CN106771179A - A kind of detection method of PRV - Google Patents
A kind of detection method of PRV Download PDFInfo
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- CN106771179A CN106771179A CN201611025747.8A CN201611025747A CN106771179A CN 106771179 A CN106771179 A CN 106771179A CN 201611025747 A CN201611025747 A CN 201611025747A CN 106771179 A CN106771179 A CN 106771179A
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- 238000001514 detection method Methods 0.000 title claims abstract description 31
- 239000007788 liquid Substances 0.000 claims abstract description 42
- 238000004140 cleaning Methods 0.000 claims abstract description 41
- 239000000243 solution Substances 0.000 claims abstract description 41
- 210000002966 serum Anatomy 0.000 claims abstract description 25
- 239000000427 antigen Substances 0.000 claims abstract description 23
- 102000036639 antigens Human genes 0.000 claims abstract description 23
- 108091007433 antigens Proteins 0.000 claims abstract description 23
- 238000011534 incubation Methods 0.000 claims abstract description 15
- 238000010790 dilution Methods 0.000 claims abstract description 11
- 239000012895 dilution Substances 0.000 claims abstract description 11
- 239000013642 negative control Substances 0.000 claims abstract description 10
- 239000013641 positive control Substances 0.000 claims abstract description 10
- 238000002156 mixing Methods 0.000 claims abstract description 7
- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 claims abstract description 6
- IOVCWXUNBOPUCH-UHFFFAOYSA-M Nitrite anion Chemical compound [O-]N=O IOVCWXUNBOPUCH-UHFFFAOYSA-M 0.000 claims description 10
- 229940005654 nitrite ion Drugs 0.000 claims description 10
- 238000000034 method Methods 0.000 claims description 8
- 102000004190 Enzymes Human genes 0.000 claims description 5
- 108090000790 Enzymes Proteins 0.000 claims description 5
- 238000002835 absorbance Methods 0.000 claims description 5
- 235000020183 skimmed milk Nutrition 0.000 claims description 5
- 239000000758 substrate Substances 0.000 claims description 5
- 238000005406 washing Methods 0.000 claims description 5
- 230000003612 virological effect Effects 0.000 claims description 4
- 239000011248 coating agent Substances 0.000 claims 1
- 238000000576 coating method Methods 0.000 claims 1
- 241000701093 Suid alphaherpesvirus 1 Species 0.000 description 12
- 241000282898 Sus scrofa Species 0.000 description 5
- 239000000203 mixture Substances 0.000 description 4
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 3
- 238000007789 sealing Methods 0.000 description 3
- 241001465754 Metazoa Species 0.000 description 2
- 241000700605 Viruses Species 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 208000009305 pseudorabies Diseases 0.000 description 2
- 229960005486 vaccine Drugs 0.000 description 2
- 208000031504 Asymptomatic Infections Diseases 0.000 description 1
- 208000035473 Communicable disease Diseases 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 206010029719 Nonspecific reaction Diseases 0.000 description 1
- 208000003251 Pruritus Diseases 0.000 description 1
- 230000004520 agglutination Effects 0.000 description 1
- 230000000840 anti-viral effect Effects 0.000 description 1
- 230000002238 attenuated effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 201000002491 encephalomyelitis Diseases 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 239000004816 latex Substances 0.000 description 1
- 229920000126 latex Polymers 0.000 description 1
- 230000008774 maternal effect Effects 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- 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
- G01N2333/01—DNA viruses
- G01N2333/03—Herpetoviridae, e.g. pseudorabies virus
- G01N2333/032—Pseudorabies virus, i.e. Aujetzky virus
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- 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)
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- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention discloses a kind of detection method of PRV, comprise the following steps：By sample by diluted, quantitatively it is coated in ELISA96 orifice plates, the μ L of serum-dilution to be checked 50 ~ 100 that will have been diluted are added in antigen coated microplate, while setting positive and negative control wells, respectively set 2 holes, per the μ L of hole 100,4 DEG C overnight；The antigen being not associated with is cleaned with PBST cleaning solutions；Negative serum, positive serum and serum to be checked are added in corresponding detection hole respectively, slight vibrations make the liquid blending in reaction plate；37 DEG C of incubations are placed in after micro-reaction plate is sealed.The detection method of PRV of the present invention enormously simplify detecting step, reduce operation difficulty, improve accuracy of detection.
Description
Technical field
The present invention relates to a kind of detection method, specifically a kind of detection method of PRV.
Background technology
Pseudoabies (Pseudorabies, PR) is caused by pseudorabies virus (Pseudorabies Virus, PrV)
A kind of including domestic animal and various wild animals to generate heat, strange itch and encephalomyelitis is the Important Infectious Diseases of symptom.The disease is given
Pig industry causes huge economic loss, and pig is this sick natural host and storage person.Prevent, control and finally eradicate the disease
It is a challenge that China currently faces.In the work anti-processed of pseudoabies, pseudo- mad dog in monitoring and detection Swine serum
The level of sick antiviral antibody is a very important link.For still nonvaccinated pig, the porcine pseudorabies disease in serum
Malicious antibody both may reflect the influence of maternal antibody, it is also possible to reflect the sign of subclinical infection.Both of these case all can shadow
Ring the effect of inoculation to attenuated live vaccine, in some instances it may even be possible to make vaccine invalid, it is impossible to produce the immunoprotection to virus, cause to be immunized
Failure.For the pig being inoculated with, the porcine pseudorabies virus antibody level in serum then reflects immune effect.
Domestic detection PRV antibody is typically using latex agglutination, indirect ELISA method etc., these methods at present
Sensitiveness is not high, higher to antigen purity requirement, and pure antigen truly is difficult to obtain.Therefore inevitably
Nonspecific reaction is produced, erroneous judgement is caused to result.
The content of the invention
It is an object of the invention to provide a kind of detection method of PRV, to solve to be carried in above-mentioned background technology
The problem for going out.
To achieve the above object, the present invention provides following technical scheme：
A kind of detection method of PRV, comprises the following steps：By sample by diluted, quantitatively it is coated on
In ELISA96 orifice plates, the μ L of serum-dilution to be checked 50 ~ 100 that will have been diluted are added in antigen coated microplate, while setting positive and negative pair
According to hole, 2 holes are respectively set, per the μ L of hole 100,4 DEG C overnight；The antigen being not associated with is cleaned with PBST cleaning solutions；Respectively by feminine gender
Serum, positive serum and serum to be checked are added in corresponding detection hole, and slight vibrations make the liquid blending in reaction plate；Will be micro-
37 DEG C of incubations are placed in after the sealing of quantitative response plate；The vertical liquid outwelled in reaction plate, and carried out carrying out each hole with cleaning solution
Cleaning；Reaction plate is closed with confining liquid, is placed in 37 DEG C of incubations；Liquid in removal reaction plate, and carried out with cleaning solution
Each hole is cleaned；The enzyme mark monoclonal antibody that will have been diluted is added in each reacting hole, is placed in 37 DEG C of incubations；Outwell reaction plate
In liquid, and carried out cleaning each hole with cleaning solution；Addition substrate, is placed in 37 DEG C and reacts；Terminate liquid is added, eventually
Only react, and 450nm absorbances are read in ELIASA, sample value/feminine gender value is judged to the positive more than 2, be feminine gender less than 1.
As further scheme of the invention：Also comprise the following steps：1）Being taken out from kit pre-coated has virus anti-
Former detection plate, the serum to be checked 1 that will have been diluted：1 100 μ L of dilution are added in antigen coated microplate, while setting positive and negative control
Hole, respectively sets 2 holes, per the μ L of hole 100；2）Gently shake sample in even hole, puts 37 DEG C and incubates 30 minutes, gets rid of the solution in plate hole, uses
Cleaning solution board-washing 5 times, 200 μ L/ holes stand outwell for 3 minutes every time, are patted dry on blotting paper for the last time；3）Per the enzyme-added mark in hole
The μ L of monoclonal antibody 100, put 37 DEG C and incubate 30 minutes, wash 5 times, and method is with step 2）, each drop 50 of nitrite ion A, nitrite ion B is added per hole
μ L, mix, and room temperature lucifuge develops the color 10 minutes, add terminate liquid one to drip 50 μ L per hole.
As further scheme of the invention：Described confining liquid is 5% skimmed milk power.
As further scheme of the invention：Described cleaning solution is the PBS solution containing 0.5 ‰ Tween-20s.
Compared with prior art, the beneficial effects of the invention are as follows：The detection method of PRV of the present invention is significantly simple
Change detecting step, reduced operation difficulty, improve accuracy of detection.
Specific embodiment
The technical scheme in the embodiment of the present invention is clearly and completely described below.
In the embodiment of the present invention, a kind of detection method of PRV is comprised the following steps：By sample by diluting
Liquid dilutes, and is quantitatively coated in ELISA96 orifice plates, and the μ L of serum-dilution to be checked 50 ~ 100 that will have been diluted add antigen coated microplate
In, while setting positive and negative control wells, 2 holes are respectively set, per the μ L of hole 100,4 DEG C are overnight；The antigen being not associated with is entered with PBST cleaning solutions
Row cleaning；Negative serum, positive serum and serum to be checked are added in corresponding detection hole respectively, slight vibrations make reaction plate
In liquid blending；37 DEG C of incubations are placed in after micro-reaction plate is sealed；The vertical liquid outwelled in reaction plate, and use cleaning solution
Each hole is cleaned；Reaction plate is closed with confining liquid, is placed in 37 DEG C of incubations；Liquid in removal reaction plate
Body, and carried out cleaning each hole with cleaning solution；The enzyme mark monoclonal antibody that will have been diluted is added in each reacting hole, is placed in 37
DEG C be incubated；The liquid in reaction plate is outwelled, and is carried out cleaning each hole with cleaning solution；Addition substrate, is placed in 37 DEG C
Reaction；Terminate liquid, terminating reaction are added, and 450nm absorbances are read in ELIASA, sample value/feminine gender value is judged to more than 2
The positive, is feminine gender less than 1.Also comprise the following steps：1）The pre-coated detection plate for having a viral antigen is taken out from kit, will be dilute
The serum to be checked 1 released：1 100 μ L of dilution are added in antigen coated microplate, while setting positive and negative control wells, 2 holes are respectively set, per hole
100μL；2）Gently shake sample in even hole, puts 37 DEG C and incubates 30 minutes, gets rid of the solution in plate hole, with cleaning solution board-washing 5 times,
200 μ L/ holes, stand outwell for 3 minutes every time, are patted dry on blotting paper for the last time；3）Per the enzyme-added mark μ L of monoclonal antibody 100 in hole, 37 are put
DEG C incubate 30 minutes, wash 5 times, method is with step 2）, nitrite ion A, nitrite ion B 50 μ L of each drop are added per hole, mix, room temperature is kept away
Light develops the color 10 minutes, adds terminate liquid one to drip 50 μ L per hole.Described confining liquid is 5% skimmed milk power.Described cleaning solution be containing
The PBS solution of 0.5 ‰ Tween-20s.
Embodiment 1：
The detection method of PRV of the present invention, comprises the following steps：By sample by diluted, quantitatively it is coated on
In ELISA96 orifice plates, the μ L of serum-dilution to be checked 50 that will have been diluted are added in antigen coated microplate, while positive and negative control wells are set,
2 holes are respectively set, per the μ L of hole 100,4 DEG C overnight；The antigen being not associated with is cleaned with PBST cleaning solutions；Respectively by negative serum,
Positive serum and serum to be checked are added in corresponding detection hole, and slight vibrations make the liquid blending in reaction plate；By micro-reaction
37 DEG C of incubations are placed in after plate sealing；The vertical liquid outwelled in reaction plate, and carried out cleaning each hole with cleaning solution；With
Confining liquid is closed to reaction plate, is placed in 37 DEG C of incubations；Liquid in removal reaction plate, and carried out to each hole with cleaning solution
Cleaned；The enzyme mark monoclonal antibody that will have been diluted is added in each reacting hole, is placed in 37 DEG C of incubations；Outwell the liquid in reaction plate
Body, and carried out cleaning each hole with cleaning solution；Addition substrate, is placed in 37 DEG C and reacts；Terminate liquid is added, terminates anti-
Should, and 450nm absorbances are read in ELIASA, sample value/feminine gender value is judged to the positive more than 2, is feminine gender less than 1.Also wrap
Include following steps：1）The pre-coated detection plate for having a viral antigen, the serum to be checked 1 that will have been diluted are taken out from kit：1 dilution
100 μ L are added in antigen coated microplate, while setting positive and negative control wells, respectively set 2 holes, per the μ L of hole 100；2）Gently shake sample in even hole
Product, put 37 DEG C and incubate 30 minutes, get rid of the solution in plate hole, and with cleaning solution board-washing 5 times, 200 μ L/ holes stand 3 minutes and fall every time
Fall, patted dry on blotting paper for the last time；3）Per the enzyme-added mark μ L of monoclonal antibody 100 in hole, put 37 DEG C and incubate 30 minutes, wash 5 times, method
With step 2）, nitrite ion A, nitrite ion B 50 μ L of each drop are added per hole, mix, room temperature lucifuge is developed the color 10 minutes, and terminate liquid is added per hole
One 50 μ L of drop.Described confining liquid is 5% skimmed milk power.Described cleaning solution is the PBS solution containing 0.5 ‰ Tween-20s.
Embodiment 2：
The detection method of PRV, comprises the following steps：By sample by diluted, quantitatively it is coated on
In ELISA96 orifice plates, the μ L of serum-dilution to be checked 100 that will have been diluted are added in antigen coated microplate, while setting positive and negative control
Hole, respectively sets 2 holes, and per the μ L of hole 100,4 DEG C overnight；The antigen being not associated with is cleaned with PBST cleaning solutions；Respectively by negative blood
Clearly, positive serum and serum to be checked are added in corresponding detection hole, and slight vibrations make the liquid blending in reaction plate；Will be micro
37 DEG C of incubations are placed in after reaction plate sealing；The vertical liquid outwelled in reaction plate, and carried out carrying out clearly each hole with cleaning solution
Wash；Reaction plate is closed with confining liquid, is placed in 37 DEG C of incubations；Removal reaction plate in liquid, and carried out with cleaning solution it is right
Each hole is cleaned；The enzyme mark monoclonal antibody that will have been diluted is added in each reacting hole, is placed in 37 DEG C of incubations；In outwelling reaction plate
Liquid, and carried out cleaning each hole with cleaning solution；Addition substrate, is placed in 37 DEG C and reacts；Terminate liquid is added, is terminated
Reaction, and 450nm absorbances are read in ELIASA, sample value/feminine gender value is judged to the positive more than 2, is feminine gender less than 1.Also
Comprise the following steps：1）The pre-coated detection plate for having a viral antigen, the serum to be checked 1 that will have been diluted are taken out from kit：1 is dilute
Release in 100 μ L addition antigen coated microplates, while setting positive and negative control wells, 2 holes are respectively set, per the μ L of hole 100；2）Gently shake in even hole
Sample, puts 37 DEG C and incubates 30 minutes, gets rid of the solution in plate hole, and with cleaning solution board-washing 5 times, 200 μ L/ holes stand 3 minutes every time
Outwell, patted dry on blotting paper for the last time；3）Per the enzyme-added mark μ L of monoclonal antibody 100 in hole, put 37 DEG C and incubate 30 minutes, wash 5 times, side
Method is with step 2）, nitrite ion A, nitrite ion B 50 μ L of each drop are added per hole, mix, room temperature lucifuge is developed the color 10 minutes, and termination is added per hole
Liquid one drips 50 μ L.Described confining liquid is 5% skimmed milk power.Described cleaning solution is the PBS solution containing 0.5 ‰ Tween-20s.
It is obvious to a person skilled in the art that the invention is not restricted to the details of above-mentioned one exemplary embodiment, Er Qie
In the case of without departing substantially from spirit or essential attributes of the invention, the present invention can be in other specific forms realized.Therefore, no matter
From the point of view of which point, embodiment all should be regarded as exemplary, and be nonrestrictive, the scope of the present invention is by appended power
Profit requires to be limited rather than described above, it is intended that all in the implication and scope of the equivalency of claim by falling
Change is included in the present invention.Moreover, it will be appreciated that although the present specification is described in terms of embodiments, not each
Implementation method only includes an independent technical scheme, and this narrating mode of specification is only this area for clarity
Specification an as entirety, the technical scheme in each embodiment should can also be formed this by technical staff through appropriately combined
Art personnel may be appreciated other embodiment.
Claims (4)
1. a kind of detection method of PRV, it is characterised in that comprise the following steps：Sample is dilute by dilution
Release, be quantitatively coated in ELISA96 orifice plates, the μ L of serum-dilution to be checked 50 ~ 100 that will have been diluted are added in antigen coated microplate, together
When set positive and negative control wells, respectively set 2 holes, per the μ L of hole 100,4 DEG C overnight；The antigen being not associated with is carried out clearly with PBST cleaning solutions
Wash；Negative serum, positive serum and serum to be checked are added in corresponding detection hole respectively, slight vibrations, in making reaction plate
Liquid blending；37 DEG C of incubations are placed in after micro-reaction plate is sealed；The vertical liquid outwelled in reaction plate, and carried out with cleaning solution
Each hole is cleaned；Reaction plate is closed with confining liquid, is placed in 37 DEG C of incubations；Liquid in removal reaction plate, and
Carried out cleaning each hole with cleaning solution；The enzyme mark monoclonal antibody that will have been diluted is added in each reacting hole, is placed in 37 DEG C and is incubated
Educate；The liquid in reaction plate is outwelled, and is carried out cleaning each hole with cleaning solution；Addition substrate, is placed in 37 DEG C and reacts；
Terminate liquid, terminating reaction are added, and 450nm absorbances are read in ELIASA, sample value/feminine gender value is judged to the positive more than 2,
It is feminine gender less than 1.
2. the detection method of PRV according to claim 1, it is characterised in that also comprise the following steps：1）
The pre-coated detection plate for having a viral antigen, the serum to be checked 1 that will have been diluted are taken out from kit：1 100 μ L of dilution add antigen
In coating plate, while setting positive and negative control wells, 2 holes are respectively set, per the μ L of hole 100；2）Gently shake sample in even hole, puts 37 DEG C of incubations
30 minutes, the solution in plate hole is got rid of, with cleaning solution board-washing 5 times, 200 μ L/ holes stand outwell for 3 minutes every time, exist for the last time
Patted dry on blotting paper；3）Per the enzyme-added mark μ L of monoclonal antibody 100 in hole, put 37 DEG C and incubate 30 minutes, wash 5 times, method is with step 2）, per hole
Plus nitrite ion A, nitrite ion B 50 μ L of each drop, mixing, room temperature lucifuge develops the color 10 minutes, adds terminate liquid one to drip 50 μ L per hole.
3. the detection method of PRV according to claim 1, it is characterised in that described confining liquid is 5%
Skimmed milk power.
4. the detection method of PRV according to claim 1, it is characterised in that described cleaning solution be containing
The PBS solution of 0.5 ‰ Tween-20s.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611025747.8A CN106771179A (en) | 2016-11-22 | 2016-11-22 | A kind of detection method of PRV |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201611025747.8A CN106771179A (en) | 2016-11-22 | 2016-11-22 | A kind of detection method of PRV |
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| CN201611025747.8A Pending CN106771179A (en) | 2016-11-22 | 2016-11-22 | A kind of detection method of PRV |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109490530A (en) * | 2018-11-21 | 2019-03-19 | 长沙金域医学检验所有限公司 | A kind of AsAb detection method |
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| CN109490530A (en) * | 2018-11-21 | 2019-03-19 | 长沙金域医学检验所有限公司 | A kind of AsAb detection method |
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Application publication date: 20170531 |