopping heating to obtain colloidal gold, adjusting the pH value of the colloidal gold to 7.5, adding 10 mu g of 2019-nCoV antibody, uniformly mixing, standing for 30min, centrifuging at 13000rpm for 30min, discarding supernatant, washing and precipitating twice with a labeled washing solution, dissolving with a gold-labeled antibody preservation solution, preserving at 4 ℃ for one week;
4) freeze-drying: spreading the labeled colloidal gold on glass fiber membrane, and spreading the solution per ml for 6cm2Freeze-drying to obtain a freeze-dried gold-labeled antibody glass fiber membrane, and storing at 4 ℃;
5) preparing a test paper board: the antigen membrane, the absorbent paper and the freeze-dried gold-labeled antibody glass fiber membrane are adhered to a plastic bottom plate to form a test paper plate, and the test paper plate is cut into the colloidal gold test paper with the width of 2.5 mm.
Example 2
A novel coronavirus colloidal gold test paper detection card comprises a base 20, a cover 10 and colloidal gold test paper of embodiment 1.
The cover 10 covers the base 20, and is preferably made of a transparent material. The lid 10 includes transparent observation window 11, application of sample hole 12 and application of sample lid, application of sample lid connect in lid 10 for imbed in order to seal in the application of sample hole 12. The cover body 10 further includes a stopper 14 corresponding to the mounting groove 21, the top of the stopper 14 is hinged to the cover body 10, and after the colloidal gold test paper rotates the stopper 14 to enter the mounting groove 21, the stopper 14 returns to the original position to seal the cover body 10. The area of the cross section of the sampling hole 12 decreases progressively along the direction away from the colloidal gold test paper, and preferably, the area of the cross section of the sampling hole 12 decreases progressively along the direction away from the colloidal gold test paper. The sample addition cover can be buckled in the sample addition hole 12 and used for blocking the sample addition hole 12. In this embodiment, the sample addition cover is connected to the cover body 10 through a connection band 131, one end of the connection band 131 is connected to the sample addition cover, and the other end is connected to the cover body 10.
Fig. 3 is a schematic structural diagram of a base 20 and a colloidal gold test paper, and as shown in fig. 3, the base 20 is provided with an installation groove 21, and the length direction of the installation groove 21 extends along the length direction of the base 20. The base 20 further includes a plurality of clamping seats 22, and the clamping seats 22 are disposed near the mounting groove 21 and used for clamping the colloidal gold test paper. The base 20 further includes a guiding portion 23 disposed at an end of the mounting groove 21 for guiding the colloidal gold test paper to move into the mounting groove 21.
The colloidal gold test paper is arranged in the mounting groove 21 and below the observation window 11, and the detection area of the colloidal gold test paper is positioned below the sample adding hole 12. The pathogen 2019-nCoV is a novel coronavirus, and has a plurality of structural proteins such as spinous process protein S (spike), matrix protein M (matrix), envelope protein E (envelope) and nucleoprotein N (nucleoprotein). Coronavirus S proteins are involved in binding of the virus to cellular receptors and in mediating fusion of the virus to host membranes. Meanwhile, the S protein is mainly responsible for inducing host immune response and virus-neutralizing antibodies. The S protein also determines the tropism of the viral tissue. The S protein is called spike glycoprotein (spike glycoprotein) and is located at the outermost layer of the new coronavirus, like a prominent "crown". The coronavirus M protein is located inside and outside the virion, and is an important component of the virus envelope and a key protein for virus release. The coronavirus nucleocapsid protein (N protein) has a structural role and is involved in RNA synthesis. The E protein has the activity of an ion channel, and when the E protein plays the role of the ion channel, a pentamer higher-order structure is formed to play a role, coronavirus E and M control the assembly of virus particles, and research proves that the mutant strain with the deletion of the E protein loses pathogenicity. Therefore, the colloidal gold test paper adopts RBD segments expressed by genetic engineering to carry out coating and respectively detects the new coronavirus IgM and IgG in serum. Different colloidal gold test paper is adopted for detecting the new coronavirus IgM and IgG respectively, so that the interference in IgM and IgG detection can be avoided. After the serum is dripped into the detection area through the sample adding hole 12, the detection result of the colloidal gold test paper is observed through the observation window 11.
When the device is used, the colloidal gold test paper is close to the shutter 14 and pushes the shutter 14 to rotate, and the colloidal gold test paper extends into the mounting groove 21 under the guidance of the guide part 23.
After the colloidal gold test paper completely enters the mounting groove 21, the shutter 14 is reset to be in a closed state under the action of gravity, so that the colloidal gold test paper is in a sealed state.
Open the application of sample lid, drip into serum through application of sample hole 12 to the colloidal gold test paper and carry out the test of novel coronavirus, cover the application of sample lid after accomplishing the application of sample, observe the test result of colloidal gold test paper through observation window 11 at last.
This novel coronavirus's colloidal gold test paper detects card is in the airtight state with the setting of colloidal gold test paper in lid 10, drips into the serum through application of sample hole 12 when the test for the serum can be detected under inclosed environment, observes the testing result through observation window 11, thereby can reduce aerosol transmission ability, reduces the infection risk of new coronavirus when detecting.
Comparative example 1
A colloidal gold test strip, substantially the same as in example 1, except that an antigen membrane was prepared by coating a recombinant S full-length antigen.
Experimental example 1
2019-nCoV RBD protein purification and confirmation
The proteins obtained in example 1 were tested by SDS-PAGE and immunoblotting, respectively, and the SDS-PAGE analysis of the purified proteins is shown in FIG. 4(A), and the immunoblotting of the purified proteins is shown in FIG. 4(B), indicating that the method of example 1 can obtain high-purity RBD proteins.
Experimental example 2
Colloidal gold testing card
The positive specimen is adopted to preliminarily verify the effectiveness of the invention, and the quality of the colloidal gold test paper detection card in the embodiment 2 is tested. The test results are shown in fig. 5, the left graph is a negative control, the right graph is a positive specimen, and the results show that the colloidal gold test paper card of the invention can effectively detect the positive specimen.
Experimental example 3
Serum samples of 96 new patients with suspected coronary disease were tested using the test card of colloidal gold test paper of example 2, and the results of the tests using immunofluorescence assay (IFA) as a control are shown in the following table:
TABLE 1 serum sample of suspected New crown patient for New crown Virus antibody test results
Figure BDA0002434052230000061
As can be seen from the above table, the specificity of the card detected by the colloidal gold test paper of example 2 was 92.9% (79/85), and the sensitivity was 90% (10/11).
Experimental example 4
The specificity of the colloidal gold test strips of example 1 (coated recombinant RBD antigen) and comparative example 1 (coated recombinant S full-length antigen) was tested using the E L ISA method, and the same sera from 12 patients were tested in duplicate in each case, and the results are shown in FIG. 6 and the following table.
TABLE 2E L ISA test results
Figure BDA0002434052230000062
Figure BDA0002434052230000071
Note: the two complex holes are negative when the numerical value is less than 0.3, and the two complex holes are positive when the numerical value is more than or equal to 0.3.
As can be seen from the above table, the negative readings are lower for example 1, the positive readings are higher, and the difference between negative and positive is greater, while the difference between negative and positive is smaller for comparative example 1.
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present invention should be subject to the appended claims.
Sequence listing
<110> Guangdong university of pharmacy affiliated first Hospital
<120> colloidal gold test paper, and preparation method and application thereof
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<213> coronavirus protein RBD fragment (coronavirus)
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Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn
1 5 10 15
Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val
20 25 30
Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser
35 40 45
Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val
50 55 60
Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp
65 70 75 80
Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln
85 90 95
Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr
100 105110
Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly
115 120 125
Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys
130 135 140
Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr
145 150 155 160
Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser
165 170 175
Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val
180 185 190
Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly
195 200 205
Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Ala
210 215 220
Asp Asp Asp Asp Lys Ala Val Pro Arg Asp Ser Gly Cys Lys Pro Cys
225 230 235 240
Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe Ile Phe Pro Pro Lys
245 250 255
Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro Lys Val Thr Cys Val
260 265270
Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val Gln Phe Ser Trp Phe
275 280 285
Val Asp Asp Val Glu Val His Thr Ala Gln Thr Gln Pro Arg Glu Glu
290 295 300
Gln Phe Asn Ser Thr Phe Arg Ser Val Ser Glu Leu Pro Ile Met His
305 310 315 320
Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg Val Asn Ser Ala
325 330 335
Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Arg
340 345 350
Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln Met
355 360 365
Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asp Phe Phe Pro
370 375 380
Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly Gln Pro Ala Glu Asn
385 390 395 400
Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe Val
405 410 415
Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp Glu Ala Gly Asn Thr
420 425 430
Phe Thr Cys Ser Val Leu His Glu Gly Leu His Asn His His Thr Glu
435 440 445
Lys Ser Leu Ser His Ser Pro Gly Lys
450 455

Claims (10)

1. The preparation method of the colloidal gold test paper is characterized by comprising the following steps of:
preparing an antigen: adopting 293T cells to express RBD antigen fragments on the S protein of 2019-nCoV, separating and purifying to obtain a recombinant antigen RBD, wherein the amino acid sequence of the recombinant antigen RBD is shown as SEQ ID No. 1;
preparing an antigen membrane: coating the antigen with a coating buffer solution, soaking in a sealing solution, drying, and taking out for later use;
preparing a gold-labeled antibody: preparing a chloroauric acid solution, heating, adding a trisodium citrate solution, continuously heating until the liquid is bright red, stopping heating to obtain colloidal gold, adjusting the pH value of the colloidal gold to 7.3-7.8, adding a 2019-nCoV antibody, uniformly mixing, centrifuging, discarding supernatant, cleaning precipitate, and dissolving by using a gold-labeled antibody preservative solution for later use;
freeze-drying: spreading the colloidal gold labeled antibody on a glass fiber membrane, and freeze-drying to obtain a freeze-dried gold labeled antibody glass fiber membrane;
preparing a test paper board: cutting the gold-labeled antibody glass fiber membrane, and sticking the antigen membrane, the absorbent paper and the freeze-dried gold-labeled antibody glass fiber membrane on the plastic bottom plate to obtain the colloidal gold test paper.
2. The method of claim 1, wherein the antigen preparation step comprises: screening RBD fragment genes of the S antigen of 2019-nCoV, connecting the RBD fragment genes with a vector, introducing the RBD fragment genes into 293T cells, culturing the 293T cells, expressing the recombinant antigen RBD, collecting culture supernatant, separating and purifying to obtain the recombinant antigen RBD.
3. A colloidal gold test paper obtained by the production method according to claim 1 or 2.
4. The utility model provides a novel coronavirus's colloidal gold test paper detects card which characterized in that includes:
the base is provided with an installation groove, and the length direction of the installation groove extends along the length direction of the base;
the cover body covers the base and comprises a transparent observation window, a sample adding hole and a sample adding cover, and the sample adding cover is connected to the cover body and is embedded into the sample adding hole to seal the sample adding hole;
the colloidal gold test paper according to claim 3, which is disposed in the mounting groove and below the observation window, wherein the detection area of the colloidal gold test paper is located below the sample addition hole, and the detection result of the colloidal gold test paper is observed through the observation window after the serum is dropped into the detection area through the sample addition hole.
5. The new coronavirus reagent card of claim 4, wherein the base further comprises a plurality of card holders, and the card holders are arranged near the mounting grooves and used for holding the reagent cards.
6. The new coronavirus reagent card of claim 5, wherein the base further comprises a guide part disposed at an end of the mounting groove for guiding the reagent to move into the mounting groove.
7. The new coronavirus reagent card of claim 6, wherein the cover further comprises a door corresponding to the mounting groove, the door is hinged to the cover, and the door returns to the original position to seal the cover after the gold reagent rotates into the mounting groove.
8. The novel coronavirus gold strip test card of claim 7, wherein the cross-sectional area of the wells decreases in a direction away from the gold strip.
9. The novel coronavirus gold strip test card of claim 8, wherein the cross-sectional area of the well has a conical shape with decreasing size in a direction away from the gold strip.
10. The new coronavirus colloidal gold test strip test card of claim 9, further comprising a connecting strip, wherein one end of the connecting strip is connected to the sample application cover, and the other end of the connecting strip is connected to the cover body.
CN202010246231.6A 2020-03-31 2020-03-31 Colloidal gold test paper and preparation method and application thereof Pending CN111426824A (en)

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Application publication date: 20200717