CN109557307A - A kind of visualization quick detection kit of O-shaped foot and mouth disease virus and preparation method thereof - Google Patents
A kind of visualization quick detection kit of O-shaped foot and mouth disease virus and preparation method thereof Download PDFInfo
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- CN109557307A CN109557307A CN201811472491.4A CN201811472491A CN109557307A CN 109557307 A CN109557307 A CN 109557307A CN 201811472491 A CN201811472491 A CN 201811472491A CN 109557307 A CN109557307 A CN 109557307A
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- 241000710198 Foot-and-mouth disease virus Species 0.000 title claims abstract description 53
- 238000001514 detection method Methods 0.000 title claims abstract description 37
- 238000002360 preparation method Methods 0.000 title claims abstract description 30
- 238000012800 visualization Methods 0.000 title claims abstract description 23
- 239000007788 liquid Substances 0.000 claims abstract description 49
- 238000004140 cleaning Methods 0.000 claims abstract description 21
- 239000000758 substrate Substances 0.000 claims abstract description 16
- 238000007865 diluting Methods 0.000 claims abstract description 14
- 239000000243 solution Substances 0.000 claims description 37
- 238000010790 dilution Methods 0.000 claims description 24
- 239000012895 dilution Substances 0.000 claims description 24
- 238000002965 ELISA Methods 0.000 claims description 22
- 238000011534 incubation Methods 0.000 claims description 19
- 241001465754 Metazoa Species 0.000 claims description 15
- 229920001213 Polysorbate 20 Polymers 0.000 claims description 12
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 claims description 12
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 claims description 12
- 239000007864 aqueous solution Substances 0.000 claims description 10
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 claims description 8
- 239000011248 coating agent Substances 0.000 claims description 8
- 238000000576 coating method Methods 0.000 claims description 8
- 239000008363 phosphate buffer Substances 0.000 claims description 7
- 102000004190 Enzymes Human genes 0.000 claims description 6
- 108090000790 Enzymes Proteins 0.000 claims description 6
- 235000020183 skimmed milk Nutrition 0.000 claims description 6
- RCEAADKTGXTDOA-UHFFFAOYSA-N OS(O)(=O)=O.CCCCCCCCCCCC[Na] Chemical compound OS(O)(=O)=O.CCCCCCCCCCCC[Na] RCEAADKTGXTDOA-UHFFFAOYSA-N 0.000 claims description 5
- 238000002649 immunization Methods 0.000 claims description 5
- 210000002966 serum Anatomy 0.000 claims description 5
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 4
- 229940098773 bovine serum albumin Drugs 0.000 claims description 4
- 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 3
- 208000030194 mouth disease Diseases 0.000 claims description 3
- 241000700605 Viruses Species 0.000 abstract description 5
- 238000003745 diagnosis Methods 0.000 abstract description 4
- 230000000007 visual effect Effects 0.000 abstract description 4
- 238000002474 experimental method Methods 0.000 abstract description 2
- 238000012360 testing method Methods 0.000 description 18
- 238000000034 method Methods 0.000 description 9
- 238000005406 washing Methods 0.000 description 6
- 241000283973 Oryctolagus cuniculus Species 0.000 description 4
- 239000000872 buffer Substances 0.000 description 4
- 201000010099 disease Diseases 0.000 description 4
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 4
- 210000000003 hoof Anatomy 0.000 description 4
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 description 3
- 229910019142 PO4 Inorganic materials 0.000 description 3
- 241000282898 Sus scrofa Species 0.000 description 3
- 239000007853 buffer solution Substances 0.000 description 3
- 235000013399 edible fruits Nutrition 0.000 description 3
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 3
- 239000010452 phosphate Substances 0.000 description 3
- 239000012153 distilled water Substances 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 239000013642 negative control Substances 0.000 description 2
- 239000002574 poison Substances 0.000 description 2
- 231100000614 poison Toxicity 0.000 description 2
- 239000013641 positive control Substances 0.000 description 2
- UIIMBOGNXHQVGW-UHFFFAOYSA-M sodium bicarbonate Substances [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 2
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 2
- CDBYLPFSWZWCQE-UHFFFAOYSA-L sodium carbonate Substances [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 2
- 229910000029 sodium carbonate Inorganic materials 0.000 description 2
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 description 2
- 229910000162 sodium phosphate Inorganic materials 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- YRNWIFYIFSBPAU-UHFFFAOYSA-N 4-[4-(dimethylamino)phenyl]-n,n-dimethylaniline Chemical compound C1=CC(N(C)C)=CC=C1C1=CC=C(N(C)C)C=C1 YRNWIFYIFSBPAU-UHFFFAOYSA-N 0.000 description 1
- 241000710777 Classical swine fever virus Species 0.000 description 1
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 206010037660 Pyrexia Diseases 0.000 description 1
- 241000701093 Suid alphaherpesvirus 1 Species 0.000 description 1
- 241001493546 Suina Species 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 150000001412 amines Chemical class 0.000 description 1
- 230000004596 appetite loss Effects 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 238000009360 aquaculture Methods 0.000 description 1
- 244000144974 aquaculture Species 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000011010 flushing procedure Methods 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 235000021266 loss of appetite Nutrition 0.000 description 1
- 208000019017 loss of appetite Diseases 0.000 description 1
- 235000013336 milk Nutrition 0.000 description 1
- 239000008267 milk Substances 0.000 description 1
- 210000004080 milk Anatomy 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 230000000644 propagated effect Effects 0.000 description 1
- 230000001850 reproductive effect Effects 0.000 description 1
- 230000000241 respiratory effect Effects 0.000 description 1
- 238000011895 specific detection Methods 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 208000011580 syndromic disease Diseases 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
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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
- G01N2333/08—RNA viruses
- G01N2333/085—Picornaviridae, e.g. coxsackie virus, echovirus, enterovirus
- G01N2333/09—Foot-and-mouth disease virus
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- Urology & Nephrology (AREA)
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- Virology (AREA)
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- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
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Abstract
The present invention provides visualization quick detection kits of a kind of O-shaped foot and mouth disease virus and preparation method thereof, belong to technical field of virus detection.The visualization quick detection kit of O-shaped foot and mouth disease virus, including coated elisa plate, cleaning solution, sample diluting liquid, substrate developing solution, ELIAS secondary antibody, terminate liquid, positive criteria product and negative standards' product, O-shaped foot and mouth disease virus antiserum is coated in the coated elisa plate；The sero-fast extension rate of O-shaped foot and mouth disease virus is 1:1000~3000.The kit is able to achieve Visual retrieval at room temperature, have the characteristics that it is easy to operate, quick, be suitable for experiment external environment in detect, for O-shaped foot and mouth disease virus it is quick detect reliable technological means is provided.Application of the visualization quick detection kit in the diagnosis of O-shaped aftosa.
Description
Technical field
The invention belongs to technical field of virus detection, and in particular to a kind of visualization of O-shaped foot and mouth disease virus quickly detects
Kit and preparation method thereof.
Background technique
Aftosa is a kind of acute, hot, highly contagious disease caused by foot and mouth disease virus (FMDV), main
Artiodactyl beast is encroached on, is often propagated in the animals such as sheep, ox, pig.There is blister in the lip of susceptible animal and hoof when morbidity, simultaneously
With symptoms such as fever, loss of appetite, infected animal weight and the output of milk decline to a great extent.Foot and mouth disease virus is easily passed by air
It broadcasts, infectiousness is strong, and popular quick, susceptible animal also results in death in the case where resistance is weaker, causes to raiser huge
Huge economic loss seriously hinders the development of aquaculture.
The diagnosis of O-shaped foot and mouth disease virus can only be completed in laboratory at present, because detection process need to rely on large size
Detecting instrument obtains testing result, and Simultaneous Detection is cumbersome, more demanding to operator, is not able to satisfy base to O-shaped mouth
Quick, the easy requirement of aphtovirus.
Summary of the invention
In view of this, the purpose of the present invention is to provide a kind of visualization quick detection kits of O-shaped foot and mouth disease virus
And preparation method thereof, the detection kit can observe by the naked eye to obtain the detection knot that O-shaped foot and mouth disease virus whether there is
Fruit realizes fast and convenient requirement.
In order to achieve the above-mentioned object of the invention, the present invention the following technical schemes are provided:
The present invention provides a kind of visualization quick detection kit of O-shaped foot and mouth disease virus, including coated elisa plate, wash
Wash liquid, sample diluting liquid, substrate developing solution, ELIAS secondary antibody, terminate liquid, positive criteria product and negative standards' product, the coating enzyme
O-shaped foot and mouth disease virus antiserum is coated in target；The sero-fast extension rate of O-shaped foot and mouth disease virus be 1:1000~
3000。
Preferably, the sero-fast preparation method of O-shaped foot and mouth disease virus, comprising the following steps:
With 0.1~0.5mgO type foot and mouth disease virus 146s first immunisation animal, after 14d, with the O-shaped mouth hoof of 0.1~0.5mg
The immune animal of epidemic disease poison 146s second after 28d, collects animal blood serum, obtains O-shaped foot and mouth disease virus antiserum.
Preferably, the phosphate that the cleaning solution is the 1mol/L of 0.01%~0.05% Tween-20 containing volumetric concentration is slow
Fliud flushing；The pH value of the cleaning solution is 7.2.
Preferably, the sample diluting liquid containing mass concentration be 20~50g/L skimmed milk power, volumetric concentration 0.01~
The 1mol/L phosphate buffer of 0.05% Tween-20.
Preferably, the substrate developing solution is the 1mol/L tetramethyl biphenyl containing 1%~5% hydrogen peroxide of volumetric concentration
Amine aqueous solution.
Preferably, the terminate liquid is the lauryl sodium sulfate aqueous solution of mass concentration 1%~5%.
Preferably, the extension rate of the ELIAS secondary antibody is 1:20000~50000.
The present invention provides the preparation method of the visualization quick detection kit, the preparation side including coated elisa plate
Method；
The preparation method of the coated elisa plate, comprising the following steps:
1) antiserum dilution will be obtained after the dilution of O-shaped foot and mouth disease virus antiserum；
2) antiserum dilution is added in each hole in ELISA Plate, primary incubation 12h is carried out under the conditions of 37 DEG C；
3) ELISA Plate after primary be incubated for is washed 3~4 times, confining liquid closing is added, carry out secondary incubate in 37 DEG C of conditions
Educate 20min；
4) ELISA Plate of secondary incubation is washed 3~4 times, dries, obtains coated elisa plate.
Preferably, the volume that the antiserum dilution is added in each hole in ELISA Plate is 100 μ l.
Preferably, the confining liquid is the Bovine serum albumin aqueous solution of mass concentration 0.1%~0.5%；The closing
The addition volume of liquid is 100 holes μ l/.
The present invention provides a kind of visualization quick detection kit of O-shaped foot and mouth disease virus, including coated elisa plate, wash
Wash liquid, sample diluting liquid, substrate developing solution, ELIAS secondary antibody, terminate liquid, positive criteria product and negative standards' product, the coating enzyme
O-shaped foot and mouth disease virus antiserum is coated in target；The sero-fast extension rate of O-shaped foot and mouth disease virus be 1:1000~
3000.Kit provided by the invention has the advantages that efficiently and repeatability is good, is able to achieve Visual retrieval at room temperature,
Independent of large-scale detecting instrument is tested, have the characteristics that easy to operate, quick, is suitable for detecting in experiment external environment, for O
The quick detection of type foot and mouth disease virus provides reliable technological means.Visualization quick detection kit provided by the invention simultaneously
It is low in cost, it is suitble to the popularization of a variety of testing conditions.
Detailed description of the invention
Fig. 1 is the testing result of test sample, wherein 1 is positive control colour developing result；2 and 3 be positive sample colour developing knot
Fruit；4,5,6 and 7 be negative sample colour developing result；8 be negative control colour developing result.
Specific embodiment
The present invention provides a kind of visualization quick detection kit of O-shaped foot and mouth disease virus, including coated elisa plate, wash
Wash liquid, sample diluting liquid, substrate developing solution, ELIAS secondary antibody, terminate liquid, positive criteria product and negative standards' product, the coating enzyme
O-shaped foot and mouth disease virus antiserum is coated in target；The sero-fast extension rate of O-shaped foot and mouth disease virus be 1:1000~
3000。
Kit provided by the invention includes coated elisa plate.In the present invention, the ELISA Plate in the coated elisa plate
Preferably transparent ELISA Plate.The specification of the ELISA Plate is preferably 96 holes.The present invention is not special to the source of the ELISA Plate
Limitation, using commodity ELISA Plate known in the art.
In the present invention, the sero-fast preparation method of O-shaped foot and mouth disease virus, preferably includes following steps: with 0.1~
0.5mgO type foot and mouth disease virus 146s first immunisation animal, after 14d, with second of the O-shaped foot and mouth disease virus 146s of 0.1~0.5mg
Immune animal after 28d, collects animal blood serum, obtains O-shaped foot and mouth disease virus antiserum.The animal is preferably rabbit.
The present invention is not particularly limited the source of the O-shaped foot and mouth disease virus 146s, routinely buys way by this field
Diameter is commercially available O-shaped foot and mouth disease virus 146s.The O-shaped foot and mouth disease virus 146s carries out first immunisation and second immune
When animal, diluted concentration is 1000~5000.
Kit provided by the invention includes cleaning solution.In the present invention, the cleaning solution preferably contains volumetric concentration
The phosphate buffer of the 1mol/L of 0.01%~0.05% Tween-20.The pH value of the cleaning solution is 7.2.The present invention is to institute
The preparation method for stating cleaning solution is not particularly limited, using the preparation program of cleaning solution known in the art.The present invention
The Tween-20 and phosphatic source are not particularly limited, using Tween-20 known in the art and phosphate commodity
?.
Kit provided by the invention includes sample diluting liquid.In the present invention, the sample diluting liquid contains mass concentration
For 20~50g/L skimmed milk power, the 1mol/L phosphate buffer of 0.01%~0.05% Tween-20 of volumetric concentration.The present invention
The preparation method of the sample diluting liquid is not particularly limited, using the preparation program of sample diluting liquid known in the art
?.The present invention is not particularly limited the skimmed milk power, Tween-20 and phosphatic source, and use is known in the art
Skimmed milk power, Tween-20 and phosphate commodity.
Kit provided by the invention includes substrate developing solution.In the present invention, the substrate developing solution preferably contains
The 1mol/L tetramethyl biphenyl amine aqueous solution of 1%~5% hydrogen peroxide of volumetric concentration.Preparation of the present invention to the substrate developing solution
Method is not particularly limited, using the preparation program of substrate developing solution known in the art.In order to guarantee display effect,
The preferred matching while using of substrate developing solution.The present invention is not particularly limited the source of the substrate developing solution, using ability
Hydrogen peroxide known to domain and tetramethyl benzidine commodity.
Kit provided by the invention includes terminate liquid.In the present invention, the terminate liquid be preferably mass concentration 1%~
5% lauryl sodium sulfate aqueous solution.The present invention is not particularly limited the source of the lauryl sodium sulfate, using this
Lauryl sodium sulfate commodity known to field.
Kit provided by the invention includes ELIAS secondary antibody.In the present invention, the extension rate of the ELIAS secondary antibody is preferred
For 1:20000~50000, more preferably 1:30000.The enzyme of the ELIAS secondary antibody is horseradish peroxidase.The present invention is to institute
The source for stating ELIAS secondary antibody is not particularly limited, and is obtained using commodity purchasing approach known in the art.In the present invention
In embodiment, the ELIAS secondary antibody is purchased from Sigma company.
Kit provided by the invention includes positive criteria product.The positive criteria product is O-shaped foot and mouth disease virus.The sun
Property standard items source be Lanzhou veterinary institute aftosa reference laboratory.
Kit provided by the invention includes negative standards' product.Negative standards' product are 1mol/L phosphate buffer.
The present invention provides the preparation methods of the visualization quick detection kit, preferably include the system of coated elisa plate
Preparation Method；
The preparation method of the coated elisa plate, preferably includes following steps:
1) antiserum dilution will be obtained after the dilution of O-shaped foot and mouth disease virus antiserum；
2) antiserum dilution is added in each hole in ELISA Plate, primary incubation 12h is carried out under the conditions of 37 DEG C；
3) ELISA Plate after primary be incubated for is washed 3~4 times, confining liquid closing is added, carry out secondary incubate in 37 DEG C of conditions
Educate 20min；
4) ELISA Plate of secondary incubation is washed 3~4 times, dries, obtains coated elisa plate.
The present invention obtains antiserum dilution after diluting O-shaped foot and mouth disease virus antiserum.The dilution is packet with solution
By liquid.The coating buffer is 0.05mol/L carbonate buffer solution.The preparation method of the 0.05mol/L carbonate buffer solution is excellent
It selects as follows: weighing Na2CO31.59g and NaHCO32.93g adds distilled water to be settled to 1000ml, and mixing obtains coating buffer.It is described
The pH value of coating buffer is preferably 9.6.The extension rate is preferably 1:1000.The present invention is not special to the diluted method
Limitation, using dilution scheme known in the art.
After dilution, antiserum dilution is added in each hole in ELISA Plate the present invention, carries out one under the conditions of 37 DEG C
Secondary incubation 12h.In the present invention, the volume that the antiserum dilution is added in each hole in ELISA Plate is preferably 50 μ l.
The primary incubation is preferred to be stood, and is conducive to the antibody in antiserum and is firmly coated on the inner wall of ELISA Plate mesoporous.
After primary incubation, the ELISA Plate after the present invention primary will be incubated for is washed 3~4 times, and confining liquid is added and closes, at 37 DEG C
Condition carries out secondary incubation 20min.
The present invention is not particularly limited the washing methods, using washing scheme known in the art.Washing
With the cleaning solution that solution is preferably in kit of the present invention.The washing is conducive to remove dissociating in antiserum or no firm
Coated antibody.
In the present invention, the confining liquid is preferably the Bovine serum albumin aqueous solution of concentration 0.1%~0.5%.This hair
The bright preparation method to the confining liquid is not particularly limited, using the preparation program of confining liquid known in the art.
The addition volume of the confining liquid is preferably 50 holes μ l/.The closed effect is to make do not have the interior of coated antibody in ELISA Plate
Upper Bovine serum albumin is coated on wall, other antibody cause testing result to be not allowed with inner wall in conjunction with when preventing subsequent detection
Really.
After secondary incubation, the present invention washs the ELISA Plate of secondary incubation 3~4 times, dries, obtains coated elisa plate.Institute
It states and dries preferred indoor natural wind and dry in the shade.
The visualization quick detection kit of the O-shaped foot and mouth disease virus provided by the invention fast, behaviour with detection speed
Make convenient and testing result to visualize.Based on this, the present invention also provides the visualization quick detection kits in mouth hoof
Application in epidemic disease diagnosis.
In the present invention, the method for the aftosa diagnosis preferably includes following steps:
A. test sample is diluted with sample diluting liquid, and the sample after dilution is added in each hole of coated elisa plate, and feminine gender is marked
Quasi- product and positive criteria product respectively add two holes as a control group, are incubated for 1h under the conditions of 20~27 DEG C；
B. the ELISA Plate after incubation is subjected to once washing with cleaning solution, adds ELIAS secondary antibody, under the conditions of 20~27 DEG C
35~55min of secondary incubation；
C. secondary washing is carried out with cleaning solution into the ELISA Plate of secondary incubation, after adding substrate developing solution, be protected from light aobvious
Terminate liquid is added in 15~25min of color；Visual color, blue are the positive.
In the present invention, the addition volume of the sample after the dilution, cleaning solution, substrate developing solution or terminate liquid is preferably
100 holes μ L/.
In the present invention, the extension rate of the test sample is preferably 40 times.The time for being protected from light colour developing is preferably
20min.The time of secondary incubation is preferably 40min.
Based on the detection method of kit provided by the invention, independent of laboratory large size detecting instrument, detection time
Testing result can be obtained in 2h, and testing result is accurately credible.
Below with reference to embodiment to a kind of visualization quick detection kit of O-shaped foot and mouth disease virus provided by the invention and
Preparation method is described in detail, but they cannot be interpreted as limiting the scope of the present invention.
Embodiment 1
1. the preparation of sample diluting liquid, cleaning solution, terminate liquid
Coating buffer is 0.05mol/L carbonate buffer solution.Preparation method is as follows: Na2CO31.59g NaHCO32.93g adding
Distilled water is settled to 1000mL, pH value 9.6.
Sample diluting liquid is the cleaning solution of the skimmed milk power containing 50g/L；
Cleaning solution is the phosphate buffer of the 0.01mol/L containing 0.05% Tween-20；PH value is 7.2.Preparation method tool
Body are as follows: NaCl8.5g, NaH2PO4·2H2O0.356g, NaH2PO4·12H2O2.772g, after being mixed, then it is water-soluble with distilling
1000mL is solved and be settled to, then 0.5mL Tween-20 is added thereto and obtains above-mentioned cleaning solution.
Terminate liquid is the SDS aqueous solution of mass concentration 1%.
2. the determination of the optimal dilution of test sample
Test sample is diluted with 1:40 times.ELIAS secondary antibody is diluted with 1:20000 times.
3, sero-fast preparation method
With 0.1mgO type foot and mouth disease virus 146s first immunisation rabbit, after 14 days, 0.1mgO type foot and mouth disease virus 146s second
Secondary immune rabbit, collected serum after 28 days.Rabbit anteserum 1:1000 dilutes coated elisa plate.
4, the preparation method of coated elisa plate:
1) antiserum dilution will be obtained after the dilution of O-shaped foot and mouth disease virus antiserum；
2) 100 μ l antiserum dilutions are added in each hole in ELISA Plate, are once incubated under the conditions of 37 DEG C
12h；
3) ELISA Plate after primary being incubated for washs 3~4 times, and the closing of 100 μ l/ hole confining liquids is added, 37 DEG C of conditions into
The secondary incubation 20min of row；
4) ELISA Plate of secondary incubation is washed 3~4 times, dries, obtains coated elisa plate.
5, specific detection method is as follows:
According to optimum operation condition determined by the above items, determining operation sequence are as follows: taking-up has been coated with and has closed
Elisa plate item, test sample dilutes according to 1:40 times, be added each hole of ELISA Plate, every 100 μ L of hole, respectively plus a hole, O-shaped mouth hoof
Epidemic disease poison is negative, the positive respectively adds two holes, and room temperature acts on 1h；Liquid in hole is discarded, every hole is washed 3 times with cleaning solution and patted, and abandons to the greatest extent
Liquid in hole；100 μ L ELIAS secondary antibodies are added in every hole, and room temperature acts on 1h；Liquid in hole is discarded, every hole washs 3 times simultaneously with cleaning solution
It pats, abandons liquid in hole to the greatest extent；After 100 μ L substrate developing solutions are added in every hole, room temperature is protected from light colour developing 20min, and it is whole that 100 μ LSDS are added
Only liquid；Visual color, blue are the positive.
Fig. 1 is the testing result of test sample, wherein 1 is positive control colour developing result；2 and 3 be positive sample colour developing knot
Fruit；4,5,6 and 7 be negative sample colour developing result；8 be negative control colour developing result.As shown in Figure 1, use is provided by the invention
Kit, testing result, which observes by the naked eye, can judge to reach qualitative inspection whether containing O-shaped foot and mouth disease virus in test sample
The purpose of survey.
Embodiment 2
The specific test of kit provided by the invention
With kit provided by the invention according to detection method in embodiment 1 detect respectively known swine fever virus (No. 4),
Pig circular ring virus (No. 5), pig parvoviral (No. 6), Pseudorabies virus (No. 7), reproductive and respiratory syndrome virus (No. 8) and O-shaped hoof-and-mouth disease
Malicious sample (No. 1-3).
As a result judgment method: feminine gender be it is colourless, the positive is blue.Kit provided by the invention is only to O-shaped hoof-and-mouth disease
Malicious sample shows blue, and other 5 kinds of viruses are illustrated as feminine gender.This shows kit tool provided by the invention by higher
Specificity.
There is above-described embodiment it is found that kit provided by the invention has efficient, spirit for detecting O-shaped foot and mouth disease virus
Quick specificity and repeated good advantage, the detection method is easy to operate, quick, low in cost, can at room temperature may be used
It is detected depending on changing, is suitable for being promoted in clinical application, provide reliable technology hand for the quick detection of O-shaped foot and mouth disease virus
Section.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Claims (10)
1. a kind of visualization quick detection kit of O-shaped foot and mouth disease virus, including the dilution of coated elisa plate, cleaning solution, sample
Liquid, substrate developing solution, ELIAS secondary antibody, terminate liquid, positive criteria product and negative standards' product, which is characterized in that the coating enzyme mark
O-shaped foot and mouth disease virus antiserum is coated in plate；The sero-fast extension rate of O-shaped foot and mouth disease virus be 1:1000~
3000。
2. visualization quick detection kit according to claim 1, which is characterized in that the O-shaped foot and mouth disease virus is anti-
The preparation method of serum, comprising the following steps:
With the O-shaped foot and mouth disease virus 146s first immunisation animal of 0.1~0.5mg, after 14d, with the O-shaped hoof-and-mouth disease of 0.1~0.5mg
The immune animal of malicious 146s second after 28d, collects animal blood serum, obtains O-shaped foot and mouth disease virus antiserum.
3. visualization quick detection kit according to claim 1, which is characterized in that the cleaning solution is dense containing volume
Spend the phosphate buffer of the 1mol/L of 0.01%~0.05% Tween-20；The pH value of the cleaning solution is 7.2.
4. visualization quick detection kit according to claim 1, which is characterized in that the sample diluting liquid contains quality
Concentration is the 1mol/L phosphate buffer of 20~50g/L skimmed milk power, 0.01~0.05% Tween-20 of volumetric concentration.
5. visualization quick detection kit according to claim 1, which is characterized in that the substrate developing solution be containing
The 1mol/L tetramethyl biphenyl amine aqueous solution of 1%~5% hydrogen peroxide of volumetric concentration.
6. visualization quick detection kit according to claim 1, which is characterized in that the terminate liquid is mass concentration
1%~5% lauryl sodium sulfate aqueous solution.
7. visualization quick detection kit described in any one according to claim 1~6, which is characterized in that the enzyme mark
The extension rate of secondary antibody is 1:20000~50000.
8. visualizing the preparation method of quick detection kit described in a kind of claim 1~7 any one, which is characterized in that
Preparation method including coated elisa plate；
The preparation method of the coated elisa plate, comprising the following steps:
1) antiserum dilution will be obtained after the dilution of O-shaped foot and mouth disease virus antiserum；
2) antiserum dilution is added in each hole in ELISA Plate, primary incubation 12h is carried out under the conditions of 37 DEG C；
3) ELISA Plate after primary be incubated for is washed 3~4 times, confining liquid closing is added, carry out secondary incubation in 37 DEG C of conditions
20min；
4) ELISA Plate of secondary incubation is washed 3~4 times, dries, obtains coated elisa plate.
9. preparation method according to claim 8, which is characterized in that each of ELISA Plate is added in the antiserum dilution
Volume in hole is 100 μ l.
10. preparation method according to claim 8 or claim 9, which is characterized in that the confining liquid be mass concentration 0.1%~
0.5% Bovine serum albumin aqueous solution；The addition volume of the confining liquid is 100 holes μ l/.
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