CN103969451A - Porcine circovirus type 2 (PCV2) IgM antibody colloidal gold immunochromatographic assay test paper, and preparation method and application thereof - Google Patents
Porcine circovirus type 2 (PCV2) IgM antibody colloidal gold immunochromatographic assay test paper, and preparation method and application thereof Download PDFInfo
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- 241001673669 Porcine circovirus 2 Species 0.000 title claims abstract description 115
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 title claims abstract description 82
- 238000013096 assay test Methods 0.000 title claims abstract description 33
- 238000002360 preparation method Methods 0.000 title claims abstract description 32
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- 238000001514 detection method Methods 0.000 claims abstract description 33
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- 239000005020 polyethylene terephthalate Substances 0.000 claims description 16
- 239000000020 Nitrocellulose Substances 0.000 claims description 15
- 229920001220 nitrocellulos Polymers 0.000 claims description 15
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- XOJVVFBFDXDTEG-UHFFFAOYSA-N pristane Chemical compound CC(C)CCCC(C)CCCC(C)CCCC(C)C XOJVVFBFDXDTEG-UHFFFAOYSA-N 0.000 claims description 6
- 238000002347 injection Methods 0.000 claims description 5
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- 206010068676 Pneumoretroperitoneum Diseases 0.000 claims description 3
- 208000005727 Retropneumoperitoneum Diseases 0.000 claims description 3
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 3
- 229930006000 Sucrose Natural products 0.000 claims description 3
- 238000012870 ammonium sulfate precipitation Methods 0.000 claims description 3
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- 238000010276 construction Methods 0.000 claims description 3
- 238000000432 density-gradient centrifugation Methods 0.000 claims description 3
- 238000013461 design Methods 0.000 claims description 3
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- 238000004108 freeze drying Methods 0.000 claims description 3
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- 241001465754 Metazoa Species 0.000 description 1
- 108700026244 Open Reading Frames Proteins 0.000 description 1
- 101710159752 Poly(3-hydroxyalkanoate) polymerase subunit PhaE Proteins 0.000 description 1
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
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Abstract
The invention discloses a porcine circovirus type 2 (PCV2) IgM antibody colloidal gold immunochromatographic assay test paper, and a preparation method and application thereof. The test paper comprises a sample absorption region, a colloidal gold labeled probe region, a curing antibody region, a water absorption region, a backboard and a shell, wherein the sample absorption region, colloidal gold labeled probe region, curing antibody region and water absorption region are sequentially attached to the backboard and arranged in the shell in a superposition mode. A purified PCV2 Cap protein is coated on the sample absorption region; a colloidal gold labeled PCV2 Cap protein monoclonal antibody is coated on the colloidal gold labeled probe region; and the curing antibody region is sequentially provided with a detection line T coated mouse anti-pig IgM monoclonal antibody and a control line C coated goat anti-mouse IgG. The test paper is simple to operate, has the advantages of favorable repetitiveness, high sensitivity, quick and visual results, simple technique and low cost, can implement mass preparation, is suitable for mass field detection for basic level, and can be used for early quick diagnosis on swinery PCV infections.
Description
Technical field
The present invention relates to a kind of Test paper card and preparation method thereof, be specifically related to a kind of porcine circovirus 2 type IgM antibody colloidal gold immunochromatographyassay assay Test paper card and its preparation method and application.
Background technology
Porcine circovirus 2 type (porcine circovirus type 2, PCV2) infect, can cause pmws, the diseases such as sow breeding difficulty, taking ill domestic animal poor growth become thin, Multi-system injuries is as principal character, since this virus self-discovery, extensively exist in the whole world, cause tremendous economic loss to global pig industry.Therefore, set up serology antibody detection method, significant to evaluating immune level and the infection state of swinery, provide new diagnostic method and detect porcine circovirus 2 type IgM antibody for this sick early diagnosis.
PCV ORF2 open reading frame coding virus nucleocapsid albumen (Cap albumen), is viral major structural protein, has four epitopes, is the desirable target antigen of detection of specific antibody.Therefore, specific detection PCV Cap protein I gM antibody can be made early diagnosis to swinery PCV2 infection conditions, effectively reduces economic loss.
This sick detection method mainly contains indirect immunofluorescence, PCR etc., although these methods can detect PCV2 antigen or IgG antibody horizontal, seldom detects IgM antibody, and the existing IgM antibody kit that detects can not be distinguished specificity PCV2 IgM antibody; And the shortcoming such as these methods exist complicated operation, need specialized equipment and personnel, detection time is long.Colloidal gold immunochromatographimethod method is the method for quick growing up on the basis of immunity percolation, the method is simple to operate, reproducible, highly sensitive, result quicklook, and can prepare in a large number, technique is simple, with low cost, being applicable to basic unit's mass field detects, therefore, utilize colloidal gold immunochromatographimethod method specific detection PCV Cap protein I gM antibody can make PCV2 and infect Rapid&Early diagnosis.
Summary of the invention
Primary and foremost purpose of the present invention is that the shortcoming that makes up existing detection technique, with not enough, provides a kind of porcine circovirus 2 type IgM antibody colloidal gold immunochromatographyassay assay Test paper card.This Test paper card is using the carrying Cap gene of porcine circovirus type 2 of absorption of sample district purifying as detectable antigens, the IgM antibody of the carrying Cap gene of porcine circovirus type 2 in specific binding serum to be checked, association colloid gold mark resisting porcine circovirus 2 type Cap protein monoclonal antibodies again, utilize the coated mouse-anti pig IgM monoclonal antibody in detection line place of curing antibody district to catch, sandwich method carries out the detection of porcine circovirus 2 type IgM antibody.
Another object of the present invention is to provide the preparation method of above-mentioned porcine circovirus 2 type IgM antibody colloidal gold immunochromatographyassay assay Test paper card.
A further object of the present invention is to provide above-mentioned porcine circovirus 2 type IgM antibody colloidal gold immunochromatographyassay assay Test paper card
Be stuck in the application in the diagnosis of swinery PCV2 early infection.
Object of the present invention is achieved through the following technical solutions:
A kind of porcine circovirus 2 type IgM antibody colloidal gold immunochromatographyassay assay Test paper card, comprise absorption of sample district, gold mark probe region, curing antibody district, suction zones, base plate and get stuck, absorption of sample district, gold mark probe region, curing antibody district and suction zones be pasted on successively base plate and overlapped pack into get stuck in; Described absorption of sample district is coated with the carrying Cap gene of porcine circovirus type 2 of purifying, and package amount is preferably 10～50 μ g; Described gold mark probe region is coated with gold mark probe, the resisting porcine circovirus 2 type Cap protein monoclonal antibodies that described gold mark probe is colloid gold label, and its labelled amount is preferably 1～30 μ g/mL, and package amount is preferably 10～50 μ L/cm; Curing antibody district has detection line T and control line C successively, and detection line T is coated with mouse-anti pig IgM monoclonal antibody, and package amount is preferably 0.1～10 μ g/cm, and control line C is coated with sheep anti-mouse igg, and package amount is preferably 0.1～10 μ g/cm.
The material in described absorption of sample district is dacron film; The material of described gold mark probe region is glass fibre membrane; The material in described curing antibody district is nitrocellulose filter; The material of described suction zones is absorbent filter; The material of described base plate is polyethylene board; Described gets stuck for getting stuck with the plastics of visual window and sample well.
The preparation method of above-mentioned porcine circovirus 2 type IgM antibody colloidal gold immunochromatographyassay assay Test paper card, comprises the steps: the carrying Cap gene of porcine circovirus type 2 of purifying to be coated on dacron film; The resisting porcine circovirus of colloid gold label 2 type Cap protein monoclonal antibodies are sprayed on glass fibre membrane; Mouse-anti pig IgM monoclonal antibody is coated in to nitrocellulose filter detection line T place, goat anti-mouse igg is coated in to nitrocellulose filter control line C place; Dacron film, glass fibre membrane, nitrocellulose filter and absorbent filter are assembled on polyethylene board, in packing into and getting stuck, obtain porcine circovirus 2 type IgM antibody colloidal gold immunochromatographyassay assay Test paper card.
Preferably, the preparation method of described porcine circovirus 2 type IgM antibody colloidal gold immunochromatographyassay assay Test paper card, comprises the steps:
(1) preparation of the PCV2-Cap albumen of purifying:
Utilize the recombinant baculovirus of baculovirus expression system construction expression PCV2-ORF2 gene, according to the gene order design Cap protein-specific primer of PCV2, primer sequence is:
Upstream primer 1:5
,-TCTGGATCCATGACGTATCCAAGGAGGCG-3
,,
Downstream primer 1:5
,-GCGAAGCTTTAAGGGTTAAGTGGGGGGTC-3
,;
Upstream primer 2:5
,-TCTCTCGAGATGACGTATCCAAGGAGGCG-3
,,
Downstream primer 2:5
,-GCGGGTACCTAAGGGTTAAGTGGGGGGTC-3
,;
Utilize the PCR method PCV2 ORF2 gene complete sequence that increases from PCV2 genome, by its subclone to obtain in pFastBac-Dual carrier the recombinant plasmid pFastBac-Dual-ORF2(2 of ORF2 Gene Double copy ×), by recombinant plasmid pFastBac-Dual-ORF2(2 ×) be converted into Escherichia coli DH10Bac, obtain recombinant baculovirus plasmid; By recombinant baculovirus plasmid transfection Sf9 cell, acquisition recombinant baculovirus rBac-ORF2(2 ×); By recombinant baculovirus rBac-ORF2(2 ×) infect Sf9 cell, make it express Cap albumen, collect Cap albumen, sucrose density gradient centrifugation obtains the Cap albumen of the virus-like particle of purifying.
(2) preparation of resisting porcine circovirus 2 type Cap protein monoclonal antibodies:
1), by aforementioned carrying Cap gene of porcine circovirus type 2 routine immunization Balb/c mouse, when tiring, mice serum ELISA reaches 1:10000 extracting spleen cell and myeloma cell's fusion; With ELISA method and indirect immunofluorescence method screening positive hybridoma cell; After adopting limiting dilution assay subclone, obtain monoclonal antibody hybridoma cell strain;
2) select female BALB/c mouse in 8 week age, every lumbar injection 0.5mL norphytane, 7 days pneumoretroperitoneum injection resisting porcine circovirus 2 type Cap protein monoclonal antibody hybridoma cell strains, inoculum concentration is 2 × 10
6cell/0.5mL/ only, treats after one week that mouse web portion expands extraction ascites, obtains the resisting porcine circovirus 2 type Cap protein monoclonal antibodies of purifying through ammonium sulfate precipitation purifying.
(3) method of colloid gold label resisting porcine circovirus 2 type Cap protein monoclonal antibodies: getting radius and be 20～40nm concentration and be 0.01% collaurum 20mL adjust pH is 8.5～9.5, add resisting porcine circovirus 2 type Cap protein monoclonal antibody 10～600 μ g, under stirring condition, make its combination, add BSA as stabilizing agent, and making BSA final concentration is 0.1～10%, centrifugal unconjugated resisting porcine circovirus 2 type Cap protein monoclonal antibodies and unstabilized colloid gold particle and the agglutinator removed, the PBS that is 7.4 by 2mL 0.01M pH value in the peony precipitation of centrifuge tube bottom dissolves, be the compound of colloid gold label resisting porcine circovirus 2 type Cap protein monoclonal antibodies.
(4) preparation of dacron film: the PBS that is 7.4 by 0.01M pH value by the PCV2-Cap protein 10～50 μ g of above-mentioned purifying is evenly coated on dacron film after dissolving, dries.
(5) preparation of glass fibre membrane: the compound of above-mentioned colloid gold label resisting porcine circovirus 2 type Cap protein monoclonal antibodies is sprayed on glass fibre membrane to 10～50 μ L/cm, freeze-drying.
(6) preparation of nitrocellulose filter: detection line T is coated with mouse-anti pig IgM monoclonal antibody, and package amount is 0.1～10 μ g/cm, and control line C is coated with sheep anti-mouse igg, and package amount is 0.1～10 μ g/cm.
(7) test card preparation: by dacron film, glass fibre membrane, nitrocellulose filter and absorbent filter successively overlapped being assembled on polyethylene board, be cut into the wide test paper of 4mm with cutting cutter, in packing into and getting stuck, obtain porcine circovirus 2 type IgM antibody colloidal gold immunochromatographyassay assay Test paper card.
Above-mentioned porcine circovirus 2 type IgM antibody colloidal gold immunochromatographyassay assay Test paper card can be used in the diagnosis of swinery PCV2 early infection, and described early infection comprises vaccine inoculation or natural infection or its combination.After PBS that it is 7.4 by 1mL 0.01M pH value that its using method comprises the steps: 3～4 of blood serum samples to be checked dilution, drip in sample well 4～5,10～15min result of determination, control line C occurs that redness shows that test card is effective, all there is redness in control line C and detection line T, show that sample IgM antibody is positive, there is redness in control line C only, shows that sample IgM antibody is negative.
The present invention has the following advantages and effect with respect to prior art tool:
(1) porcine circovirus 2 type IgM antibody colloidal gold immunochromatographyassay assay Test paper card of the present invention, adopt colloid gold label resisting porcine circovirus 2 type Cap protein monoclonal antibodies as probe, mouse-anti pig IgM monoclonal antibody is coated with nitrocellulose filter, the carrying Cap gene of porcine circovirus type 2 of purifying is as detectable antigens, sandwich method specificity is caught PCV2 IgM antibody in serum, has set up PCV2 IgM antibody colloidal gold immunochromatographyassay assay detection method.Except having the advantage of test card detection own, overcome again the low shortcoming of sensitivity that antigen purification process and purity problem etc. cause, significantly improve the specificity and the susceptibility that detect, can be used for the Rapid&Early diagnosis that swinery PCV2 infects.
(2) detect quick, easy to carry, easy and simple to handle, visual result and easily distinguish, do not need Other Instruments equipment and professional and technical personnel, only need 10～15 minutes detection time, meets the needs of basic unit size pig farm and individual Site Detection.
(3) test card can be preserved at normal temperatures, and storage life can reach 1 year, and repeatability is good.
(4) Detection accuracy and specificity are high: other susceptible pathogenic autoantibodies of test card of the present invention and pig do not have cross reaction, and detection sensitivity and ELISA are suitable.
Brief description of the drawings
Fig. 1 is the structural representation of porcine circovirus 2 type IgM antibody colloidal gold immunochromatographyassay assay Test paper card; Wherein, 1 is suction zones, and 2 is curing antibody district, and 3 is gold mark probe region, and 4 is absorption of sample district, and 5 is base plate, and 6 for getting stuck, and 7 is detection line, and 8 is control line.
Fig. 2 is test card specific test result figure; Wherein, be from left to right porcine circovirus type 2 vaccines immunity serum on the 7th, porcine circovirus type 2 infection serum on the 7th, porcine circovirus 2 type IgM negative antibody serum, porcine circovirus type 2 vaccines immunity serum on the 60th, porcine reproductive and respiratory syndrome IgM Positive Sera, porcine pseudorabies antibody IgM positive serum, pig japanese b encephalitis IgM Positive Sera, swine fever IgM Positive Sera, pig parvoviral IgM Positive Sera.
Fig. 3 is test card sensitivity tests result figure; Wherein, 1～7: known positive is from 1:50 doubling dilution to 1:3200.
Embodiment
Below in conjunction with embodiment and accompanying drawing, technical scheme of the present invention is described in detail and is illustrated, but do not limit essence of the present invention.If do not specialize, the conventional means that in embodiment, technological means used is well known to those skilled in the art.
Embodiment 1
The structural representation of porcine circovirus 2 type IgM antibody colloidal gold immunochromatographyassay assay Test paper card as shown in Figure 1, test strips surface level is from left to right followed successively by suction zones 1, curing antibody district 2, gold mark probe region 3, absorption of sample district 4 in test card, mutually partly overlaps to be successively assembled on base plate 5, to be cut into that packing into after the wide test strips of 4mm gets stuck in 6 is porcine circovirus 2 type IgM antibody colloidal gold immunochromatographyassay assay Test paper card; Suction zones 1, curing antibody district 2, gold mark probe region 3, absorption of sample district 4, the material of base plate 5 is respectively absorbent filter, nitrocellulose filter, glass fibre membrane, dacron film and polyethylene board, gets stuck 6 for to get stuck with the plastics of visual window and sample well.2 detection line T 7 places of curing antibody district are coated with mouse-anti pig IgM monoclonal antibody, and package amount is 4 μ g/cm, and control line C 8 places are coated with sheep anti-mouse igg, and package amount is 5 μ g/cm.Gold mark probe region 3 is coated with the resisting porcine circovirus 2 type Cap protein monoclonal antibodies of colloid gold label, and labelled amount is 15 μ g monoclonal antibody/mL collaurums, and package amount is 20 μ L/cm.Absorption of sample district 4 is coated with the carrying Cap gene of porcine circovirus type 2 antigen of purifying, and package amount is 20 μ g.
Above-mentioned porcine circovirus 2 type IgM antibody colloidal gold immunochromatographyassay assay Test paper blocking Preparation Method is as follows:
(1) preparation of the PCV2-Cap albumen of purifying:
Utilize the recombinant baculovirus of baculovirus expression system construction expression PCV2-ORF2 gene, according to the gene order design Cap protein-specific primer of PCV2, primer sequence is:
Upstream primer 1:5
,-TCTGGATCCATGACGTATCCAAGGAGGCG-3
,,
Downstream primer 1:5
,-GCGAAGCTTTAAGGGTTAAGTGGGGGGTC-3
,;
Upstream primer 2:5
,-TCTCTCGAGATGACGTATCCAAGGAGGCG-3
,,
Downstream primer 2:5
,-GCGGGTACCTAAGGGTTAAGTGGGGGGTC-3
,;
Utilize the PCR method PCV2 ORF2 gene complete sequence that increases from PCV2 genome, by its subclone to obtain in pFastBac-Dual carrier the recombinant plasmid pFastBac-Dual-ORF2(2 of ORF2 Gene Double copy ×), by recombinant plasmid pFastBac-Dual-ORF2(2 ×) be converted into Escherichia coli DH10Bac, obtain recombinant baculovirus plasmid; By recombinant baculovirus plasmid transfection Sf9 cell, acquisition recombinant baculovirus rBac-ORF2(2 ×); By recombinant baculovirus rBac-ORF2(2 ×) infect Sf9 cell, make it express Cap albumen, collect Cap albumen, sucrose density gradient centrifugation obtains the Cap albumen of the virus-like particle of purifying.
(2) preparation of resisting porcine circovirus 2 type Cap protein monoclonal antibodies:
1), by aforementioned carrying Cap gene of porcine circovirus type 2 routine immunization Balb/c mouse, when tiring, mice serum ELISA reaches 1:10000 extracting spleen cell and myeloma cell's fusion; With ELISA method and indirect immunofluorescence method screening positive hybridoma cell; After adopting limiting dilution assay subclone, obtain monoclonal antibody hybridoma cell strain;
2) select female BALB/c mouse in 8 week age, every lumbar injection 0.5mL norphytane, 7 days pneumoretroperitoneum injection resisting porcine circovirus 2 type Cap protein monoclonal antibody hybridoma cell strains, inoculum concentration is 2 × 10
6cell/0.5mL/ only, treats after one week that mouse web portion expands extraction ascites, obtains the mouse-anti PRV monoclonal antibody of purifying through ammonium sulfate precipitation purifying.
(3) colloid gold label resisting porcine circovirus 2 type Cap protein monoclonal antibodies: the collaurum 20mL adjust pH that the concentration of getting radius and be 25nm is 0.01% is 9.0, add resisting porcine circovirus 2 type Cap protein monoclonal antibody 300 μ g, under stirring condition, make its combination, add BSA as stabilizing agent, and making BSA final concentration is 5%, centrifugal unconjugated resisting porcine circovirus 2 type Cap protein monoclonal antibodies and unstabilized colloid gold particle and the agglutinator removed, peony precipitation in centrifuge tube bottom is dissolved with the PBS of 2mL 0.01M pH 7.4, be the compound of colloid gold label resisting porcine circovirus 2 type Cap protein monoclonal antibodies.
(4) preparation of dacron film: the PBS that is 7.4 by 0.01M pH value by the PCV2-Cap protein 20 μ g of above-mentioned purifying is evenly coated on dacron film after dissolving, dries.
(5) preparation of glass fibre membrane: the compound of above-mentioned colloid gold label resisting porcine circovirus 2 type Cap protein monoclonal antibodies is sprayed on glass fibre membrane to 20 μ L/cm, freeze-drying.
(6) preparation of nitrocellulose filter: detection line T is coated with mouse-anti pig IgM monoclonal antibody, and package amount is 4 μ g/cm, and control line C is coated with sheep anti-mouse igg, and package amount is 5 μ g/cm.
(7) test card preparation: by dacron film, glass fibre membrane, nitrocellulose filter and absorbent filter successively overlapped being assembled on polyethylene board, be cut into the wide test paper of 4mm with cutting cutter, in packing into and getting stuck, obtain porcine circovirus 2 type IgM antibody colloidal gold immunochromatographyassay assay Test paper card.
Embodiment 2
Except the amount of detection line T 7 coated mouse-anti pig IgM monoclonal antibodies is 3 μ g/cm, the amount of control line C 8 coated goat anti-mouse iggs is 7 μ g/cm, the labelled amount that gold is marked the resisting porcine circovirus 2 type Cap protein monoclonal antibodies of the coated colloid gold label in probe region 3 is 12 μ g/mL, and package amount is 30 μ L/cm, the method of colloid gold label resisting porcine circovirus 2 type Cap protein monoclonal antibodies: getting radius is that 40nm pH value is that 9.2 concentration are 0.01% collaurum 20mL, add resisting porcine circovirus 2 type Cap protein monoclonal antibody 240 μ g, under stirring condition, make its combination, add BSA to final concentration be 7%, centrifugal unconjugated resisting porcine circovirus 2 type Cap protein monoclonal antibodies and unstabilized colloid gold particle and the agglutinator removed, peony precipitation in centrifuge tube bottom is dissolved with the PBS of 2mL0.01M pH7.4, be outside the compound of colloid gold label resisting porcine circovirus 2 type Cap protein monoclonal antibodies, all the other are identical with embodiment 1.
Embodiment 3
The using method of above-mentioned porcine circovirus 2 type IgM antibody colloidal gold immunochromatographyassay assay Test paper card:
After the PBS that is 7.4 by 1mL 0.01M pH value by 3～4 of blood serum samples to be checked dilution, drip in sample well 4～5,10～15min result of determination, control line C occurs that redness shows that test card is effective, all there is redness in control line C and detection line T, show that sample IgM antibody is positive, there is redness in control line C only, shows that sample IgM antibody is negative.
Embodiment 4
Specificity and the sensitivity tests of porcine circovirus 2 type IgM antibody colloidal gold immunochromatographyassay assay Test paper card of the present invention:
Two kinds of porcine circovirus 2 type IgM antibody colloidal gold immunochromatographyassay assay Test paper cards of Application Example 1 and 2 preparations detect respectively known porcine circovirus type 2 vaccines immunity serum on the 7th, porcine circovirus type 2 infection serum on the 7th, porcine circovirus 2 type IgM negative antibody serum, porcine circovirus type 2 vaccines immunity serum on the 60th, porcine reproductive and respiratory syndrome IgM Positive Sera, porcine pseudorabies antibody IgM positive serum, pig japanese b encephalitis IgM Positive Sera, swine fever IgM Positive Sera, pig parvoviral IgM Positive Sera.After the PBS that is 7.4 by 1mL 0.01M pH value by 3～4 of blood serum samples to be checked dilution, drip in sample well 4～5,10～15min result of determination.Result shows (seeing Fig. 2 and table 1): porcine circovirus type 2 vaccines immunity serum on the 7th and porcine circovirus type 2 infection serum testing result on the 7th are positive, and other serum testing result is negative, and specificity is good; Illustrate that test card of the present invention can be used for comprising the diagnosis of porcine circovirus type 2 vaccines inoculation or natural infection or its combination early infection.
Table 1 specific detection result of the present invention
| Sample number | Sample title | Testing result |
| 1 | Porcine circovirus type 2 vaccines immunity serum on the 7th | + |
| 2 | Porcine circovirus type 2 infection serum on the 7th | + |
| 3 | Circovurus type 2 IgM negative antibody serum | - |
| 4 | Porcine circovirus type 2 vaccines immunity serum on the 60th | - |
| 5 | Porcine reproductive and respiratory syndrome virus IgM Positive Sera | |
| 6 | Porcine pseudorabies IgM Positive Sera | - |
| 7 | Pig japanese b encephalitis IgM Positive Sera | - |
| 8 | Swine fever IgM Positive Sera | - |
| 9 | Pig parvoviral IgM Positive Sera | - |
The PBS that the positive serum that ELISA method detection PCV2 IgM antibody titer is 1:800 is 7.4 by 0.01M pH value is from 1:50 successively doubling dilution to 1:3200, porcine circovirus 2 type IgM antibody colloidal gold immunochromatographyassay assay Test paper card prepared by Application Example 1 detects, 4～5 of the blood serum samples to be checked of PBS dilution are dripped in sample well to 10～15min result of determination.Result shows (seeing Fig. 3 and table 2): the highly diluted multiple that test card of the present invention detects the positive serum that known IgM antibody titer is 1:800 is 1:800, suitable with ELISA testing result, shows that susceptibility is good.
Table 2 sensitivity Detection result of the present invention
| Sample number | Serum diluting multiple | Testing result |
| 1 | 1:50 | + |
| 2 | 1:100 | + |
| 3 | 1:200 | + |
| 4 | 1:400 | + |
| 5 | 1:800 | + |
| 6 | 1:1600 | - |
| 7 | 1:3200 | - |
Above-described embodiment is preferably embodiment of the present invention; but embodiments of the present invention are not restricted to the described embodiments; other any do not deviate from change, the modification done under Spirit Essence of the present invention and principle, substitutes, combination, simplify; all should be equivalent substitute mode, within being included in protection scope of the present invention.
SEQUENCE LISTING
<110> Wuhan Chopper Biology Co., Ltd.
<120> porcine circovirus 2 type IgM antibody colloidal gold immunochromatographyassay assay Test paper card and its preparation method and application
<130> 1
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 29
<212> DNA
<213> Artificial Sequence
<220>
<223> upstream primer 1
<400> 1
tctggatcca tgacgtatcc aaggaggcg 29
<210> 2
<211> 29
<212> DNA
<213> Artificial Sequence
<220>
<223> downstream primer 1
<400> 2
gcgaagcttt aagggttaag tggggggtc 29
<210> 3
<211> 29
<212> DNA
<213> Artificial Sequence
<220>
<223> upstream primer 2
<400> 3
tctctcgaga tgacgtatcc aaggaggcg 29
<210> 4
<211> 29
<212> DNA
<213> Artificial Sequence
<220>
<223> downstream primer 2
<400> 4
gcgggtacct aagggttaag tggggggtc 29
Claims (7)
1. a porcine circovirus 2 type IgM antibody colloidal gold immunochromatographyassay assay Test paper card, is characterized in that: comprise absorption of sample district, gold mark probe region, curing antibody district, suction zones, base plate and get stuck; Absorption of sample district, gold mark probe region, curing antibody district and suction zones be pasted on successively base plate and overlapped pack into get stuck in; Described absorption of sample district is coated with the carrying Cap gene of porcine circovirus type 2 of purifying; Described gold mark probe region is coated with gold mark probe, the resisting porcine circovirus 2 type Cap protein monoclonal antibodies that described gold mark probe is colloid gold label; Described curing antibody district has detection line T and control line C successively, and described detection line T is coated with mouse-anti pig IgM monoclonal antibody, and described control line C is coated with sheep anti-mouse igg.
2. porcine circovirus 2 type IgM antibody colloidal gold immunochromatographyassay assay Test paper card according to claim 1, is characterized in that: the package amount of the carrying Cap gene of porcine circovirus type 2 of described purifying is 10～50 μ g; The labelled amount of described gold mark probe is 1～30 μ g/mL, and package amount is 10 ~ 50 μ L/cm; The package amount of described mouse-anti pig IgM monoclonal antibody is 0.1～10 μ g/cm; The package amount of described sheep anti-mouse igg is 0.1～10 μ g/cm.
3. porcine circovirus 2 type IgM antibody colloidal gold immunochromatographyassay assay Test paper card according to claim 1, is characterized in that: the material in described absorption of sample district is dacron film; The material of described gold mark probe region is glass fibre membrane; The material in described curing antibody district is nitrocellulose filter; The material of described suction zones is absorbent filter; The material of described base plate is polyethylene board; Described gets stuck for getting stuck with the plastics of visual window and sample well.
4. the preparation method of the porcine circovirus 2 type IgM antibody colloidal gold immunochromatographyassay assay Test paper card described in claim 1-3 any one, is characterized in that comprising the steps: the PCV2-Cap albumen of purifying to be coated on dacron film; The resisting porcine circovirus of colloid gold label 2 type Cap protein monoclonal antibodies are sprayed on glass fibre membrane; Coated mouse-anti pig IgM monoclonal antibody, the coated sheep anti-mouse igg in control line C place at nitrocellulose filter detection line T place; The dacron film, glass fibre membrane, the nitrocellulose filter that is coated with detection line T and control line C and the absorbent filter that is coated with the resisting porcine circovirus 2 type Cap protein monoclonal antibodies of colloid gold label of the PCV2-Cap albumen that is coated with purifying are assembled on polyethylene board successively, in packing into and getting stuck, obtain porcine circovirus 2 type IgM antibody colloidal gold immunochromatographyassay assay Test paper card.
5. the preparation method of porcine circovirus 2 type IgM antibody colloidal gold immunochromatographyassay assay Test paper card according to claim 4, is characterized in that comprising the steps:
(1) preparation of the PCV2-Cap albumen of purifying:
Utilize the recombinant baculovirus of baculovirus expression system construction expression PCV2-ORF2 gene, according to the gene order design Cap protein-specific primer of PCV2, primer sequence is:
Upstream primer 1:5
,-TCTGGATCCATGACGTATCCAAGGAGGCG-3
,,
Downstream primer 1:5
,-GCGAAGCTTTAAGGGTTAAGTGGGGGGTC-3
,;
Upstream primer 2:5
,-TCTCTCGAGATGACGTATCCAAGGAGGCG-3
,,
Downstream primer 2:5
,-GCGGGTACCTAAGGGTTAAGTGGGGGGTC-3
,;
Utilize the PCR method PCV2 ORF2 gene complete sequence that increases from PCV2 genome, by its subclone to obtain in pFastBac-Dual carrier the recombinant plasmid pFastBac-Dual-ORF2(2 of ORF2 Gene Double copy ×), by recombinant plasmid pFastBac-Dual-ORF2(2 ×) be converted into Escherichia coli DH10Bac, obtain recombinant baculovirus plasmid; By recombinant baculovirus plasmid transfection Sf9 cell, acquisition recombinant baculovirus rBac-ORF2(2 ×); By recombinant baculovirus rBac-ORF2(2 ×) infect Sf9 cell, make it express Cap albumen, collect Cap albumen, sucrose density gradient centrifugation obtains the Cap albumen of the virus-like particle of purifying;
(2) preparation of resisting porcine circovirus 2 type Cap protein monoclonal antibodies:
1), by aforementioned carrying Cap gene of porcine circovirus type 2 routine immunization Balb/c mouse, when tiring, mice serum ELISA reaches 1:10000 extracting spleen cell and myeloma cell's fusion; With ELISA method and indirect immunofluorescence method screening positive hybridoma cell; After adopting limiting dilution assay subclone, obtain monoclonal antibody hybridoma cell strain;
2) select female BALB/c mouse in 8 week age, every lumbar injection 0.5mL norphytane, within 7 days, pneumoretroperitoneum is injected anti-circovurus type 2 Cap protein monoclonal antibody hybridoma cell strain, and inoculum concentration is 2 × 10
6cell/0.5mL/ only, treats after one week that mouse web portion expands extraction ascites, obtains the resisting porcine circovirus 2 type Cap protein monoclonal antibodies of purifying through ammonium sulfate precipitation purifying;
(3) method of colloid gold label resisting porcine circovirus 2 type Cap protein monoclonal antibodies: the collaurum 20mL adjust pH of getting respectively radius and be 20～40nm is 8.5～9.5, add resisting porcine circovirus 2 type Cap protein monoclonal antibody 10～600 μ g, under stirring condition, make its combination, add BSA as stabilizing agent, and making BSA final concentration is 0.1～10%, centrifugal unconjugated resisting porcine circovirus 2 type Cap protein monoclonal antibodies and unstabilized colloid gold particle and the agglutinator removed, the PBS that is 7.4 by 2mL 0.01M pH value in the peony precipitation of centrifuge tube bottom dissolves, be the compound of colloid gold label resisting porcine circovirus 2 type Cap protein monoclonal antibodies,
(4) preparation of dacron film: the PBS that is 7.4 by 0.01M pH value by the PCV2-Cap protein 10～50 μ g of above-mentioned purifying is evenly coated on dacron film after dissolving, dries;
(5) preparation of glass fibre membrane: the compound of above-mentioned colloid gold label resisting porcine circovirus 2 type Cap protein monoclonal antibodies is sprayed on glass fibre membrane to 10～50 μ L/cm, freeze-drying;
(6) preparation of nitrocellulose filter: detection line T is coated with mouse-anti pig IgM monoclonal antibody, and package amount is 0.1～10 μ g/cm, and control line C is coated with sheep anti-mouse igg, and package amount is 0.1～10 μ g/cm;
(7) test card preparation: by dacron film, glass fibre membrane, nitrocellulose filter and absorbent filter successively overlapped being assembled on polyethylene board, be cut into the wide test paper of 4mm with cutting cutter, in packing into and getting stuck, obtain porcine circovirus 2 type IgM antibody colloidal gold immunochromatographyassay assay Test paper card.
6. the porcine circovirus 2 type IgM antibody colloidal gold immunochromatographyassay assay Test paper described in claim 1-3 any one is stuck in the application in the diagnosis of swinery PCV2 early infection, it is characterized in that: described early infection comprises vaccine inoculation or natural infection or its combination.
7. the using method of the described porcine circovirus 2 type IgM antibody colloidal gold immunochromatographyassay assay Test paper card described in claim 1-3 any one, it is characterized in that comprising the steps: 3～4 of blood serum samples to be checked dripping after the PBS that is 7.4 by 1mL 0.01M pH value dilution in sample well 4～5,10～15min result of determination, control line C occurs that redness shows that test card is effective, all there is redness in control line C and detection line T, show that sample IgM antibody is positive, there is redness in control line C only, shows that sample IgM antibody is negative.
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Application publication date: 20140806 |