CN103698517A - Kit for rapidly detecting infectious bronchitis viruses - Google Patents
Kit for rapidly detecting infectious bronchitis viruses Download PDFInfo
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- CN103698517A CN103698517A CN201310744847.6A CN201310744847A CN103698517A CN 103698517 A CN103698517 A CN 103698517A CN 201310744847 A CN201310744847 A CN 201310744847A CN 103698517 A CN103698517 A CN 103698517A
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- infectious bronchitis
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- quick detection
- nitrite ion
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- 241000711450 Infectious bronchitis virus Species 0.000 title claims abstract description 30
- 238000001514 detection method Methods 0.000 claims abstract description 18
- 239000007788 liquid Substances 0.000 claims abstract description 18
- 102000004190 Enzymes Human genes 0.000 claims abstract description 13
- 108090000790 Enzymes Proteins 0.000 claims abstract description 13
- 238000005406 washing Methods 0.000 claims abstract description 5
- 241000287828 Gallus gallus Species 0.000 claims description 32
- 239000000243 solution Substances 0.000 claims description 28
- IOVCWXUNBOPUCH-UHFFFAOYSA-M Nitrite anion Chemical compound [O-]N=O IOVCWXUNBOPUCH-UHFFFAOYSA-M 0.000 claims description 24
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 24
- 229940005654 nitrite ion Drugs 0.000 claims description 24
- 206010006451 bronchitis Diseases 0.000 claims description 20
- 208000015181 infectious disease Diseases 0.000 claims description 20
- 230000002458 infectious effect Effects 0.000 claims description 20
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 claims description 16
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 claims description 16
- 239000000872 buffer Substances 0.000 claims description 16
- 210000002966 serum Anatomy 0.000 claims description 14
- 229920001213 Polysorbate 20 Polymers 0.000 claims description 12
- 238000004140 cleaning Methods 0.000 claims description 12
- 238000007865 diluting Methods 0.000 claims description 12
- 238000010790 dilution Methods 0.000 claims description 12
- 239000012895 dilution Substances 0.000 claims description 12
- 239000013642 negative control Substances 0.000 claims description 12
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 claims description 12
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 claims description 12
- 239000013641 positive control Substances 0.000 claims description 12
- 239000011780 sodium chloride Substances 0.000 claims description 12
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims description 8
- 238000001914 filtration Methods 0.000 claims description 8
- 239000012530 fluid Substances 0.000 claims description 8
- 239000008363 phosphate buffer Substances 0.000 claims description 8
- 238000007789 sealing Methods 0.000 claims description 8
- WBODDOZXDKQEFS-UHFFFAOYSA-N 1,2,3,4-tetramethyl-5-phenylbenzene Chemical group CC1=C(C)C(C)=CC(C=2C=CC=CC=2)=C1C WBODDOZXDKQEFS-UHFFFAOYSA-N 0.000 claims description 4
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 4
- GHCZTIFQWKKGSB-UHFFFAOYSA-N 2-hydroxypropane-1,2,3-tricarboxylic acid;phosphoric acid Chemical compound OP(O)(O)=O.OC(=O)CC(O)(C(O)=O)CC(O)=O GHCZTIFQWKKGSB-UHFFFAOYSA-N 0.000 claims description 4
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 4
- 238000002965 ELISA Methods 0.000 claims description 4
- DVKFVGVMPLXLKC-PUGXJXRHSA-N [(2s,3r,4s,5s,6r)-2-[(2s,3s,4s,5r)-3,4-dihydroxy-2,5-bis(hydroxymethyl)oxolan-2-yl]-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl] dihydrogen phosphate Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@]1(CO)[C@@]1(OP(O)(O)=O)[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 DVKFVGVMPLXLKC-PUGXJXRHSA-N 0.000 claims description 4
- 238000002835 absorbance Methods 0.000 claims description 4
- 239000007864 aqueous solution Substances 0.000 claims description 4
- 229940098773 bovine serum albumin Drugs 0.000 claims description 4
- 239000011248 coating agent Substances 0.000 claims description 4
- 238000000576 coating method Methods 0.000 claims description 4
- 238000013016 damping Methods 0.000 claims description 4
- 230000036039 immunity Effects 0.000 claims description 4
- 238000002360 preparation method Methods 0.000 claims description 4
- AQLJVWUFPCUVLO-UHFFFAOYSA-N urea hydrogen peroxide Chemical compound OO.NC(N)=O AQLJVWUFPCUVLO-UHFFFAOYSA-N 0.000 claims description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 4
- 230000035945 sensitivity Effects 0.000 abstract description 3
- 108090000623 proteins and genes Proteins 0.000 abstract description 2
- 102000004169 proteins and genes Human genes 0.000 abstract description 2
- 201000010099 disease Diseases 0.000 description 5
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 5
- 102000011931 Nucleoproteins Human genes 0.000 description 3
- 108010061100 Nucleoproteins Proteins 0.000 description 3
- 241000271566 Aves Species 0.000 description 2
- 238000000105 evaporative light scattering detection Methods 0.000 description 2
- 208000003322 Coinfection Diseases 0.000 description 1
- 241000711573 Coronaviridae Species 0.000 description 1
- 108090001074 Nucleocapsid Proteins Proteins 0.000 description 1
- 101710141454 Nucleoprotein Proteins 0.000 description 1
- 208000018569 Respiratory Tract disease Diseases 0.000 description 1
- 206010039438 Salmonella Infections Diseases 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 230000005875 antibody response Effects 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 210000003719 b-lymphocyte Anatomy 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 230000007969 cellular immunity Effects 0.000 description 1
- 230000001684 chronic effect Effects 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 102000035122 glycosylated proteins Human genes 0.000 description 1
- 108091005608 glycosylated proteins Proteins 0.000 description 1
- 230000004727 humoral immunity Effects 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 230000003053 immunization Effects 0.000 description 1
- 238000002649 immunization Methods 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000000034 method Methods 0.000 description 1
- 231100001028 renal lesion Toxicity 0.000 description 1
- 210000004994 reproductive system Anatomy 0.000 description 1
- 230000000241 respiratory effect Effects 0.000 description 1
- 210000002345 respiratory system Anatomy 0.000 description 1
- 238000003757 reverse transcription PCR Methods 0.000 description 1
- 206010039447 salmonellosis Diseases 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
Classifications
-
- 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
-
- 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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6875—Nucleoproteins
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
- G01N2333/08—RNA viruses
- G01N2333/165—Coronaviridae, e.g. avian infectious bronchitis virus
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Urology & Nephrology (AREA)
- Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Hematology (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Virology (AREA)
- Pathology (AREA)
- Food Science & Technology (AREA)
- Microbiology (AREA)
- Analytical Chemistry (AREA)
- Cell Biology (AREA)
- Biochemistry (AREA)
- Physics & Mathematics (AREA)
- General Physics & Mathematics (AREA)
- Tropical Medicine & Parasitology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a kit for rapidly detecting infectious bronchitis viruses. The kit comprises an infectious bronchitis virus N-recombined protein enveloped microporous reaction plate/strip, terminating liquid, a male contrast, a female contrast, an enzyme composition, a sample diluted solution, concentrated washing liquid, a color-developing solution A and a color-developing solution B. The kit is applied to large-batch detection and one-by-one/group-by-group manual detection and has the characteristics of high sensitivity and accuracy and high reproducibility.
Description
Technical field
The present invention relates to avian infectious bronchitis virus detection technique field, particularly relate to a kind of quick detection kit of avian infectious bronchitis virus.
Background technology
Avian infectious bronchitis (Avian Infectious Bronchitis, IB) be avian infectious bronchitis virus (the Avian Infectious Bronchitis Virus by coronaviridae coronavirus genus, IBV) a kind of acute, the height contagious disease of the chicken causing, IBV mainly encroaches on respiratory tract, alimentary canal, reproductive system and the kidney of chicken, and IBV produces harm in various degree to the chicken of different days.It is characterized by disease chicken and occur respiratory symptom, egg production and quality decline and renal lesions etc.IBV also often causes mixed infection (as ewcastle disease, chronic respiratory tract disease etc.), and can secondary bacteriosis as colibacillosis, salmonellosis etc., thereby increase the weight of the harm to chicken group.
IBV nucleocapsid protein claims again nucleoprotein (N albumen), has good antigenicity and immunogenicity, in cellular immunity and humoral immunity, plays an important role.The N albumen of vivoexpression can cause immune response, can make immune chicken speedup produce anti-IBV antibody, the immanoprotection action that can induce chicken to infect IBV tracheae.With after N protein immunization chicken, can activate T auxiliary cell, can strengthen the ability that B lymphocyte produces antibody simultaneously.Add the high conservative property of having of N albumen, this makes N albumen and antibody thereof demonstrate advantage aspect IBV diagnosis.It is large that nucleoprotein accounts for viral total protein concentration, and nucleoprotein is non-glycosylated protein, because it can detect group antibody response, can be used as IBV group specificity diagnostic antigen.
At present, the most length consuming time of the detection method of avian infectious bronchitis virus, complex operation, susceptibility, poor stability.PCR method has the advantages such as high specificity, highly sensitive and speed be fast, but is difficult to be promoted because of shortcomings such as the easy cross pollution of RT-PCR, complex operation, the heat circulating equipment that needs are expensive.
Summary of the invention
Object of the present invention is exactly that a kind of quick detection kit of avian infectious bronchitis virus is provided for the defect of above-mentioned existence.Be applied to during mass detection and one by one/group manually detects, there is sensitivity, accuracy is high, the feature that repeatability is good.
The quick detection kit technical scheme of a kind of avian infectious bronchitis virus of the present invention is that this kit includes the coated micro reaction plate/bar of infectious bronchitis of chicken N recombinant protein, stop buffer, positive control and negative control, enzyme conjugates, sample diluting liquid, concentrated cleaning solution, nitrite ion A and nitrite ion B.
Coated micro reaction plate/bar the preparation method of infectious bronchitis of chicken N recombinant protein is: with the 0.1mol/L Tris-HCl damping fluid of pH8, make coating buffer, by the dilution of infectious bronchitis of chicken N recombinant protein, be 5 μ g/mL, by 50 μ L/ holes, add in ELISA reaction plate, 37 ℃ of sealing 2h, 4 ℃ of coated spending the night, pat dry, with 37 ℃ of sealing 1-3h of 1% bovine serum albumin(BSA), 20%NaCl solution washing with containing Tween-20, pats dry, then adds 20% sucrose phosphate buffer room temperature to keep 2h.
Described sample diluting liquid is the 10%NaCl solution containing Tween-20; Described concentrated cleaning solution is the 20%NaCl solution containing Tween-20.
Described enzyme conjugates is the anti-chicken IgG of HRP-rabbit.
The tetramethyl biphenyl amine aqueous solution that described nitrite ion A is 0.2mg/L, nitrite ion B is the citric acid-phosphate buffer containing hydrogen peroxide urea.
Stop buffer is 3mol/L sulfuric acid solution.
Positive control is the standard positive serum obtaining through infectious bronchitis of chicken recombinant protein immunity, and then its OD450nm >=1.0 add the streptomysin of 5mg/mL, after aseptic filtration, obtain; Negative control is SPF chicken standard female serum, and its OD450nm≤0.25 adds the streptomysin of 5mg/mL, after aseptic filtration, obtains.
Serum to be checked is done to 1:500 dilution with sample diluting liquid, by 25-100 μ L/ hole, add in antibody test plate, establish negative control, positive control simultaneously, hatch 30-60min for 37 ℃; Discard the liquid in reacting hole, every hole adds the concentrated cleaning solution 200 μ L after pure water 1:20 dilution, washs 5 times, pats dry; Every hole adds, and the enzyme conjugates working fluid of 50-150 μ L is hatched 30-60min for 37 ℃; Wash 5 times, pat dry; Add successively 50 μ L nitrite ion A and 50 μ L nitrite ion B, 37 ℃ of lucifuges are hatched 15min; Add 50 μ L stop buffers, by microplate reader, under 450nm wavelength, measure each hole absorbance A value; Ratio with sample OD450nm value to be checked and standard female OD450nm value is greater than 2, and sample OD450nm value to be checked is greater than 0.5 and is judged to the positive.
Beneficial effect of the present invention is: kit of the present invention is easy to use, and sensing range is broad, highly sensitive.Be applied to during mass detection and one by one/group manually detects, there is sensitivity, accuracy is high, the feature that repeatability is good.
embodiment:
In order to understand better the present invention, with instantiation, describe technical scheme of the present invention in detail below.
Embodiment 1
The quick detection kit of a kind of avian infectious bronchitis virus of the present invention comprises: infectious bronchitis of chicken N recombinant protein is coated with micro reaction plate/bar, stop buffer, positive control and negative control, enzyme conjugates, sample diluting liquid, concentrated cleaning solution, nitrite ion A and nitrite ion B.
Coated micro reaction plate/bar the preparation method of infectious bronchitis of chicken N recombinant protein is: with the 0.1mol/L Tris-HCl damping fluid of pH8, make coating buffer, by the dilution of infectious bronchitis of chicken N recombinant protein, be 5 μ g/mL, by 50 μ L/ holes, add in ELISA reaction plate, 37 ℃ of sealing 2h, 4 ℃ of coated spending the night, pat dry, with 37 ℃ of sealing 3h of 1% bovine serum albumin(BSA), 20%NaCl solution washing with containing Tween-20, pats dry, then adds 20% sucrose phosphate buffer room temperature to keep 2h.
Described sample diluting liquid is the 10%NaCl solution containing Tween-20; Described concentrated cleaning solution is the 20%NaCl solution containing Tween-20.
Described enzyme conjugates is the anti-chicken IgG of HRP-rabbit.
The tetramethyl biphenyl amine aqueous solution that described nitrite ion A is 0.2mg/L, nitrite ion B is the citric acid-phosphate buffer containing hydrogen peroxide urea.
Stop buffer is 3mol/L sulfuric acid solution.
Positive control is the standard positive serum obtaining through infectious bronchitis of chicken recombinant protein immunity, and then its OD450nm >=1.0 add the streptomysin of 5mg/mL, after aseptic filtration, obtain; Negative control is SPF chicken standard female serum, and its OD450nm≤0.25 adds the streptomysin of 5mg/mL, after aseptic filtration, obtains.
Serum to be checked is done to 1:500 dilution with sample diluting liquid, by 50 μ L/ holes, add in antibody test plate, establish negative control, positive control simultaneously, hatch 30-60min for 37 ℃; Discard the liquid in reacting hole, every hole adds the concentrated cleaning solution 200 μ L after pure water 1:20 dilution, washs 5 times, pats dry; Every hole adds, and the enzyme conjugates working fluid of 100 μ L is hatched 30-60min for 37 ℃; Wash 5 times, pat dry; Add successively 50 μ L nitrite ion A and 50 μ L nitrite ion B, 37 ℃ of lucifuges are hatched 15min; Add 50 μ L stop buffers, by microplate reader, under 450nm wavelength, measure each hole absorbance A value; Ratio with sample OD450nm value to be checked and standard female OD450nm value is greater than 2, and sample OD450nm value to be checked is greater than 0.5 and is judged to the positive.
Apply blood serum sample that kit of the present invention infects disease chicken to 50 parts of doubtful avian infectious bronchitis viruses and carried out infectious bronchitis of chicken antibody horizontal and detect, positive rate reaches 100%.
Embodiment 2
The quick detection kit of a kind of avian infectious bronchitis virus of the present invention comprises: infectious bronchitis of chicken N recombinant protein is coated with micro reaction plate/bar, stop buffer, positive control and negative control, enzyme conjugates, sample diluting liquid, concentrated cleaning solution, nitrite ion A and nitrite ion B.
Coated micro reaction plate/bar the preparation method of infectious bronchitis of chicken N recombinant protein is: with the 0.1mol/L Tris-HCl damping fluid of pH8, make coating buffer, by the dilution of infectious bronchitis of chicken N recombinant protein, be 5 μ g/mL, by 50 μ L/ holes, add in ELISA reaction plate, 37 ℃ of sealing 2h, 4 ℃ of coated spending the night, pat dry, with 37 ℃ of sealing 2h of 1% bovine serum albumin(BSA), 20%NaCl solution washing with containing Tween-20, pats dry, then adds 20% sucrose phosphate buffer room temperature to keep 2h.
Described sample diluting liquid is the 10%NaCl solution containing Tween-20; Described concentrated cleaning solution is the 20%NaCl solution containing Tween-20.
Described enzyme conjugates is the anti-chicken IgG of HRP-rabbit.
The tetramethyl biphenyl amine aqueous solution that described nitrite ion A is 0.2mg/L, nitrite ion B is the citric acid-phosphate buffer containing hydrogen peroxide urea.
Stop buffer is 3mol/L sulfuric acid solution.
Positive control is the standard positive serum obtaining through infectious bronchitis of chicken recombinant protein immunity, and then its OD450nm >=1.0 add the streptomysin of 5mg/mL, after aseptic filtration, obtain; Negative control is SPF chicken standard female serum, and its OD450nm≤0.25 adds the streptomysin of 5mg/mL, after aseptic filtration, obtains.
Serum to be checked is done to 1:500 dilution with sample diluting liquid, by 100 μ L/ holes, add in antibody test plate, establish negative control, positive control simultaneously, hatch 30-60min for 37 ℃; Discard the liquid in reacting hole, every hole adds the concentrated cleaning solution 200 μ L after pure water 1:20 dilution, washs 5 times, pats dry; Every hole adds, and the enzyme conjugates working fluid of 100 μ L is hatched 30-60min for 37 ℃; Wash 5 times, pat dry; Add successively 50 μ L nitrite ion A and 50 μ L nitrite ion B, 37 ℃ of lucifuges are hatched 15min; Add 50 μ L stop buffers, by microplate reader, under 450nm wavelength, measure each hole absorbance A value; Ratio with sample OD450nm value to be checked and standard female OD450nm value is greater than 2, and sample OD450nm value to be checked is greater than 0.5 and is judged to the positive.
Apply blood serum sample that kit of the present invention infects disease chicken to 50 parts of doubtful avian infectious bronchitis viruses and carried out infectious bronchitis of chicken antibody horizontal and detect, positive rate reaches 99.6%.
Claims (8)
1. the quick detection kit of an avian infectious bronchitis virus, it is characterized in that, this kit includes the coated micro reaction plate/bar of infectious bronchitis of chicken N recombinant protein, stop buffer, positive control and negative control, enzyme conjugates, sample diluting liquid, concentrated cleaning solution, nitrite ion A and nitrite ion B.
2. the quick detection kit of a kind of avian infectious bronchitis virus according to claim 1, it is characterized in that, coated micro reaction plate/bar the preparation method of infectious bronchitis of chicken N recombinant protein is: with the 0.1mol/L Tris-HCl damping fluid of pH8, make coating buffer, by the dilution of infectious bronchitis of chicken N recombinant protein, be 5 μ g/mL, by 50 μ L/ holes, add in ELISA reaction plate, 37 ℃ of sealing 2h, 4 ℃ of coated spending the night, pat dry, with 37 ℃ of sealing 1-3h of 1% bovine serum albumin(BSA), with the 20%NaCl solution washing containing Tween-20, pat dry, add again 20% sucrose phosphate buffer room temperature to keep 2h.
3. the quick detection kit of a kind of avian infectious bronchitis virus according to claim 1, is characterized in that, described sample diluting liquid is the 10%NaCl solution containing Tween-20; Described concentrated cleaning solution is the 20%NaCl solution containing Tween-20.
4. the quick detection kit of a kind of avian infectious bronchitis virus according to claim 1, is characterized in that, described enzyme conjugates is the anti-chicken IgG of HRP-rabbit.
5. the quick detection kit of a kind of avian infectious bronchitis virus according to claim 1, it is characterized in that, the tetramethyl biphenyl amine aqueous solution that described nitrite ion A is 0.2mg/L, nitrite ion B is the citric acid-phosphate buffer containing hydrogen peroxide urea.
6. the quick detection kit of a kind of avian infectious bronchitis virus according to claim 1, is characterized in that, stop buffer is 3mol/L sulfuric acid solution.
7. the quick detection kit of a kind of avian infectious bronchitis virus according to claim 1, it is characterized in that, positive control is the standard positive serum obtaining through the immunity of infectious bronchitis of chicken recombinant protein, its OD450nm >=1.0, then the streptomysin that adds 5mg/mL obtains after aseptic filtration; Negative control is SPF chicken standard female serum, and its OD450nm≤0.25 adds the streptomysin of 5mg/mL, after aseptic filtration, obtains.
8. according to the quick detection kit of the arbitrary described a kind of avian infectious bronchitis virus of claim 1-7, it is characterized in that, serum to be checked is done to 1:500 dilution with sample diluting liquid, by 25-100 μ L/ hole, add in antibody test plate, establish negative control, positive control simultaneously, hatch 30-60min for 37 ℃; Discard the liquid in reacting hole, every hole adds the concentrated cleaning solution 200 μ L after pure water 1:20 dilution, washs 5 times, pats dry; Every hole adds, and the enzyme conjugates working fluid of 50-150 μ L is hatched 30-60min for 37 ℃; Wash 5 times, pat dry; Add successively 50 μ L nitrite ion A and 50 μ L nitrite ion B, 37 ℃ of lucifuges are hatched 15min; Add 50 μ L stop buffers, by microplate reader, under 450nm wavelength, measure each hole absorbance A value; Ratio with sample OD450nm value to be checked and standard female OD450nm value is greater than 2, and sample OD450nm value to be checked is greater than 0.5 and is judged to the positive.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310744847.6A CN103698517B (en) | 2013-12-30 | 2013-12-30 | A kind of quick detection kit of avian infectious bronchitis virus |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201310744847.6A CN103698517B (en) | 2013-12-30 | 2013-12-30 | A kind of quick detection kit of avian infectious bronchitis virus |
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| Publication Number | Publication Date |
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| CN103698517A true CN103698517A (en) | 2014-04-02 |
| CN103698517B CN103698517B (en) | 2015-09-09 |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105092839A (en) * | 2015-08-20 | 2015-11-25 | 中国农业科学院哈尔滨兽医研究所 | Detection kit for chicken infectious bronchitis indirect ELISA antibody |
| CN105785049A (en) * | 2016-04-16 | 2016-07-20 | 浙江大学 | Infectious bronchitis virus (IBV) nsp5 (non-structural protein) ELISA (enzyme-linked immunosorbent assay) antibody detection kit and application thereof |
| CN111766389A (en) * | 2020-07-28 | 2020-10-13 | 郑州大学 | ELISA antibody detection kit based on chicken infectious bronchitis virus recombinant N protein |
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| KR20100101217A (en) * | 2009-03-09 | 2010-09-17 | 대한민국(관리부서 : 농림수산식품부 국립수의과학검역원) | Avian infectious bronchitis virus and vaccine for avian infectious bronchitis comprising the same |
| CN102093999A (en) * | 2010-12-14 | 2011-06-15 | 山东省农业科学院家禽研究所 | Indirect ELISA kit for detecting avian infectious bronchitis virus antibody |
| CN102721812A (en) * | 2012-06-20 | 2012-10-10 | 江苏省农业科学院 | Indirect ELISA (enzyme-linked immuno-sorbent assay) kit for detecting nephropathogenic avian infectious bronchitis virus and antibody thereof |
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2013
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|---|---|---|---|---|
| CN2578831Y (en) * | 2002-11-21 | 2003-10-08 | 陈福勇 | Antibody inspection reagent box of virus of infectious fowl bronchitis |
| RU2380708C1 (en) * | 2008-12-05 | 2010-01-27 | Государственное образовательное учреждение высшего профессионального образования "Московская государственная академия тонкой химической технологии имени М.В. Ломоносова" | Method for making antibody test system for latex fixation test |
| KR20100101217A (en) * | 2009-03-09 | 2010-09-17 | 대한민국(관리부서 : 농림수산식품부 국립수의과학검역원) | Avian infectious bronchitis virus and vaccine for avian infectious bronchitis comprising the same |
| CN102093999A (en) * | 2010-12-14 | 2011-06-15 | 山东省农业科学院家禽研究所 | Indirect ELISA kit for detecting avian infectious bronchitis virus antibody |
| CN102721812A (en) * | 2012-06-20 | 2012-10-10 | 江苏省农业科学院 | Indirect ELISA (enzyme-linked immuno-sorbent assay) kit for detecting nephropathogenic avian infectious bronchitis virus and antibody thereof |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105092839A (en) * | 2015-08-20 | 2015-11-25 | 中国农业科学院哈尔滨兽医研究所 | Detection kit for chicken infectious bronchitis indirect ELISA antibody |
| CN105785049A (en) * | 2016-04-16 | 2016-07-20 | 浙江大学 | Infectious bronchitis virus (IBV) nsp5 (non-structural protein) ELISA (enzyme-linked immunosorbent assay) antibody detection kit and application thereof |
| CN111766389A (en) * | 2020-07-28 | 2020-10-13 | 郑州大学 | ELISA antibody detection kit based on chicken infectious bronchitis virus recombinant N protein |
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| CN103698517B (en) | 2015-09-09 |
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Denomination of invention: A rapid detection kit for infectious bronchitis virus in chickens Effective date of registration: 20231011 Granted publication date: 20150909 Pledgee: Weihai Commercial Bank Co.,Ltd. Binzhou Branch Pledgor: SHANDONG BINZHOU BOLAIWEI BIOTECH Co.,Ltd. Registration number: Y2023980060827 |