CN103675275A - Duck flavivirus detection reagent kit - Google Patents
Duck flavivirus detection reagent kit Download PDFInfo
- Publication number
- CN103675275A CN103675275A CN201310742908.5A CN201310742908A CN103675275A CN 103675275 A CN103675275 A CN 103675275A CN 201310742908 A CN201310742908 A CN 201310742908A CN 103675275 A CN103675275 A CN 103675275A
- Authority
- CN
- China
- Prior art keywords
- duck flavivirus
- duck
- detection kit
- nitrite ion
- add
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
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
- 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
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Virology (AREA)
- Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- Urology & Nephrology (AREA)
- Molecular Biology (AREA)
- Hematology (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Biotechnology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a duck flavivirus detection reagent kit, which comprises a duck flavivirus E recombinant protein coating micropore reaction plate/strip, stop solution, a positive control part, a negative control part, enzyme bonders, sample diluent, concentrated washing liquid, color developing liquid A and color developing liquid B. The detection reagent kit disclosed by the invention has the advantages that the sensitivity is good, the specificity is high, accuracy and reliability are realized, and the detection reagent kit can effectively help farmers to reduce the loss and to improve the culture benefits.
Description
Technical field
The present invention relates to duck flavivirus detection technique field, particularly relate to a kind of duck flavivirus detection kit.
Background technology
Duck flavivirus (Bai yang dian virus, BYD) has another name called duck tembusu virus, mainly causes that kind of duck and egg duck appetite sharply go down, lay eggs sharply to decline, and ovarian follicle sex change, membrana follicularis is hemorrhage is principal character.
Therefore in the urgent need to setting up a kind of duck flavivirus detection method of quick sensitivity, just can detect these viruses in early days infecting, thereby strive for the valuable time for the control of these diseases.Traditional detection method exist length consuming time, susceptibility lower, be difficult for the shortcomings such as standardization, there is in actual applications certain limitation.Quantitative fluorescent PCR technology realized to template quantitatively, and have sensitivity, special, accurately and reliably, can realize multiple reaction and the feature such as real-time is good.In practical application, when sample size is very large, there is certain inferior position in substance fluorescent PCR aspect cost and time, in the urgent need to a kind of high flux, low cost, high efficiency method, carries out fast detecting in batches.
Summary of the invention
Object of the present invention is exactly to provide a kind of duck flavivirus detection kit for the defect of above-mentioned existence.This detection kit susceptibility is good, specificity is high, accurately and reliably, can effectively help raiser to reduce the loss, improve culture benefit.
A kind of duck flavivirus detection kit technical scheme of the present invention is that this kit includes the coated micro reaction plate/bar of duck flavivirus E 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 duck flavivirus E recombinant protein is: with the 0.05mol/L Tris-HCl damping fluid of pH8.5, make coating buffer, by the dilution of duck flavivirus E recombinant protein, be 5 μ g/mL, by 50-100 μ L/ hole, 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), phosphate buffer washing with the 0.1mol/L pH7.4 containing 0.5% Tween-20, pat dry, then add 20% sucrose phosphate buffer room temperature to keep 2h.
Described sample diluting liquid is the phosphate buffer of the 0.1mol/L pH7.4 containing 0.05% Tween-20; Described concentrated cleaning solution is the phosphate buffer of the 0.5mol/L pH7.4 containing 0.5% Tween-20.
Described enzyme conjugates is goat-anti chicken IgG.
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 duck flavivirus 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≤0.20 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 the enzyme conjugates working fluid of 50-150 μ L, hatches 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; Negative and positive critical value is 0.40.
Beneficial effect of the present invention is: a kind of duck flavivirus detection kit of the present invention, susceptibility is good, specificity is high, accurately and reliably, can effectively help raiser to reduce the loss, improve culture benefit.
embodiment:
In order to understand better the present invention, with instantiation, describe technical scheme of the present invention in detail below.
Embodiment 1
Kit includes the coated micro reaction plate/bar of duck flavivirus E 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 duck flavivirus E recombinant protein is: with the 0.05mol/L Tris-HCl damping fluid of pH8.5, make coating buffer, by the dilution of duck flavivirus E recombinant protein, be 5 μ g/mL, 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), phosphate buffer washing with the 0.1mol/L pH7.4 containing 0.5% Tween-20, pats dry, then adds 20% sucrose phosphate buffer room temperature to keep 2h.
Described sample diluting liquid is the phosphate buffer of the 0.1mol/L pH7.4 containing 0.05% Tween-20; Described concentrated cleaning solution is the phosphate buffer of the 0.5mol/L pH7.4 containing 0.5% Tween-20.
Described enzyme conjugates is goat-anti chicken IgG.
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 duck flavivirus 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≤0.20 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 the enzyme conjugates working fluid of 100 μ L, hatches 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;
Apply the blood serum sample that kit of the present invention infects disease duck to 50 parts of doubtful duck flavivirus and detect, positive rate reaches 100%.
Embodiment 2
Kit includes the coated micro reaction plate/bar of duck flavivirus E 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 duck flavivirus E recombinant protein is: with the 0.05mol/L Tris-HCl damping fluid of pH8.5, make coating buffer, by the dilution of duck flavivirus E recombinant protein, be 5 μ g/mL, by 50-100 μ L/ hole, 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), phosphate buffer washing with the 0.1mol/L pH7.4 containing 0.5% Tween-20, pat dry, then add 20% sucrose phosphate buffer room temperature to keep 2h.
Described sample diluting liquid is the phosphate buffer of the 0.1mol/L pH7.4 containing 0.05% Tween-20; Described concentrated cleaning solution is the phosphate buffer of the 0.5mol/L pH7.4 containing 0.5% Tween-20.
Described enzyme conjugates is goat-anti chicken IgG.
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 duck flavivirus 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≤0.20 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 the enzyme conjugates working fluid of 100 μ L, hatches 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;
Apply the blood serum sample that kit of the present invention infects disease duck to 50 parts of doubtful duck flavivirus and detect, positive rate reaches 99.8%.
Claims (8)
1. a duck flavivirus detection kit, it is characterized in that, this kit includes the coated micro reaction plate/bar of duck flavivirus E 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. a kind of duck flavivirus detection kit according to claim 1, it is characterized in that, coated micro reaction plate/bar the preparation method of duck flavivirus E recombinant protein is: with the 0.05mol/L Tris-HCl damping fluid of pH8.5, make coating buffer, by the dilution of duck flavivirus E recombinant protein, be 5 μ g/mL, by 50-100 μ L/ hole, 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), phosphate buffer washing with the 0.1mol/L pH7.4 containing 0.5% Tween-20, pat dry, add again 20% sucrose phosphate buffer room temperature to keep 2h.
3. a kind of duck flavivirus detection kit according to claim 1, is characterized in that, described sample diluting liquid is the phosphate buffer of the 0.1mol/L pH7.4 containing 0.05% Tween-20; Described concentrated cleaning solution is the phosphate buffer of the 0.5mol/L pH7.4 containing 0.5% Tween-20.
4. a kind of duck flavivirus detection kit according to claim 1, is characterized in that, described enzyme conjugates is goat-anti chicken IgG.
5. a kind of duck flavivirus detection kit according to claim 1, is characterized in that, the tetramethyl biphenyl amine aqueous solution that described nitrite ion A is 0.2mg/L, and nitrite ion B is the citric acid-phosphate buffer containing hydrogen peroxide urea.
6. a kind of duck flavivirus detection kit according to claim 1, is characterized in that, stop buffer is 3mol/L sulfuric acid solution.
7. a kind of duck flavivirus detection kit according to claim 1, it is characterized in that, positive control is the standard positive serum obtaining through the immunity of duck flavivirus 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≤0.20 adds the streptomysin of 5mg/mL, after aseptic filtration, obtains.
8. according to the arbitrary described a kind of duck flavivirus detection kit 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 the enzyme conjugates working fluid of 50-150 μ L, hatches 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; Negative and positive critical value is 0.40.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310742908.5A CN103675275A (en) | 2013-12-30 | 2013-12-30 | Duck flavivirus detection reagent kit |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310742908.5A CN103675275A (en) | 2013-12-30 | 2013-12-30 | Duck flavivirus detection reagent kit |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN103675275A true CN103675275A (en) | 2014-03-26 |
Family
ID=50313461
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310742908.5A Pending CN103675275A (en) | 2013-12-30 | 2013-12-30 | Duck flavivirus detection reagent kit |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103675275A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103901213A (en) * | 2014-03-31 | 2014-07-02 | 山东农业大学 | Detection method for distinguishing generation of antibody through activated vaccine and inactivated vaccine of tembusu virus |
| CN108169496A (en) * | 2018-03-14 | 2018-06-15 | 山东省农业科学院家禽研究所 | A kind of DHAV-3 types polypeptide indirect ELISA antibody assay kit and its application |
| CN108445208A (en) * | 2018-03-14 | 2018-08-24 | 山东省农业科学院家禽研究所 | A kind of universal DHAV polypeptides indirect ELISA antibody assay kit and its application |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2004040263A2 (en) * | 2002-10-31 | 2004-05-13 | Health Research, Inc. | Diagnostic test for west nile virus |
| EP1757934A1 (en) * | 2005-08-26 | 2007-02-28 | Buddhist Tzu Chi General Hospital | Method for screening compounds against flaviviruses by using persistent virus-infected cell system |
| CN102323411A (en) * | 2011-08-15 | 2012-01-18 | 福建省农业科学院畜牧兽医研究所 | Indirect enzyme-linked immunosorbent assay (ELISA) method for detecting egg yolk antibody of duck flavivirus |
| CN102384975A (en) * | 2011-08-15 | 2012-03-21 | 福建省农业科学院畜牧兽医研究所 | Indirect enzyme-linked immunosorbent assay (ELISA) method for detecting duck flavivirus serum antibody |
| CN102533668A (en) * | 2010-12-20 | 2012-07-04 | 中国农业科学院上海兽医研究所 | Duck flavivirus, and vaccine and kit thereof |
| CN102618557A (en) * | 2012-03-20 | 2012-08-01 | 江苏省农业科学院 | Recombinant avian flavivirus E protein and application thereof |
-
2013
- 2013-12-30 CN CN201310742908.5A patent/CN103675275A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2004040263A2 (en) * | 2002-10-31 | 2004-05-13 | Health Research, Inc. | Diagnostic test for west nile virus |
| EP1757934A1 (en) * | 2005-08-26 | 2007-02-28 | Buddhist Tzu Chi General Hospital | Method for screening compounds against flaviviruses by using persistent virus-infected cell system |
| CN102533668A (en) * | 2010-12-20 | 2012-07-04 | 中国农业科学院上海兽医研究所 | Duck flavivirus, and vaccine and kit thereof |
| CN102323411A (en) * | 2011-08-15 | 2012-01-18 | 福建省农业科学院畜牧兽医研究所 | Indirect enzyme-linked immunosorbent assay (ELISA) method for detecting egg yolk antibody of duck flavivirus |
| CN102384975A (en) * | 2011-08-15 | 2012-03-21 | 福建省农业科学院畜牧兽医研究所 | Indirect enzyme-linked immunosorbent assay (ELISA) method for detecting duck flavivirus serum antibody |
| CN102618557A (en) * | 2012-03-20 | 2012-08-01 | 江苏省农业科学院 | Recombinant avian flavivirus E protein and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| 郝明飞: "鸭坦布苏病毒巢式PCR检测方法及其重组E蛋白间接ELISA诊断方法的研究", 《中国优秀硕士学位论文全文数据库》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103901213A (en) * | 2014-03-31 | 2014-07-02 | 山东农业大学 | Detection method for distinguishing generation of antibody through activated vaccine and inactivated vaccine of tembusu virus |
| CN103901213B (en) * | 2014-03-31 | 2016-04-13 | 山东农业大学 | A kind of tembusu virus poison of living of distinguishing produces the detection method of antibody with inactivated vaccine |
| CN108169496A (en) * | 2018-03-14 | 2018-06-15 | 山东省农业科学院家禽研究所 | A kind of DHAV-3 types polypeptide indirect ELISA antibody assay kit and its application |
| CN108445208A (en) * | 2018-03-14 | 2018-08-24 | 山东省农业科学院家禽研究所 | A kind of universal DHAV polypeptides indirect ELISA antibody assay kit and its application |
| CN108169496B (en) * | 2018-03-14 | 2022-06-28 | 山东省农业科学院家禽研究所 | DHAV-3 type polypeptide indirect ELISA antibody detection kit and application thereof |
| CN108445208B (en) * | 2018-03-14 | 2022-06-28 | 山东省农业科学院家禽研究所 | Universal DHAV polypeptide indirect ELISA antibody detection kit and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN109655621B (en) | Indirect ELISA antibody detection method for swine T-type coronavirus N protein and kit thereof | |
| CN103675275A (en) | Duck flavivirus detection reagent kit | |
| CN103308684A (en) | ELISA (enzyme-linked immuno sorbent assay) detection kit of porcine pseudorabies virus IgM antibody | |
| CN103217414A (en) | Chemiluminescent immunoassay method for 3,5,3'-triiodothyronine in blood serum | |
| CN105388282A (en) | Coating method for indirect antigen-coated elisa plate of ELISA kit | |
| CN101936997B (en) | Human anti-rabies virus IgG antibody ELISA test kit | |
| CN111413507A (en) | Method for evaluating plasma antiviral ability in convalescent period by detecting S protein RBD antibody | |
| CN103399148A (en) | Human respiratory syncytial virus detection kit based on nanogold signal amplification | |
| CN103698517B (en) | A kind of quick detection kit of avian infectious bronchitis virus | |
| CN101477117B (en) | Visible protein chip for detecting poultry disease serum antibody, its preparation method and application | |
| CN109239348A (en) | Gastrin-releasing peptide precursor detection kit antibody and kit | |
| CN103665098B (en) | Diphasic column membrane protein microreactor and application thereof | |
| CN117229367A (en) | African swine fever virus epitope polypeptide and ELISA antibody detection kit | |
| CN105510580A (en) | Polypeptide-ELISA (enzyme-linked immunosorbent assay) kit for detecting specific antibody of N (nucleocapsid) protein of SFTSV (severe fever with thrombocytopenia syndrome virus) | |
| CN105974127A (en) | Human neutrophil apolipoprotein heterodimer quantifying device based on enzyme-linked immune adsorption technology | |
| CN102433339B (en) | HCV Nucleic acid aptamer and application thereof in preparing HCV-cAg detection kit | |
| CN105758847A (en) | Chemiluminescence enzyme linked immunoassay kit for detecting sulfonamides | |
| CN206002549U (en) | A kind of human neutrophil apolipoprotein heterodimer proportioning device based on Enzyme-linked Immunosorbent Assay technology | |
| EP2905615B1 (en) | Method for measuring hemagglutinin from influenza virus | |
| CN101900736A (en) | Detect a kind of ELISA kit of sequence-specific epitope antibodies | |
| CN105606816A (en) | Polypeptide-ELISA kit for detecting specific antibody against envelope glycoprotein of severe fever with thrombocytopenia syndrome virus | |
| CN107478831B (en) | Anti-idiotype detection method | |
| JP2018072045A (en) | Method for inspection of human blood and human blood inspection kit | |
| CN105486855B (en) | Improve indirect enzyme-linked immunosorbent assay | |
| CN105541983A (en) | pfHRP-2 recombinant protein and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C12 | Rejection of a patent application after its publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20140326 |