CN116359498B - Immune chromatography test paper card for joint inspection of foot-and-mouth disease, african swine fever, swine fever and porcine reproductive and respiratory syndrome virus antibodies - Google Patents
Immune chromatography test paper card for joint inspection of foot-and-mouth disease, african swine fever, swine fever and porcine reproductive and respiratory syndrome virus antibodies Download PDFInfo
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—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
- G01N33/559—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody through a gel, e.g. Ouchterlony technique
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Chemical & Material Sciences (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Food Science & Technology (AREA)
- Physics & Mathematics (AREA)
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- Pathology (AREA)
- Virology (AREA)
- Biotechnology (AREA)
- Medicinal Chemistry (AREA)
- Microbiology (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Tropical Medicine & Parasitology (AREA)
- Dispersion Chemistry (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
The invention discloses an immunochromatography test paper card for joint inspection of foot-and-mouth disease, african swine fever, porcine reproductive and respiratory syndrome virus antibodies. The test paper card comprises a colloidal gold immune test strip for detecting the foot-and-mouth disease O-type virus antibody, a colloidal gold immune test strip for detecting the African swine fever virus antibody, a colloidal gold immune test strip for detecting the porcine reproductive and respiratory syndrome virus antibody and a shell; the colloidal gold immune test strips are all positioned in the shell, the sample pad of each colloidal gold immune test strip is partially overlapped, the rest parts are mutually separated in a radial mode, and the shell is provided with a sample adding hole corresponding to the overlapping part of the sample pad of each colloidal gold immune test strip. The test paper card can simultaneously detect four virus disease antibodies of foot-and-mouth disease type O, african swine fever, swine fever and porcine reproductive and respiratory syndrome virus at one time, and has the advantages of high sensitivity and specificity, simple and convenient use method, easy operation and good stability.
Description
Technical Field
The invention belongs to the field of animal epidemic disease diagnosis, and particularly relates to an immunochromatography test paper card, in particular to an immunochromatography test paper card for joint inspection of foot-and-mouth disease, african swine fever, pig reproduction and respiratory syndrome virus antibodies.
Background
The common viral diseases in pig raising have great influence on pig raising, and generally, the non-specific viral diseases do not lead to death of pigs, but because the viral diseases directly attack immune organs and immune cells, the immune system of pigs is disturbed, cross infection is further caused, once the pigs are ill, the loss caused by the viral diseases is serious, and if the diseased pork flows into the market, the pig raising is greatly restricted from developing. At present, common viral diseases in large-scale pig farms are foot-and-mouth disease, african swine fever, pig breeding and respiratory syndrome and the like, and the diseases are clinically difficult to distinguish between clinical symptoms and anatomical changes and the like. When a pig group is infected with a certain viral disease, vaccine immunity is adopted while the drug treatment is carried out, and the situation of the pig group is judged through antibody detection, so that the elimination measure is favorably executed.
However, the existing detection methods can not realize the on-site detection of the farm, and can not obtain the infection and immunity conditions in time. In conclusion, it is important to establish a detection method which is accurate and suitable for on-site detection and wide-range epidemiological investigation, so that measures can be taken in time, and the method has great significance in achieving complete eradication.
Diagnostic methods commonly used in the laboratory include virus neutralization assay (VNT), reverse transcription-polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay (ELISA), immunochromatography (immunochromatographytrips; ICT), and the like. Among them, immunochromatography is a rapid diagnostic technique which has been developed in recent years, and has the advantages of simple operation, rapid response, visual and clear result judgment, and no dependence on professional technical experience. Various immunochromatographic products for diagnosing animal epidemic diseases are currently available and widely used in laboratories and on site. However, the test strips can only detect single disease species, consume a large amount of serum and simultaneously repeatedly operate, bring a plurality of inconveniences to the diagnosis of the basic level site, and are not beneficial to the timely formulation and implementation of symptomatic treatment and epidemic control strategies of infected livestock groups.
Disclosure of Invention
The invention aims to solve the technical problem of how to detect four virus disease antibodies of foot-and-mouth disease virus, african swine fever virus, swine fever virus and porcine reproductive and respiratory syndrome virus simultaneously.
In a first aspect, the present invention provides an immunochromatographic test paper card.
The immunochromatography test paper card comprises a colloidal gold immune test paper strip for detecting foot-and-mouth disease O-type virus antibodies, a colloidal gold immune test paper strip for detecting African swine fever virus antibodies, a colloidal gold immune test paper strip for detecting porcine reproductive and respiratory syndrome virus antibodies and a shell;
The colloidal gold immune test strip consists of a sample pad, an immune colloidal gold probe pad, a nitrocellulose membrane and a water absorption pad which are sequentially connected and fixed on a bottom plate;
the immune colloidal gold probe pads are coated with colloidal gold markers;
The nitrocellulose membrane is coated with a detection line and a quality control line, and the detection line and the quality control line are mutually separated; the detection line is positioned at one end of the nitrocellulose membrane, which is close to the sample pad; the quality control line is positioned at one end of the nitrocellulose membrane, which is close to the water absorption pad;
the colloidal gold immune test strips are all positioned in the shell, the sample pad part of each colloidal gold immune test strip is overlapped, the rest parts are mutually separated in a radial way, the shell is provided with a sample adding hole corresponding to the sample pad overlapped part of each colloidal gold immune test strip, and the shell is provided with four observation windows corresponding to the detection line part and the quality control line part of each colloidal gold immune test strip respectively;
The colloidal gold marker in the colloidal gold immune test strip for detecting the foot-and-mouth disease O-type virus antibody consists of a foot-and-mouth disease O-type virus antigen 146S marked by colloidal gold and a rabbit anti-IgG marked by colloidal gold, and the detection line is coated with the foot-and-mouth disease O-type virus antigen 146S;
The colloidal gold marker in the colloidal gold immune test strip for detecting the African swine fever virus antibody consists of a colloidal gold-labeled African swine fever virus recombinant p30 protein and a colloidal gold-labeled rabbit anti-IgG, and the detection line is coated with the African swine fever virus recombinant p30 protein;
the colloidal gold marker in the colloidal gold immune test strip for detecting the swine fever virus antibody consists of a swine fever virus recombinant E2 protein marked by colloidal gold and a rabbit anti-IgG marked by colloidal gold, and the detection line is coated with the swine fever virus recombinant E2 protein;
The colloidal gold marker in the colloidal gold immune test strip for detecting the antibodies of the porcine reproductive and respiratory syndrome virus consists of a colloidal gold-labeled porcine reproductive and respiratory syndrome virus N protein and a colloidal gold-labeled rabbit anti-IgG, and the detection line is coated with the porcine reproductive and respiratory syndrome virus N protein.
In the immunochromatography test paper card, in the colloidal gold label of the colloidal gold immune test paper strip for detecting the foot-and-mouth disease O-type virus antibody, the volume ratio of the colloidal gold labeled foot-and-mouth disease O-type virus antigen 146S to the colloidal gold labeled rabbit anti-IgG is 9:1.
In the colloidal gold marker of the colloidal gold immune test strip for detecting the African swine fever virus antibody, the volume ratio of the colloidal gold-labeled African swine fever virus recombinant p30 protein to the colloidal gold-labeled rabbit anti-IgG is 9:1.
In the colloidal gold marker of the colloidal gold immune test strip for detecting the swine fever virus antibody, the volume ratio of the colloidal gold-labeled swine fever virus recombinant E2 protein to the colloidal gold-labeled rabbit anti-IgG is 9:1.
In the colloidal gold marker of the colloidal gold immune test strip for detecting the porcine reproductive and respiratory syndrome virus antibody, the volume ratio of the colloidal gold-labeled porcine reproductive and respiratory syndrome virus N protein to the colloidal gold-labeled rabbit anti-IgG is 9:1.
In the immunochromatography test paper card, the coating concentration of the foot-and-mouth disease O-type virus antigen 146S on the detection line of the colloidal gold immune test paper strip for detecting the foot-and-mouth disease O-type virus antibody can be 0.2-0.25 mg/mL, and specifically 0.2mg/mL.
The coating concentration of the African swine fever virus recombinant p30 protein on the detection line of the colloidal gold immune test strip for detecting the African swine fever virus antibody is 0.1-0.15 mg/mL, specifically 0.1mg/mL.
The coating concentration of the swine fever virus recombinant E2 protein on the detection line of the colloidal gold immune test strip for detecting the swine fever virus antibody is 0.55-0.6 mg/mL, specifically 0.57mg/mL.
The coating concentration of the N protein of the porcine reproductive and respiratory syndrome virus on the detection line of the colloidal gold immune test strip for detecting the porcine reproductive and respiratory syndrome virus antibody is 0.4-0.45 mg/mL, specifically 0.42mg/mL.
In the immunochromatography test paper card, the quality control lines of the colloidal gold immune test paper strip are coated with goat anti-rabbit IgG.
The coating concentration of the goat anti-rabbit IgG can be 0.2-0.25 mg/mL, and can be specifically 0.2mg/mL.
And the spraying amounts of the coating solution on the detection line and the quality control line are 1 mu L/cm.
In the immunochromatography test paper card, the shell is fan-shaped.
The included angle between two adjacent colloidal gold immune test strips is 35 degrees.
In a second aspect, the present invention provides a method for preparing the immunochromatographic test paper card.
The preparation method of the immunochromatography test paper card disclosed by the invention comprises the following steps of:
1) Respectively preparing a colloidal gold immune test strip for detecting the foot-and-mouth disease O-type virus antibody, a colloidal gold immune test strip for detecting the African swine fever virus antibody, a colloidal gold immune test strip for detecting the swine fever virus antibody and a colloidal gold immune test strip for detecting the porcine reproductive and respiratory syndrome virus antibody;
The colloidal gold immune test strip consists of a sample pad, an immune colloidal gold probe pad, a nitrocellulose membrane and a water absorption pad which are sequentially connected and fixed on a bottom plate;
the colloidal gold binding pads are coated with colloidal gold labels;
The nitrocellulose membrane is coated with a detection line and a quality control line, and the detection line and the quality control line are mutually separated; the detection line is positioned at one end of the nitrocellulose membrane, which is close to the sample pad; the quality control line is positioned at one end of the nitrocellulose membrane, which is close to the water absorption pad;
The colloidal gold marker in the colloidal gold immune test strip for detecting the foot-and-mouth disease O-type virus antibody consists of a foot-and-mouth disease O-type virus antigen 146S marked by colloidal gold and a rabbit anti-IgG marked by colloidal gold, and the detection line is coated with the foot-and-mouth disease O-type virus antigen 146S;
The colloidal gold marker in the colloidal gold immune test strip for detecting the African swine fever virus antibody consists of a colloidal gold-labeled African swine fever virus recombinant p30 protein and a colloidal gold-labeled rabbit anti-IgG, and the detection line is coated with the African swine fever virus recombinant p30 protein;
the colloidal gold marker in the colloidal gold immune test strip for detecting the swine fever virus antibody consists of a swine fever virus recombinant E2 protein marked by colloidal gold and a rabbit anti-IgG marked by colloidal gold, and the detection line is coated with the swine fever virus recombinant E2 protein;
The colloidal gold marker in the colloidal gold immune test strip for detecting the antibodies of the porcine reproductive and respiratory syndrome virus consists of a colloidal gold-labeled porcine reproductive and respiratory syndrome virus N protein and a colloidal gold-labeled rabbit anti-IgG, and the detection line is coated with the porcine reproductive and respiratory syndrome virus N protein;
2) Placing the colloidal gold immune test strip for detecting the foot-and-mouth disease O-type virus antibody, the colloidal gold immune test strip for detecting the African swine fever virus antibody, the colloidal gold immune test strip for detecting the swine fever virus antibody and the colloidal gold immune test strip for detecting the porcine reproductive and respiratory syndrome virus antibody in a shell, so that the sample pads of the colloidal gold immune test strip are partially overlapped, and the rest parts are mutually separated in a radial manner; the shell is provided with a sample adding hole corresponding to the overlapping part of the sample pads of each colloidal gold immune test strip, and four observation windows corresponding to the detection line part and the quality control line part of each colloidal gold immune test strip respectively.
In a third aspect, the present invention provides a novel use of the immunochromatographic test paper card described above or the immunochromatographic test paper card prepared according to the method described above.
The invention provides an application of the immunochromatography test paper card or the immunochromatography test paper card prepared by the method in detecting whether a sample to be detected contains foot-and-mouth disease O-type virus antibodies and/or African swine fever virus antibodies and/or porcine reproductive and respiratory syndrome virus antibodies.
The invention also provides an application of the immunochromatography test paper card or the immunochromatography test paper card prepared by the method in preparing a product for detecting whether a sample to be detected contains foot-and-mouth disease O-type virus antibodies and/or African swine fever virus antibodies and/or porcine reproductive and respiratory syndrome virus antibodies.
The invention also protects the application of the immunochromatography test paper card or the immunochromatography test paper card prepared by the method in the diagnosis, prevention and control of foot-and-mouth disease and/or african swine fever and/or porcine reproductive and respiratory syndrome.
The invention also provides an application of the immunochromatography test paper card or the immunochromatography test paper card prepared by the method in preparing products for diagnosing, preventing and controlling foot-and-mouth disease and/or african swine fever and/or porcine reproductive and respiratory syndrome.
In a fourth aspect, the invention provides a method for detecting whether a sample to be tested contains aftosa O-type virus antibodies and/or african swine fever virus antibodies and/or porcine reproductive and respiratory syndrome virus antibodies.
The method for detecting whether the sample to be detected contains foot-and-mouth disease O-type virus antibody and/or African swine fever virus antibody and/or porcine reproductive and respiratory syndrome virus antibody comprises the following steps: adding the immunochromatography test paper card or a sample adding hole of the immunochromatography test paper card prepared according to the method into a sample to be detected, then dripping an equivalent amount of expanding liquid into the sample adding hole, and observing the color development condition in an observation window in the immunochromatography test paper card after 10 minutes;
if the quality control zone and the detection zone in the observation window corresponding to a certain virus are developed, the sample to be detected contains the virus antibody; if the quality control zone in the observation window corresponding to a certain virus is colored and the detection zone is not colored, the sample to be detected does not contain the virus antibody.
In any of the above immunochromatographic test paper cards, the method or the application, the aftosa O-type virus antigen 146S is an aftosa O-type virus purified antigen 146S, and the aftosa O-type virus purified antigen 146S is described in literature "BARTELINGSJ,MELOENRH.A simple method for the quantification of 140S particles of foot and mouth disease virus(FMDV)[J].Arch Gesamte Virusforsch,1974,45(4):362-364.", and can be specifically prepared according to the method described below.
The amino acid sequence of the African swine fever virus recombinant p30 protein is shown as a sequence 1.
The amino acid sequence of the swine fever virus recombinant E2 protein is shown as a sequence 2.
The amino acid sequence of the N protein of the porcine reproductive and respiratory syndrome virus is shown as a sequence 3.
The invention has the following advantages:
(1) Can detect various common virus antibodies of pigs simultaneously: the immunochromatography test paper card can detect four virus disease antibodies of foot-and-mouth disease, african swine fever, porcine reproductive and respiratory syndrome viruses at one time.
(2) Sensitivity and specificity are high: the sensitivity of the immunochromatography test paper card for detecting the foot-and-mouth disease type O virus antibody is 91.8%, and the specificity is 96.1%; the sensitivity of the antibody for detecting African swine fever virus is 92.3%, and the specificity is 92%; the sensitivity of the antibody for detecting the swine fever virus is 98.9%, and the specificity is 100%; the sensitivity and specificity of the antibodies for detecting the porcine reproductive and respiratory syndrome virus are 87.9 percent.
(3) The use method is simple and convenient and is easy to operate: the immunochromatography test paper card has the advantages of simple and quick use method, visual and clear result judgment, no need of special instruments and special technical experience, operation by non-professional technicians according to specifications, and suitability for on-site and basic-level use.
(4) The stability is good: the immunochromatographic test paper card has the advantages of good stability, easy preservation, convenient carrying and low cost, and is suitable for clinical and on-site use.
Drawings
FIG. 1 is a schematic diagram of an immunochromatographic test strip for joint inspection of virus antibodies of foot-and-mouth disease, african swine fever, porcine reproductive and respiratory syndrome. Wherein 1 is a clamping shell, 2 is a reaction zone (comprising a C line and a T line), and 3 is a sample adding zone.
FIG. 2 is a side view of a colloidal gold immunochromatographic test strip. Wherein, 4 is a substrate, 5 is a sample absorption pad, 6 is an immune colloidal gold probe pad, 7 is a nitrocellulose membrane, and 8 is a water absorption pad.
Fig. 3 is a transmission electron microscope image and a scanning electron microscope image of colloidal gold particles. Wherein, the left image is a transmission electron microscope image; the right graph is a wavelength graph.
FIG. 4 is a schematic diagram of the results of antibody detection. Wherein, the left graph is a schematic diagram of the positive result of the antibody; the right panel is a schematic representation of antibody negative results.
Detailed Description
The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.
The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
The solvent (pH 7.5) of the gold colloid buffer in the following examples was water, and the solutes and concentrations thereof were as follows: 10mMPB, 0.25% Tween-20, 10% sucrose, 5% BSA.
The formulation of the extension liquid in the following examples is as follows: the domestic analysis pure disodium hydrogen phosphate 35.8g, sodium dihydrogen phosphate 1.56g, adding ultrapure water to 1000ml (pH 7.4), then adding 0.1ml Tween-20 and 500 μl Proclin300.
The foot-and-mouth disease O virus purified antigen 146S in the following examples is described in literature "BARTELINGSJ,MELOENRH.A simple method for the quantification of 140S particles of foot and mouth disease virus(FMDV)[J].Arch Gesamte Virusforsch,1974,45(4):362-364." and is specifically prepared as follows:
1. propagation and inactivation of the virus seed: inoculating O/MYA98/JSCJ/2010 cell virus seed into a well-grown BHK-21 monolayer cell rotating bottle according to the amount of 5% -10% of a maintenance solution, culturing for 8-12 hours at 37 ℃, when the cell lesion reaches more than 90%, regulating the pH value to 7.6-7.8 by using 7.5% NaHCO 3 solution, adding penicillin (100 IU/mL) and streptomycin (100 IU/mL), inactivating virus solution by using 2mmol/L of diethyl imine at 30 ℃ for 24 hours, adding 2% sodium thiosulfate for blocking, centrifuging at 4000r/min and 2-8 ℃ for 30 minutes, taking supernatant (O/MYA 98/JSCJ/2010 inactivated vaccine virus antigen solution), and freezing at 20 ℃.
2. Degreasing and concentration of antigen: (1) 10000mL of O/MYA98/JSCJ/2010 inactivated vaccine virus antigen solution stored at the temperature of minus 20 ℃ is added with PEG 6000 and 40g/L NaCl according to 80g/L, and the mixture is fully stirred for 4 hours at the temperature of 2-8 ℃ overnight; (2) Centrifuging the virus solution 8000r/min obtained in the step (1) for 45min, and discarding the supernatant; (3) PB buffer (0.2 mol/L Na 2HPO4,0.2mol/L NaH2PO4, pH 7.4) the pellet obtained in step (2) (on ice operation), the homogenizer was repeatedly ground to a final volume of 100mL; (4) Adding trichloroethylene with the volume of 2 times into the solution obtained in the step (3) in a closed container, carrying out intense shaking and full emulsification, centrifuging at the temperature of 2-8 ℃ for 30 minutes at the speed of 8000r/min, and collecting an upper layer solution; (5) Centrifuging the supernatant obtained in the step (4) for 180 minutes at the temperature of 2-8 ℃ and the speed of 45000r/min, and discarding the supernatant; (6) Suspending and precipitating PB buffer solution, and grinding in a homogenizer to a final volume of 6mL; (7) Adding 1% sodium deoxycholate solution into the solution obtained in the step (6), and shaking uniformly, wherein the temperature is 2-8 ℃ overnight, thus obtaining the concentrated virus antigen.
3. Sucrose density gradient centrifugation: preparing 50% sucrose mother liquor; preparing four concentration gradient sucrose solutions of 45%, 35%, 25% and 15% by using 50% sucrose; preparing sucrose gradient, centrifuging, adding 2.5mL of 45% sucrose solution into a centrifuge tube, sequentially stacking 2.5mL of 35%, 25% and 15% sucrose solution, preparing 6 sucrose gradient columns, and standing at 2-8 ℃ for balancing overnight; the next day 6mL of concentrated virus antigen solution was added to the top of 6 sucrose gradient columns (1 mL for each column) and centrifuged at 35000r/min for 150min at 2-8deg.C.
4. Antigen 146S collection: after centrifugation, 1 tube of the above gradient centrifugation purified antigen was taken, the gradient centrifugation antigen was aspirated from top to bottom, 1 fraction was collected every 0.5mL, and the collected fractions were sequentially numbered, and 22 fractions were collected in total. The remaining 5-branch separation tubes repeat the above operation and the same-layer collection fractions are combined into 22 fraction tubes, respectively. 22 fractions OD 259nm were measured by ultraviolet spectrophotometry (Shimadzu-UV 260) and fractions with an OD 259nm greater than 0.4 were pooled as foot-and-mouth disease virus O purified antigen 146S according to the regional analysis of the possible presence of FMDV whole virus 146S when significant absorption peaks appear in the fractions collected at 15-19.
The preparation method of the African swine fever virus recombinant p30 protein (the amino acid sequence is shown as the sequence 1) in the following example is as follows: the gene sequence of African swine fever virus gene II Georgia 2008/1 strain p30 (Genebank number: MH 910495) in GenBank database is used as a reference sequence for expressing a target gene, and the gene synthesis method is utilized to synthesize the p30 gene sequence from Kirschner Biotechnology, inc., and the p30 gene sequence is introduced into pGEM T-easy vector to construct recombinant plasmid pGEM-p30 with the p30 gene. The recombinant plasmid pGEM-p30 is subjected to double digestion by using restriction enzymes EcoR I and Not I, the target fragment of the p30 gene is recovered, and the pET-28a vector recovered after the same double digestion is connected overnight at 16 ℃. And transforming DH5 alpha competent cells, screening positive strains, and extracting positive plasmids. The plasmid is verified by double digestion of EcoR I and Not I, and the result shows that two obvious bands appear and the size of the target fragment is about 585bp, which accords with the expectations; the PCR amplification (T7 general primer pair T7P:5'-TAATACGACTCACTATAGGG-3'; T7T:5'-GCTAGTTATTGCT CAGCGG-3') shows a gene fragment with the size of about 884bp, the plasmid with consistent enzyme digestion and PCR identification is subjected to sequencing identification according to the expectations, the sequencing result shows that the homology of the P30 gene sequence of the African Swine Fever Virus (ASFV) gene II type Georgia 2008/1 strain is 100%, and the plasmid with correct sequencing is named as pET-28a-P30 recombinant positive plasmid. pET-28a-p30 recombinant positive plasmid was transformed into Rosetta competent cells. The single clone was picked and sent to the biological engineering (Shanghai) Co., ltd for sequencing. Bacterial liquid 1 with correct sequencing: 100 is added into a 2 XYT culture medium for expansion culture until the OD 600nm is about 3.0, IPTG with the final concentration of 0.1mmol/L is added for induction expression for 4h at 37 ℃, the sediment is collected after centrifugation for 10min at 12000r/min, the sediment is frozen at-80 ℃ after PBS is resuspended, and then thawed, repeatedly frozen and thawed for 3 times, and ultrasonically crushed until the liquid is clear (ultrasonic condition: voltage 200V, work for 3s, pause for 3 s); centrifugation was performed at 12000r/min for 10min, and the pellet and supernatant were collected, respectively, and protein expression was analyzed by SDS-PAGE. The precipitate was cleaved with 8mol/L urea and then renatured with 3mol/L guanidine hydrochloride, and purified by Ni column method. The purification method comprises the following steps: the hybrid protein was eluted 3 times with 10mL each with 20mmol/L and 50mmol/L imidazole, respectively, followed by elution of the target protein with 200mmol/L imidazole.
The amino acid sequence of the recombinant E2 protein of the classical swine fever virus in the following examples is shown as a sequence 2, and is a product of Shandong blue all Biotech Co., ltd., product number: 20210816.
The amino acid sequence of the N protein of the porcine reproductive and respiratory syndrome virus in the following examples is shown in the sequence 3, and is a product of Shandong blue all Biotechnology Co., ltd., product number: AT01PRRSVA.
The rabbit anti-IgG in the following examples is a product from Shanghai Jie Biotechnology Inc., cat#: 161025.
Sheep anti-rabbit IgG in the following examples is a product from Shanghai Jie Biotechnology Inc., cat No.: 161125.
Example 1 preparation of colloidal gold immunoassay test strip for foot-and-mouth disease O-type Virus antibody
1. Preparation of colloidal gold solution
1ML of 1% chloroauric acid aqueous solution is taken and added with 99mL of distilled water to prepare 0.01% chloroauric acid aqueous solution, the solution is heated to boiling, 1.5mL of trisodium citrate aqueous solution with the mass percentage concentration of 1% is added, the solution is continuously boiled until the liquid turns into wine red, and a colloidal gold solution is obtained and is cooled for standby.
2. Preparation of immune colloidal gold
Preparation of immune colloidal gold (1): adjusting the pH value of the colloidal gold solution to 7.5 by using 0.02M K 2CO3 solution; then adding the foot-and-mouth disease O-type virus purified antigen 146S according to the optimal labeling amount of 1.1 mug/mL, and magnetically stirring for 40min; adding 100g/L BSA to a final concentration of 10g/L, and magnetically stirring for 30min; and finally, centrifuging at 10000r/min for 40min, discarding supernatant, and suspending and dissolving the precipitate with a gold colloid buffer solution for later use.
Preparation of immune colloidal gold (2): adjusting the pH value of the colloidal gold solution to 8.5 by using 0.02M K 2CO3 solution; then adding rabbit anti-IgG according to the optimal labeling amount of 4 mug/mL, and magnetically stirring for 40min; adding 100g/L BSA to a final concentration of 10g/L, and magnetically stirring for 30min; and finally, centrifuging at 10000r/min for 40min, discarding supernatant, and suspending and dissolving the precipitate with a gold colloid buffer solution for later use.
And mixing the immune colloidal gold (1) and the immune colloidal gold (2) in a fixed ratio of 9:1 to obtain the foot-and-mouth disease O-type virus immune colloidal gold solution.
3. Preparation of immune colloidal gold probe pad
The immune colloidal gold solution of foot-and-mouth disease type O virus was sprayed onto a glass fiber pad (Ahlstrom Co.) having a length of 270mm and a width of 10mm, and dried at 37℃for 0.5 hours to obtain an immune colloidal gold probe pad.
4. Preparation of nitrocellulose membrane containing quality control line (C line) and detection line (T line)
And (3) adjusting the foot-and-mouth disease O-type virus purified antigen 146S to 0.2mg/mL, adjusting the goat anti-rabbit antibody to the working concentration of 0.2mg/mL, respectively spraying on a nitrocellulose membrane to form a detection zone and a quality control zone, drying at 37 ℃ for 0.5 hour, and preserving for later use.
5. Assembly of test paper
And sequentially and mutually bonding a sample pad, an immune colloidal gold probe pad, a nitrocellulose membrane containing a quality control line (C line) and a detection line (T line) and absorbent paper on a substrate (PVC material), and cutting into test paper with the width of 3.8mm according to requirements to obtain the foot-and-mouth disease O-type virus antibody colloidal gold immunochromatographic test paper (figure 2).
6. Sample detection
Taking immunochromatography test paper, simultaneously dripping 30 mu L of sample and an equivalent amount of extension liquid on an absorption pad of the test paper, timing for 10min after finishing the reaction on a nitrocellulose membrane, and observing the result.
7. Result determination
Positive: the quality control belt and the detection belt are both developed; negative: the quality control band develops color and the detection band does not develop color.
Example 2 preparation of African swine fever Virus antibody colloidal gold immunoassay test strip
1. Preparation of colloidal gold solution
1ML of 1% chloroauric acid aqueous solution is taken and added with 99mL of distilled water to prepare 0.01% chloroauric acid aqueous solution, the solution is heated to boiling, 1.5mL of trisodium citrate aqueous solution with the mass percentage concentration of 1% is added, the solution is continuously boiled until the liquid turns into wine red, and a colloidal gold solution is obtained and is cooled for standby.
2. Preparation of immune colloidal gold
Preparation of immune colloidal gold (1): adjusting the pH value of the colloidal gold solution to 9.0 by using 0.02M K 2CO3 solution; then adding the African swine fever virus recombinant p30 protein according to the optimal labeling amount of 2.7 mug/mL, and magnetically stirring for 40min; adding 100g/L BSA to a final concentration of 10g/L, and magnetically stirring for 30min; and finally, centrifuging at 10000r/min for 40min, discarding supernatant, and suspending and dissolving the precipitate with a gold colloid buffer solution for later use.
Preparation of immune colloidal gold (2): adjusting the pH value of the colloidal gold solution to 8.5 by using 0.02M K 2CO3 solution; then adding rabbit anti-IgG according to the optimal labeling amount of 4 mug/mL, and magnetically stirring for 40min; adding 100g/L BSA to a final concentration of 10g/L, and magnetically stirring for 30min; and finally, centrifuging at 10000r/min for 40min, discarding supernatant, and suspending and dissolving the precipitate with a gold colloid buffer solution for later use.
And mixing the immune colloidal gold (1) and the immune colloidal gold (2) in a fixed ratio of 9:1 to obtain the African swine fever virus immune colloidal gold solution.
3. Preparation of immune colloidal gold probe pad
The immune colloidal gold solution of African swine fever virus was sprayed onto a glass fiber pad (Ahlstrom Co.) having a length of 270mm and a width of 10mm, and dried at 37℃for 0.5 hours to obtain an immune colloidal gold probe pad.
4. Preparation of nitrocellulose membrane containing quality control line (C line) and detection line (T line)
The recombinant p30 protein of African swine fever virus is regulated to 0.1mg/mL, goat anti-rabbit antibody is regulated to the working concentration of 0.2mg/mL, and the working concentration is respectively sprayed on a nitrocellulose membrane to form a detection zone and a quality control zone, and the detection zone and the quality control zone are dried at 37 ℃ for 0.5 hour and stored for standby.
5. Assembly of test paper
And sequentially and mutually bonding a sample pad, an immune colloidal gold probe pad, a nitrocellulose membrane containing a quality control line (C line) and a detection line (T line) and absorbent paper on a substrate (PVC material), and cutting into test paper with the width of 3.8mm according to requirements to obtain the African swine fever virus antibody colloidal gold immunochromatographic test paper (figure 2).
6. Sample detection
Taking immunochromatography test paper, simultaneously dripping 30 mu L of sample and an equivalent amount of extension liquid on an absorption pad of the test paper, timing for 10min after finishing the reaction on a nitrocellulose membrane, and observing the result.
7. Result determination
Positive: the quality control belt and the detection belt are both developed; negative: the quality control band develops color and the detection band does not develop color.
Example 3 preparation of colloidal gold immunoassay test strip for antibodies to classical swine fever Virus
1. Preparation of colloidal gold solution
1ML of 1% chloroauric acid aqueous solution is taken and added with 99mL of distilled water to prepare 0.01% chloroauric acid aqueous solution, the solution is heated to boiling, 1.5mL of trisodium citrate aqueous solution with the mass percentage concentration of 1% is added, the solution is continuously boiled until the liquid turns into wine red, and a colloidal gold solution is obtained and is cooled for standby.
2. Preparation of immune colloidal gold
Preparation of immune colloidal gold (1): adjusting the pH value of the colloidal gold solution to 7.5 by using 0.02M K 2CO3 solution; then adding the recombinant E2 protein of the swine fever virus according to the optimal labeling amount of 34 mug/mL, and magnetically stirring for 40min; adding 100g/L BSA to a final concentration of 10g/L, and magnetically stirring for 30min; and finally, centrifuging at 10000r/min for 40min, discarding supernatant, and suspending and dissolving the precipitate with a gold colloid buffer solution for later use.
Preparation of immune colloidal gold (2): adjusting the pH value of the colloidal gold solution to 8.5 by using 0.02M K 2CO3 solution; then adding rabbit anti-IgG according to the optimal labeling amount of 4 mug/mL, and magnetically stirring for 40min; adding 100g/L BSA to a final concentration of 10g/L, and magnetically stirring for 30min; and finally, centrifuging at 10000r/min for 40min, discarding supernatant, and suspending and dissolving the precipitate with a gold colloid buffer solution for later use.
And mixing the immune colloidal gold (1) and the immune colloidal gold (2) in a fixed ratio of 9:1 to obtain the swine fever virus immune colloidal gold solution.
3. Preparation of immune colloidal gold probe pad
The immune colloidal gold solution of the swine fever virus was sprayed onto a glass fiber pad (Ahlstrom Co.) having a length of 270mm and a width of 10mm, and dried at 37℃for 0.5 hour to obtain an immune colloidal gold probe pad.
4. Preparation of nitrocellulose membrane containing quality control line (C line) and detection line (T line)
The recombinant E2 protein of the hog cholera virus is regulated to 0.57mg/mL, the goat anti-rabbit antibody is regulated to the working concentration of 0.2mg/mL, and the goat anti-rabbit antibody is respectively sprayed on a nitrocellulose membrane to form a detection zone and a quality control zone, dried for 0.5 hour at 37 ℃ and preserved for standby.
5. Assembly of test paper
And sequentially and mutually bonding a sample pad, an immune colloidal gold probe pad, a nitrocellulose membrane containing a quality control line (C line) and a detection line (T line) and absorbent paper on a substrate (PVC material), and cutting into test paper with the width of 3.8mm according to requirements to obtain the swine fever virus antibody colloidal gold immunochromatographic test paper (figure 2).
6. Sample detection
Taking immunochromatography test paper, simultaneously dripping 30 mu L of sample and an equivalent amount of extension liquid on an absorption pad of the test paper, timing for 10min after finishing the reaction on a nitrocellulose membrane, and observing the result.
7. Result determination
Positive: the quality control belt and the detection belt are both developed; negative: the quality control band develops color and the detection band does not develop color.
Example 4 preparation of colloidal gold immunoassay test strip for antibodies to porcine reproductive and respiratory syndrome Virus
1. Preparation of colloidal gold solution
1ML of 1% chloroauric acid aqueous solution is taken and added with 99mL of distilled water to prepare 0.01% chloroauric acid aqueous solution, the solution is heated to boiling, 1.5mL of trisodium citrate aqueous solution with the mass percentage concentration of 1% is added, the solution is continuously boiled until the liquid turns into wine red, and a colloidal gold solution is obtained and is cooled for standby.
2. Preparation of immune colloidal gold
Preparation of immune colloidal gold (1): adjusting the pH value of the colloidal gold solution to 7.5 by using 0.02M K 2CO3 solution; then adding N protein of porcine reproductive and respiratory syndrome virus according to the optimal marking amount of 4 mug/mL, and magnetically stirring for 40min; adding 100g/L BSA to a final concentration of 10g/L, and magnetically stirring for 30min; and finally, centrifuging at 10000r/min for 40min, discarding supernatant, and suspending and dissolving the precipitate with a gold colloid buffer solution for later use.
Preparation of immune colloidal gold (2): adjusting the pH value of the colloidal gold solution to 8.5 by using 0.02M K 2CO3 solution; then adding rabbit anti-IgG according to the optimal labeling amount of 4 mug/mL, and magnetically stirring for 40min; adding 100g/L BSA to a final concentration of 10g/L, and magnetically stirring for 30min; and finally, centrifuging at 10000r/min for 40min, discarding supernatant, and suspending and dissolving the precipitate with a gold colloid buffer solution for later use.
And mixing the immune colloidal gold (1) and the immune colloidal gold (2) in a fixed ratio of 9:1 to obtain the immune colloidal gold solution of the porcine reproductive and respiratory syndrome virus.
3. Preparation of immune colloidal gold probe pad
The immune colloidal gold solution of the porcine reproductive and respiratory syndrome virus is sprayed on a glass fiber pad (Ahlstrom company) with the length of 270mm and the width of 10mm, and dried for 0.5 hour at 37 ℃ to obtain the immune colloidal gold probe pad.
4. Preparation of nitrocellulose membrane containing quality control line (C line) and detection line (T line)
And (3) regulating the N protein of the porcine reproductive and respiratory syndrome virus to 0.42mg/mL, regulating the goat anti-rabbit antibody to the working concentration of 0.2mg/mL, respectively spraying on a nitrocellulose membrane to form a detection zone and a quality control zone, drying at 37 ℃ for 0.5 hour, and preserving for later use.
5. Assembly of test paper
And sequentially and mutually bonding a sample pad, an immune colloidal gold probe pad, a nitrocellulose membrane containing a quality control line (C line) and a detection line (T line) and absorbent paper on a substrate (PVC material), and cutting into test paper with the width of 3.8mm according to requirements to obtain the colloidal gold immunochromatographic test paper (figure 2) for antibodies of the porcine reproductive and respiratory syndrome virus.
6. Sample detection
Taking immunochromatography test paper, simultaneously dripping 30 mu L of sample and an equivalent amount of extension liquid on an absorption pad of the test paper, timing for 10min after finishing the reaction on a nitrocellulose membrane, and observing the result.
7. Result determination
Positive: the quality control belt and the detection belt are both developed; negative: the quality control band develops color and the detection band does not develop color.
Example 5 preparation of immunochromatographic test paper card for Joint detection of foot-and-mouth disease type O (FMD), african Swine Fever (ASF), swine fever (CSF), porcine Reproductive and Respiratory Syndrome (PRRS) virus antibody
The test strips prepared in examples 1-4 are put into a fan-shaped shell (1 in fig. 1), the part of the shell corresponding to each test strip sample pad is provided with a sample adding hole (3 in fig. 1), the 4 test strip sample pads are partially overlapped and are provided with sample adding holes at the overlapping part of the sample pads, the rest parts are mutually separated in a radial way, the included angles between two adjacent test strips are 35 degrees, finally, the fan shape is presented, the positions corresponding to the detection line and the quality control line of the test strip are provided with observation windows (2 in fig. 1), the positions of the detection line and the quality control line are respectively marked with T and C, and then, after drying agent is added, the test strip is sealed and stored in a single package, thus obtaining the foot-and-mouth disease O type (FMD), african Swine Fever (ASF), swine fever (CSF), porcine Reproductive and Respiratory Syndrome (PRRS) virus antibody joint detection immunochromatography test paper card.
Example 6 application of Joint detection immunochromatography test paper card for foot-and-mouth disease type O (FMD), african Swine Fever (ASF), swine fever (CSF), porcine Reproductive and Respiratory Syndrome (PRRS) virus antibody
1. Experimental materials
Foot-and-mouth disease type O virus (FMDV) antibody positive serum (61 parts) and negative serum (52 parts) are both provided by the national foot-and-mouth disease reference laboratory.
African Swine Fever Virus (ASFV) antibody positive serum (52 parts) and negative serum (50 parts) were both supplied by the national African swine fever area laboratory (Lanzhou).
Swine Fever (CSFV) antibody positive serum (92 parts) and negative serum (40 parts), and Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) antibody positive serum (66 parts) and negative serum (83 parts) were all provided by the animal institute of Lanzhou, national academy of agricultural sciences.
The sample should be detected as soon as possible after collection, if the sample can not be detected in time, the sample can be stored for 3 days at 2-8 ℃, if the sample needs to be stored for a long time, the sample needs to be stored below-20 ℃, and repeated freezing and thawing are avoided. The sample needs to be transported under vacuum or other device conditions containing dry ice or ice bags.
2. Experimental method
And sucking 30 mu L of serum sample to be detected, adding the serum sample into a sample adding hole in an immunochromatography test paper card, slowly dripping an equivalent amount of extension liquid, and observing the color development condition on the nitrocellulose membrane in an observation window after 10 minutes.
If the quality control belt and the detection belt are both developed, the serum sample to be detected is positive; if the quality control band is colored and the detection band is not colored, the serum sample to be detected is negative.
3. Experimental results
The results show that: the sensitivity of the colloidal gold immunochromatographic test paper card for rapidly diagnosing the swine virus disease antibody for detecting the foot-and-mouth disease type O virus antibody is 91.8%, and the specificity is 96.1%; the sensitivity of the antibody for detecting African swine fever virus is 92.3%, and the specificity is 92%; the sensitivity of the antibody for detecting the swine fever virus is 98.9%, and the specificity is 100%; the sensitivity and specificity of the antibodies for detecting the porcine reproductive and respiratory syndrome virus are 87.9 percent (table 1).
The detection result shows that the colloidal gold immunochromatography test paper card can be used for rapidly detecting the common virus diseases of pigs.
Table 1, results of the detection of serum samples by the joint test strips are summarized
Note that: sensitivity% = positive detection number/detection serum number; specific% = negative detection number/detection serum number.
The present application is described in detail above. It will be apparent to those skilled in the art that the present application can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the application and without undue experimentation. While the application has been described with respect to specific embodiments, it will be appreciated that the application may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the application following, in general, the principles of the application and including such departures from the present disclosure as come within known or customary practice within the art to which the application pertains. The application of some of the basic features may be done in accordance with the scope of the claims that follow.
Claims (7)
1. An immunochromatography test paper card comprises a colloidal gold immune test paper strip for detecting foot-and-mouth disease O-type virus antibodies, a colloidal gold immune test paper strip for detecting African swine fever virus antibodies, a colloidal gold immune test paper strip for detecting porcine reproductive and respiratory syndrome virus antibodies and a shell;
The colloidal gold immune test strip consists of a sample pad, an immune colloidal gold probe pad, a nitrocellulose membrane and a water absorption pad which are sequentially connected and fixed on a bottom plate;
the immune colloidal gold probe pads are coated with colloidal gold markers;
The nitrocellulose membrane is coated with a detection line and a quality control line, and the detection line and the quality control line are mutually separated; the detection line is positioned at one end of the nitrocellulose membrane, which is close to the sample pad; the quality control line is positioned at one end of the nitrocellulose membrane, which is close to the water absorption pad;
the colloidal gold immune test strips are all positioned in the shell, the sample pad part of each colloidal gold immune test strip is overlapped, the rest parts are mutually separated in a radial way, the shell is provided with a sample adding hole corresponding to the sample pad overlapped part of each colloidal gold immune test strip, and the shell is provided with four observation windows corresponding to the detection line part and the quality control line part of each colloidal gold immune test strip respectively;
The colloidal gold marker in the colloidal gold immune test strip for detecting the foot-and-mouth disease O-type virus antibody consists of a foot-and-mouth disease O-type virus antigen 146S marked by colloidal gold and a rabbit anti-IgG marked by colloidal gold, and the detection line is coated with the foot-and-mouth disease O-type virus antigen 146S; the preparation method of the colloidal gold labeled foot-and-mouth disease O-type virus antigen 146S comprises the following steps: adjusting the pH value of the colloidal gold solution to 7.5 by using 0.02M K 2CO3 solution; then adding foot-and-mouth disease O-type virus purified antigen 146S according to the mark amount of 1.1 mug/mL, and magnetically stirring for 40 min; then 100 g/L BSA is added to the final concentration of 10 g/L, and the mixture is magnetically stirred for 30 min; finally, 10000 r/min is centrifuged for 40 min, the supernatant is discarded, and the precipitate is suspended and dissolved by gold colloid buffer solution;
The colloidal gold marker in the colloidal gold immune test strip for detecting the African swine fever virus antibody consists of a colloidal gold-labeled African swine fever virus recombinant p30 protein and a colloidal gold-labeled rabbit anti-IgG, and the detection line is coated with the African swine fever virus recombinant p30 protein; the preparation method of the colloidal gold labeled African swine fever virus recombinant p30 protein comprises the following steps: adjusting the pH value of the colloidal gold solution to 9.0 by using 0.02M K 2CO3 solution; then adding the African swine fever virus recombinant p30 protein according to the mark amount of 2.7 mug/mL, and magnetically stirring for 40 min; adding 100 g/L BSA to a final concentration of 10 g/L, and magnetically stirring for 30min; finally, 10000 r/min is centrifuged for 40 min, the supernatant is discarded, and the precipitate is suspended and dissolved by gold colloid buffer solution;
The colloidal gold marker in the colloidal gold immune test strip for detecting the swine fever virus antibody consists of a swine fever virus recombinant E2 protein marked by colloidal gold and a rabbit anti-IgG marked by colloidal gold, and the detection line is coated with the swine fever virus recombinant E2 protein; the preparation method of the colloidal gold labeled swine fever virus recombinant E2 protein comprises the following steps: adjusting the pH value of the colloidal gold solution to 7.5 by using 0.02M K 2CO3 solution; then adding the recombinant E2 protein of the swine fever virus according to the mark amount of 34 mug/mL, and magnetically stirring for 40 min; then 100 g/L BSA is added to the final concentration of 10 g/L, and the mixture is magnetically stirred for 30 min; finally, 10000 r/min is centrifuged for 40 min, the supernatant is discarded, and the precipitate is suspended and dissolved by gold colloid buffer solution;
The colloidal gold marker in the colloidal gold immune test strip for detecting the antibodies of the porcine reproductive and respiratory syndrome virus consists of a colloidal gold-labeled porcine reproductive and respiratory syndrome virus N protein and a colloidal gold-labeled rabbit anti-IgG, and the detection line is coated with the porcine reproductive and respiratory syndrome virus N protein; the preparation method of the colloidal gold labeled porcine reproductive and respiratory syndrome virus N protein comprises the following steps: adjusting the pH value of the colloidal gold solution to 7.5 by using 0.02M K 2CO3 solution; then adding N protein of porcine reproductive and respiratory syndrome virus according to the marking amount of 4 mug/mL, and magnetically stirring for 40 min; then 100 g/L BSA is added to the final concentration of 10 g/L, and the mixture is magnetically stirred for 30 min; finally, 10000 r/min is centrifuged for 40 min, the supernatant is discarded, and the precipitate is suspended and dissolved by gold colloid buffer solution;
The preparation method of the colloidal gold labeled rabbit anti-IgG comprises the following steps: adjusting the pH value of the colloidal gold solution to 8.5 by using 0.02M K 2CO3 solution; then adding rabbit anti-IgG according to the marked amount of 4 mug/mL, and magnetically stirring for 40min; then 100g/L BSA is added to the final concentration of 10g/L, and the mixture is magnetically stirred for 30min; finally, 10000r/min is centrifuged for 40min, the supernatant is discarded, and the precipitate is suspended and dissolved by gold colloid buffer solution;
In the colloidal gold marker of the colloidal gold immune test strip for detecting the foot-and-mouth disease O-type virus antibody, the volume ratio of the colloidal gold labeled foot-and-mouth disease O-type virus antigen 146S to the colloidal gold labeled rabbit anti-IgG is 9:1;
in the colloidal gold marker of the colloidal gold immune test strip for detecting the African swine fever virus antibody, the volume ratio of the colloidal gold-labeled African swine fever virus recombinant p30 protein to the colloidal gold-labeled rabbit anti-IgG is 9:1;
in the colloidal gold marker of the colloidal gold immune test strip for detecting the swine fever virus antibody, the volume ratio of the colloidal gold-labeled swine fever virus recombinant E2 protein to the colloidal gold-labeled rabbit anti-IgG is 9:1;
In the colloidal gold marker of the colloidal gold immune test strip for detecting the porcine reproductive and respiratory syndrome virus antibody, the volume ratio of the colloidal gold-labeled porcine reproductive and respiratory syndrome virus N protein to the colloidal gold-labeled rabbit anti-IgG is 9:1;
The coating concentration of the foot-and-mouth disease O-type virus antigen 146S on the detection line of the colloidal gold immune test strip for detecting the foot-and-mouth disease O-type virus antibody is 0.2 mg/mL;
the coating concentration of the African swine fever virus recombinant p30 protein on the detection line of the colloidal gold immune test strip for detecting the African swine fever virus antibody is 0.1 mg/mL;
The coating concentration of the swine fever virus recombinant E2 protein on the detection line of the colloidal gold immune test strip for detecting the swine fever virus antibody is 0.57 mg/mL;
The coating concentration of the N protein of the porcine reproductive and respiratory syndrome virus on the detection line of the colloidal gold immune test strip for detecting the porcine reproductive and respiratory syndrome virus antibody is 0.42 mg/mL;
The quality control lines of the colloidal gold immune test strips are coated with goat anti-rabbit IgG;
The coating concentration of the goat anti-rabbit IgG is 0.2 mg/mL.
2. The immunochromatographic test paper card according to claim 1, wherein: and the spraying amounts of the coating solution on the detection line and the quality control line are 1 mu L/cm.
3. The immunochromatographic test paper card according to claim 1 or 2, characterized in that: the shell is fan-shaped;
the included angle between two adjacent colloidal gold immune test strips is 35 degrees.
4. A method for preparing the immunochromatographic test paper card of any one of claims 1 to 3, comprising the steps of:
1) Respectively preparing a colloidal gold immune test strip for detecting the foot-and-mouth disease O-type virus antibody, a colloidal gold immune test strip for detecting the African swine fever virus antibody, a colloidal gold immune test strip for detecting the swine fever virus antibody and a colloidal gold immune test strip for detecting the porcine reproductive and respiratory syndrome virus antibody;
The colloidal gold immune test strip consists of a sample pad, an immune colloidal gold probe pad, a nitrocellulose membrane and a water absorption pad which are sequentially connected and fixed on a bottom plate;
the colloidal gold binding pads are coated with colloidal gold labels;
The nitrocellulose membrane is coated with a detection line and a quality control line, and the detection line and the quality control line are mutually separated; the detection line is positioned at one end of the nitrocellulose membrane, which is close to the sample pad; the quality control line is positioned at one end of the nitrocellulose membrane, which is close to the water absorption pad;
The colloidal gold marker in the colloidal gold immune test strip for detecting the foot-and-mouth disease O-type virus antibody consists of a foot-and-mouth disease O-type virus antigen 146S marked by colloidal gold and a rabbit anti-IgG marked by colloidal gold, and the detection line is coated with the foot-and-mouth disease O-type virus antigen 146S; the preparation method of the colloidal gold labeled foot-and-mouth disease O-type virus antigen 146S comprises the following steps: adjusting the pH value of the colloidal gold solution to 7.5 by using 0.02M K 2CO3 solution; then adding foot-and-mouth disease O-type virus purified antigen 146S according to the mark amount of 1.1 mug/mL, and magnetically stirring for 40 min; then 100 g/L BSA is added to the final concentration of 10 g/L, and the mixture is magnetically stirred for 30 min; finally, 10000 r/min is centrifuged for 40 min, the supernatant is discarded, and the precipitate is suspended and dissolved by gold colloid buffer solution;
The colloidal gold marker in the colloidal gold immune test strip for detecting the African swine fever virus antibody consists of a colloidal gold-labeled African swine fever virus recombinant p30 protein and a colloidal gold-labeled rabbit anti-IgG, and the detection line is coated with the African swine fever virus recombinant p30 protein; the preparation method of the colloidal gold labeled African swine fever virus recombinant p30 protein comprises the following steps: adjusting the pH value of the colloidal gold solution to 9.0 by using 0.02M K 2CO3 solution; then adding the African swine fever virus recombinant p30 protein according to the mark amount of 2.7 mug/mL, and magnetically stirring for 40 min; adding 100 g/L BSA to a final concentration of 10 g/L, and magnetically stirring for 30min; finally, 10000 r/min is centrifuged for 40 min, the supernatant is discarded, and the precipitate is suspended and dissolved by gold colloid buffer solution;
The colloidal gold marker in the colloidal gold immune test strip for detecting the swine fever virus antibody consists of a swine fever virus recombinant E2 protein marked by colloidal gold and a rabbit anti-IgG marked by colloidal gold, and the detection line is coated with the swine fever virus recombinant E2 protein; the preparation method of the colloidal gold labeled swine fever virus recombinant E2 protein comprises the following steps: adjusting the pH value of the colloidal gold solution to 7.5 by using 0.02M K 2CO3 solution; then adding the recombinant E2 protein of the swine fever virus according to the mark amount of 34 mug/mL, and magnetically stirring for 40 min; then 100 g/L BSA is added to the final concentration of 10 g/L, and the mixture is magnetically stirred for 30 min; finally, 10000 r/min is centrifuged for 40 min, the supernatant is discarded, and the precipitate is suspended and dissolved by gold colloid buffer solution;
The colloidal gold marker in the colloidal gold immune test strip for detecting the antibodies of the porcine reproductive and respiratory syndrome virus consists of a colloidal gold-labeled porcine reproductive and respiratory syndrome virus N protein and a colloidal gold-labeled rabbit anti-IgG, and the detection line is coated with the porcine reproductive and respiratory syndrome virus N protein; the preparation method of the colloidal gold labeled porcine reproductive and respiratory syndrome virus N protein comprises the following steps: adjusting the pH value of the colloidal gold solution to 7.5 by using 0.02M K 2CO3 solution; then adding N protein of porcine reproductive and respiratory syndrome virus according to the marking amount of 4 mug/mL, and magnetically stirring for 40 min; then 100 g/L BSA is added to the final concentration of 10 g/L, and the mixture is magnetically stirred for 30 min; finally, 10000 r/min is centrifuged for 40 min, the supernatant is discarded, and the precipitate is suspended and dissolved by gold colloid buffer solution;
The preparation method of the colloidal gold labeled rabbit anti-IgG comprises the following steps: adjusting the pH value of the colloidal gold solution to 8.5 by using 0.02M K 2CO3 solution; then adding rabbit anti-IgG according to the marked amount of 4 mug/mL, and magnetically stirring for 40min; then 100g/L BSA is added to the final concentration of 10g/L, and the mixture is magnetically stirred for 30min; finally, 10000r/min is centrifuged for 40min, the supernatant is discarded, and the precipitate is suspended and dissolved by gold colloid buffer solution;
In the colloidal gold marker of the colloidal gold immune test strip for detecting the foot-and-mouth disease O-type virus antibody, the volume ratio of the colloidal gold labeled foot-and-mouth disease O-type virus antigen 146S to the colloidal gold labeled rabbit anti-IgG is 9:1;
in the colloidal gold marker of the colloidal gold immune test strip for detecting the African swine fever virus antibody, the volume ratio of the colloidal gold-labeled African swine fever virus recombinant p30 protein to the colloidal gold-labeled rabbit anti-IgG is 9:1;
in the colloidal gold marker of the colloidal gold immune test strip for detecting the swine fever virus antibody, the volume ratio of the colloidal gold-labeled swine fever virus recombinant E2 protein to the colloidal gold-labeled rabbit anti-IgG is 9:1;
In the colloidal gold marker of the colloidal gold immune test strip for detecting the porcine reproductive and respiratory syndrome virus antibody, the volume ratio of the colloidal gold-labeled porcine reproductive and respiratory syndrome virus N protein to the colloidal gold-labeled rabbit anti-IgG is 9:1;
The coating concentration of the foot-and-mouth disease O-type virus antigen 146S on the detection line of the colloidal gold immune test strip for detecting the foot-and-mouth disease O-type virus antibody is 0.2 mg/mL;
the coating concentration of the African swine fever virus recombinant p30 protein on the detection line of the colloidal gold immune test strip for detecting the African swine fever virus antibody is 0.1 mg/mL;
The coating concentration of the swine fever virus recombinant E2 protein on the detection line of the colloidal gold immune test strip for detecting the swine fever virus antibody is 0.57 mg/mL;
The coating concentration of the N protein of the porcine reproductive and respiratory syndrome virus on the detection line of the colloidal gold immune test strip for detecting the porcine reproductive and respiratory syndrome virus antibody is 0.42 mg/mL;
The quality control lines of the colloidal gold immune test strips are coated with goat anti-rabbit IgG;
the coating concentration of the goat anti-rabbit IgG is 0.2 mg/mL;
2) Placing the colloidal gold immune test strip for detecting the foot-and-mouth disease O-type virus antibody, the colloidal gold immune test strip for detecting the African swine fever virus antibody, the colloidal gold immune test strip for detecting the swine fever virus antibody and the colloidal gold immune test strip for detecting the porcine reproductive and respiratory syndrome virus antibody in a shell, so that the sample pads of the colloidal gold immune test strip are partially overlapped, and the rest parts are mutually separated in a radial manner; the shell is provided with a sample adding hole corresponding to the overlapping part of the sample pads of each colloidal gold immune test strip, and four observation windows corresponding to the detection line part and the quality control line part of each colloidal gold immune test strip respectively.
5. Use of an immunochromatographic test paper card according to any one of claims 1 to 3 or an immunochromatographic test paper card prepared according to the method of claim 4 for preparing a product for detecting whether a sample to be detected contains any one or more of an african swine fever virus antibody, a porcine reproductive and respiratory syndrome virus antibody.
6. Use of an immunochromatographic test paper card according to any one of claims 1 to 3 or an immunochromatographic test paper card prepared according to the method of claim 4 for the preparation of a product for diagnosis and control of any one or more of foot-and-mouth disease, african swine fever, porcine reproductive and respiratory syndrome.
7. A method for detecting whether a sample to be tested contains any one or more of african swine fever virus antibody, swine fever virus antibody and porcine reproductive and respiratory syndrome virus antibody, comprising the steps of: adding a sample to be detected into a sample adding hole of the immunochromatography test paper card of any one of claims 1-3 or the immunochromatography test paper card prepared according to the method of claim 4, then dripping an equivalent amount of extension liquid into the sample adding hole, and observing the color development condition in an observation window in the immunochromatography test paper card after 10 minutes;
if the quality control zone and the detection zone in the observation window corresponding to a certain virus are developed, the sample to be detected contains the virus antibody; if the quality control zone in the observation window corresponding to a certain virus is colored and the detection zone is not colored, the sample to be detected does not contain the virus antibody;
The formula of the expanding liquid is as follows: disodium hydrogen phosphate 35.8 g, sodium dihydrogen phosphate 1.56 g, adding ultrapure water to a volume of 1000 ml, and then adding 0.1 ml Tween-20 and 500 μl Proclin300;
The methods are for non-disease diagnosis and treatment purposes.
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Citations (28)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1348104A (en) * | 2001-10-09 | 2002-05-08 | 上海生物芯片有限公司 | Fast test biochip for systemic lupus erythematosus |
| CN2837844Y (en) * | 2005-09-27 | 2006-11-15 | 黄熠 | Test paper structure for multi-item joint detection |
| CN101216489A (en) * | 2008-01-10 | 2008-07-09 | 中国农业科学院哈尔滨兽医研究所 | Test paper for rapidly detecting hog cholera antibody and method for making same |
| JP2009501901A (en) * | 2005-07-05 | 2009-01-22 | リージェンツ オブ ザ ユニバーシティ オブ ミネソタ | Diagnostic test kit |
| CN101363858A (en) * | 2008-05-26 | 2009-02-11 | 北京庄笛浩禾生物医学科技有限公司 | Test paper strip for detecting PRRSV antibody colloidal gold, method for making same and applications |
| CN101373187A (en) * | 2008-08-25 | 2009-02-25 | 深圳市菲鹏生物股份有限公司 | Immune chromatography reagent kit for screening multiple great infectious diseases simultaneously |
| CN201203619Y (en) * | 2008-05-16 | 2009-03-04 | 郑州博赛生物技术股份有限公司 | Combined detection card capable of detecting AIDS, hepatitis B, hepatitis C, and syphilis |
| CN201464479U (en) * | 2009-07-17 | 2010-05-12 | 郑州安图绿科生物工程有限公司 | Immune body immunochromatography joint inspection kit for EA- IgG and VCA- IgA of EB viruses |
| CN101762705A (en) * | 2010-01-21 | 2010-06-30 | 中国农业科学院哈尔滨兽医研究所 | Colloidal gold immunochromatographic test strip for detecting wild-type classical swine fever virus |
| CN201555849U (en) * | 2009-11-18 | 2010-08-18 | 吉林大学第一医院 | Disc type collaurum immunochromatography joint detection plate |
| US7892729B1 (en) * | 2005-06-23 | 2011-02-22 | Iowa State University Research Foundation, Inc. | Universal and differential serologic assay for swine influenza virus |
| CN202013354U (en) * | 2011-01-05 | 2011-10-19 | 苏州浩欧博生物医药有限公司 | Detection box used for immune chromatography detection technology |
| CN202018459U (en) * | 2011-04-11 | 2011-10-26 | 深圳市博卡生物技术有限公司 | Colloidal-gold detecting and collecting card |
| CN202583199U (en) * | 2011-12-13 | 2012-12-05 | 中华人民共和国北京出入境检验检疫局 | Synchronous detection card structure |
| CN202676705U (en) * | 2012-02-28 | 2013-01-16 | 中国农业科学院生物技术研究所 | Combined insect-resistant BT protein gold mark fast detection reagent kit |
| CN203224492U (en) * | 2013-03-08 | 2013-10-02 | 北京海瑞祥天生物科技有限公司 | Test strip and kit for in-vitro joint inspection and diagnosis |
| CN205538992U (en) * | 2016-03-30 | 2016-08-31 | 珠海华澳生物科技有限公司 | Test paper dish and test paper tray salver |
| CN106706893A (en) * | 2015-11-17 | 2017-05-24 | 重庆市涪陵三海兰陵有限责任公司 | Assay kit for immunochromatographic assay technique |
| CN206420883U (en) * | 2017-01-19 | 2017-08-18 | 北京亿森宝生物科技有限公司 | Detect aflatoxin B1, zearalenone, the detection card of vomitoxin |
| CN108061800A (en) * | 2017-12-08 | 2018-05-22 | 重庆市畜牧科学院 | Hog cholera antibody colloidal-gold detecting-card and preparation method thereof |
| CN109001461A (en) * | 2018-06-26 | 2018-12-14 | 安阳工学院 | The immune quick testing reagent box of PCV-3 immunity evaluation and wild virus infection diagnosis |
| CN210269868U (en) * | 2019-03-28 | 2020-04-07 | 刘红蕾 | Joint detection mechanism and female fertility detection device |
| CN213023170U (en) * | 2019-11-27 | 2021-04-20 | 北京勤邦生物技术有限公司 | Card shell test paper for realizing one-time sample application detection of multiple drugs |
| CN214225182U (en) * | 2020-11-10 | 2021-09-17 | 基因赛奥(大连)生物科技发展有限公司 | Triple quick test card shell of immunochromatography product |
| CN113514633A (en) * | 2021-08-04 | 2021-10-19 | 博迪泰（厦门）生物科技有限公司 | Rapid detection card and detection kit |
| CN215493637U (en) * | 2021-08-04 | 2022-01-11 | 博迪泰（厦门）生物科技有限公司 | Rapid detection card and detection kit |
| CN115166235A (en) * | 2021-04-02 | 2022-10-11 | 洛阳普泰生物技术有限公司 | African swine fever virus double-antigen sandwich antibody detection kit and preparation method thereof |
| CN218512457U (en) * | 2022-09-08 | 2023-02-21 | 华测检测认证集团股份有限公司 | Colloidal gold detection card |
-
2023
- 2023-02-23 CN CN202310158152.3A patent/CN116359498B/en active Active
Patent Citations (28)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1348104A (en) * | 2001-10-09 | 2002-05-08 | 上海生物芯片有限公司 | Fast test biochip for systemic lupus erythematosus |
| US7892729B1 (en) * | 2005-06-23 | 2011-02-22 | Iowa State University Research Foundation, Inc. | Universal and differential serologic assay for swine influenza virus |
| JP2009501901A (en) * | 2005-07-05 | 2009-01-22 | リージェンツ オブ ザ ユニバーシティ オブ ミネソタ | Diagnostic test kit |
| CN2837844Y (en) * | 2005-09-27 | 2006-11-15 | 黄熠 | Test paper structure for multi-item joint detection |
| CN101216489A (en) * | 2008-01-10 | 2008-07-09 | 中国农业科学院哈尔滨兽医研究所 | Test paper for rapidly detecting hog cholera antibody and method for making same |
| CN201203619Y (en) * | 2008-05-16 | 2009-03-04 | 郑州博赛生物技术股份有限公司 | Combined detection card capable of detecting AIDS, hepatitis B, hepatitis C, and syphilis |
| CN101363858A (en) * | 2008-05-26 | 2009-02-11 | 北京庄笛浩禾生物医学科技有限公司 | Test paper strip for detecting PRRSV antibody colloidal gold, method for making same and applications |
| CN101373187A (en) * | 2008-08-25 | 2009-02-25 | 深圳市菲鹏生物股份有限公司 | Immune chromatography reagent kit for screening multiple great infectious diseases simultaneously |
| CN201464479U (en) * | 2009-07-17 | 2010-05-12 | 郑州安图绿科生物工程有限公司 | Immune body immunochromatography joint inspection kit for EA- IgG and VCA- IgA of EB viruses |
| CN201555849U (en) * | 2009-11-18 | 2010-08-18 | 吉林大学第一医院 | Disc type collaurum immunochromatography joint detection plate |
| CN101762705A (en) * | 2010-01-21 | 2010-06-30 | 中国农业科学院哈尔滨兽医研究所 | Colloidal gold immunochromatographic test strip for detecting wild-type classical swine fever virus |
| CN202013354U (en) * | 2011-01-05 | 2011-10-19 | 苏州浩欧博生物医药有限公司 | Detection box used for immune chromatography detection technology |
| CN202018459U (en) * | 2011-04-11 | 2011-10-26 | 深圳市博卡生物技术有限公司 | Colloidal-gold detecting and collecting card |
| CN202583199U (en) * | 2011-12-13 | 2012-12-05 | 中华人民共和国北京出入境检验检疫局 | Synchronous detection card structure |
| CN202676705U (en) * | 2012-02-28 | 2013-01-16 | 中国农业科学院生物技术研究所 | Combined insect-resistant BT protein gold mark fast detection reagent kit |
| CN203224492U (en) * | 2013-03-08 | 2013-10-02 | 北京海瑞祥天生物科技有限公司 | Test strip and kit for in-vitro joint inspection and diagnosis |
| CN106706893A (en) * | 2015-11-17 | 2017-05-24 | 重庆市涪陵三海兰陵有限责任公司 | Assay kit for immunochromatographic assay technique |
| CN205538992U (en) * | 2016-03-30 | 2016-08-31 | 珠海华澳生物科技有限公司 | Test paper dish and test paper tray salver |
| CN206420883U (en) * | 2017-01-19 | 2017-08-18 | 北京亿森宝生物科技有限公司 | Detect aflatoxin B1, zearalenone, the detection card of vomitoxin |
| CN108061800A (en) * | 2017-12-08 | 2018-05-22 | 重庆市畜牧科学院 | Hog cholera antibody colloidal-gold detecting-card and preparation method thereof |
| CN109001461A (en) * | 2018-06-26 | 2018-12-14 | 安阳工学院 | The immune quick testing reagent box of PCV-3 immunity evaluation and wild virus infection diagnosis |
| CN210269868U (en) * | 2019-03-28 | 2020-04-07 | 刘红蕾 | Joint detection mechanism and female fertility detection device |
| CN213023170U (en) * | 2019-11-27 | 2021-04-20 | 北京勤邦生物技术有限公司 | Card shell test paper for realizing one-time sample application detection of multiple drugs |
| CN214225182U (en) * | 2020-11-10 | 2021-09-17 | 基因赛奥(大连)生物科技发展有限公司 | Triple quick test card shell of immunochromatography product |
| CN115166235A (en) * | 2021-04-02 | 2022-10-11 | 洛阳普泰生物技术有限公司 | African swine fever virus double-antigen sandwich antibody detection kit and preparation method thereof |
| CN113514633A (en) * | 2021-08-04 | 2021-10-19 | 博迪泰（厦门）生物科技有限公司 | Rapid detection card and detection kit |
| CN215493637U (en) * | 2021-08-04 | 2022-01-11 | 博迪泰（厦门）生物科技有限公司 | Rapid detection card and detection kit |
| CN218512457U (en) * | 2022-09-08 | 2023-02-21 | 华测检测认证集团股份有限公司 | Colloidal gold detection card |
Non-Patent Citations (9)
| Title |
|---|
| AHA83252.1.GenBank.2013,序列. * |
| AYE67020.1.GenBank.2018,序列. * |
| A型口蹄疫病毒抗体金标检测试纸条的研制;孙燕燕 等;中国兽医科学;20160930;第46卷(第9期);第1123-1127页 * |
| QTZ19803.1.GenBank.2021,序列. * |
| 免疫层析技术在口蹄疫POCT中的应用研究;蒋韬;中国博士学位论文全文数据库 农业科技辑(月刊);20121115;2012年(第11期);摘要，正文第55-71页 * |
| 基于不同微球标记非洲猪瘟病毒和口蹄疫A型病毒免疫层析检测方法的建立;孙燕燕;中国博士学位论文全文数据库 基础科学辑(月刊);20220115;2022年(第1期);摘要，正文第16-49页 * |
| 猪瘟抗体胶体金快速定量检测卡的研制;李成洪 等;中国兽药杂志;20180731;第52卷(第7期);第22-27页 * |
| 猪瘟病毒抗体胶体金免疫层析快速检测试纸条的研制及初步应用;尹梅;中国优秀硕士学位论文全文数据库 农业科技辑(月刊);20140915;2014年(第9期);摘要，正文第31-44页 * |
| 猪瘟病毒抗体胶体金免疫层析试纸条的研制及初步应用;万颖 等;中国兽医科学;20210806;第52卷(第1期);第1-10页 * |
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