AU2021105824A4 - Visual Rapid Detection Kit for O-type Foot-and-Mouth Disease Virus and Preparation Method Thereof - Google Patents
Visual Rapid Detection Kit for O-type Foot-and-Mouth Disease Virus and Preparation Method Thereof Download PDFInfo
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- AU2021105824A4 AU2021105824A4 AU2021105824A AU2021105824A AU2021105824A4 AU 2021105824 A4 AU2021105824 A4 AU 2021105824A4 AU 2021105824 A AU2021105824 A AU 2021105824A AU 2021105824 A AU2021105824 A AU 2021105824A AU 2021105824 A4 AU2021105824 A4 AU 2021105824A4
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- mouth disease
- disease virus
- type foot
- elisa plate
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- 241000710198 Foot-and-mouth disease virus Species 0.000 title claims abstract description 58
- 238000001514 detection method Methods 0.000 title claims abstract description 54
- 238000002360 preparation method Methods 0.000 title claims abstract description 34
- 230000000007 visual effect Effects 0.000 title claims abstract description 29
- 239000000243 solution Substances 0.000 claims abstract description 68
- 238000002965 ELISA Methods 0.000 claims abstract description 55
- 238000005406 washing Methods 0.000 claims abstract description 29
- 238000010790 dilution Methods 0.000 claims abstract description 27
- 239000012895 dilution Substances 0.000 claims abstract description 27
- 239000000126 substance Substances 0.000 claims abstract description 17
- 239000000758 substrate Substances 0.000 claims abstract description 17
- 102000004190 Enzymes Human genes 0.000 claims abstract description 16
- 108090000790 Enzymes Proteins 0.000 claims abstract description 16
- 239000003085 diluting agent Substances 0.000 claims abstract description 14
- 239000012089 stop solution Substances 0.000 claims abstract description 14
- 241001465754 Metazoa Species 0.000 claims description 17
- 230000000903 blocking effect Effects 0.000 claims description 14
- 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
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 claims description 8
- 239000008055 phosphate buffer solution Substances 0.000 claims description 8
- 239000007864 aqueous solution Substances 0.000 claims description 7
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 claims description 6
- 239000007788 liquid Substances 0.000 claims description 6
- 239000000843 powder Substances 0.000 claims description 6
- 235000020183 skimmed milk Nutrition 0.000 claims description 6
- 238000007865 diluting Methods 0.000 claims description 5
- 210000002966 serum Anatomy 0.000 claims description 5
- 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 claims description 4
- 235000019333 sodium laurylsulphate Nutrition 0.000 claims description 4
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 3
- 229940098773 bovine serum albumin Drugs 0.000 claims description 3
- 241000700605 Viruses Species 0.000 abstract description 13
- 208000007212 Foot-and-Mouth Disease Diseases 0.000 abstract description 8
- 238000003745 diagnosis Methods 0.000 abstract description 4
- 230000009286 beneficial effect Effects 0.000 abstract description 2
- 239000000523 sample Substances 0.000 description 20
- 238000012360 testing method Methods 0.000 description 9
- 238000000034 method Methods 0.000 description 8
- 201000010099 disease Diseases 0.000 description 7
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 7
- 239000011248 coating agent Substances 0.000 description 6
- 238000000576 coating method Methods 0.000 description 6
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 4
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 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
- 239000007853 buffer solution Substances 0.000 description 3
- 239000012153 distilled water Substances 0.000 description 3
- 230000000694 effects Effects 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
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- 241000283973 Oryctolagus cuniculus Species 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 description 2
- 238000007605 air drying Methods 0.000 description 2
- 239000012470 diluted sample Substances 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 239000013642 negative control Substances 0.000 description 2
- 239000013641 positive control Substances 0.000 description 2
- 229910052708 sodium Inorganic materials 0.000 description 2
- 239000011734 sodium Substances 0.000 description 2
- 235000017557 sodium bicarbonate Nutrition 0.000 description 2
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 2
- 235000017550 sodium carbonate Nutrition 0.000 description 2
- 229910000029 sodium carbonate Inorganic materials 0.000 description 2
- 108010088751 Albumins Proteins 0.000 description 1
- 102000009027 Albumins Human genes 0.000 description 1
- 241000283690 Bos taurus Species 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
- 241000202347 Porcine circovirus Species 0.000 description 1
- 241000702619 Porcine parvovirus Species 0.000 description 1
- 206010037660 Pyrexia Diseases 0.000 description 1
- 241000701093 Suid alphaherpesvirus 1 Species 0.000 description 1
- 241000282887 Suidae Species 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 230000004596 appetite loss Effects 0.000 description 1
- 238000009360 aquaculture Methods 0.000 description 1
- 244000144974 aquaculture Species 0.000 description 1
- 230000037396 body weight Effects 0.000 description 1
- 239000012888 bovine serum Substances 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000003113 dilution method Methods 0.000 description 1
- 210000000003 hoof Anatomy 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
- 238000011017 operating method Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
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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
- G01N33/52—Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements
- G01N33/521—Single-layer analytical elements
- G01N33/523—Single-layer analytical elements the element being adapted for a specific analyte
-
- 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
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2469/00—Immunoassays for the detection of microorganisms
- G01N2469/10—Detection of antigens from microorganism in sample from host
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2470/00—Immunochemical assays or immunoassays characterised by the reaction format or reaction type
- G01N2470/04—Sandwich assay format
Abstract
The present invention provides a visual rapid detection kit for an 0-type foot-and-mouth
disease virus and a preparation method thereof, and belongs to the technical field of virus
detection. The visual rapid detection kit for 0-type foot-and-mouth disease virus comprises a
coated ELISA plate, a washing solution, a sample diluent, a substrate color developing solution,
an enzyme-labeled secondary antibody, a stop solution, a positive standard substance and a
negative standard substance, wherein 0-type foot-and-mouth disease virus antiserum is coated
in the coated ELISA plate; and the dilution ratio of the 0-type foot-and-mouth disease virus
antiserum is 1: (1,000-3,000). The kit has the beneficial effects that the visual detection at the
room temperature can be realized, has the characteristics of being simple, convenient and rapid
in operation and is suitable for detection in the environment outside a laboratory, thereby
providing a reliable technical means for the rapid detection of the 0-type foot-and-mouth
disease virus. The visual rapid detection kit is applied to the diagnosis of an 0-type foot-and
mouth disease.
Description
Visual Rapid Detection Kit for O-type Foot-and-Mouth Disease Virus and
Preparation Method Thereof
The present invention belongs to the technical field of virus detection, and
specifically relates to a visual rapid detection kit for an0-type foot-and-mouth
disease virus and a preparation method thereof.
Foot-and-mouth disease is an acute, febrile, and highly contagious disease
caused by a Foot-and-Mouth Disease Virus (FMDV), mainly encroaches on hoofed
beasts and is often transmitted in animals such as sheep, cattle, and pigs. At the time
of onset, blisters appear in lips and hoofs of the animals, accompanied by symptoms
such as fever and loss of appetite, and therefore, the body weight and the milk yield of
the diseased animals are greatly reduced. The FMDV is easily transmitted through the
air, is highly contagious, and is popular. The susceptible animals may also die in the
case of weak resistance, causing huge economic losses to the farmers and seriously
hindering the development of the aquaculture industry.
At present, the diagnosis of FMDV can only be done in a laboratory; the
detection process relies on large-scale detection instruments to obtain the detection
results, and the detection method is complex and is high in requirements on operators,
which cannot meet the rapid and simple requirements on the FMDV at basic level.
In view of the above, an objective of the present invention is to provide a visual
rapid detection kit for an 0-type foot-and-mouth disease virus and a preparation
method thereof. The detection kit can quickly and easily detect the presence or
absence of 0-type foot-and-mouth disease virus by visual observation, thereby realizing the rapid and simple requirements.
To achieve the invention objective, the present invention adopts the technical
solution:
A visual rapid detection kit for an0-type foot-and-mouth disease virus provided
in the present invention includes a coated ELISA plate, a washing solution, a sample
diluent, a substrate color developing solution, an enzyme-labeled secondary antibody,
a stop solution, a positive standard substance and a negative standard substance,
where 0-type foot-and-mouth disease virus antiserum is coated in the coated ELISA
plate; and the dilution ratio of the0-type foot-and-mouth disease virus antiserum is 1:
(1,000-3,000).
Preferably, a preparation method for the 0-type foot-and-mouth disease virus
antiserum includes the following steps.
The animals are immunized with 0.1-0.5 mg of the0-type foot-and-mouth
disease virus 146s for the first time; after 14 days, the animals are immunized with
0.1-0.5 mg of the 0-type foot-and-mouth disease virus 146s for the second time; and
after 28 days, animal serum is collected to obtain the0-type foot-and-mouth disease
virus antiserum.
Preferably, the washing solution is a 1 mol/L phosphate buffer solution
containing Tween-20 with a volume concentration of 0.01% to 0.05%, and the pH
value of the washing solution is 7.2.
Preferably, the sample diluent is a 1 mol/L phosphate buffer solution containing
skim milk powder with a mass concentration of 20-50 g/L and Tween-20 with a
volume concentration of 0.01% to 0.05%.
Preferably, the substrate color developing solution is a 1 mol/L
tetramethylbenzidine solution containing hydrogen peroxide with a volume concentration of 1% to 5%.
Preferably, the stop solution is an aqueous solution of sodium lauryl sulfate with
a mass concentration of 1% to 5%.
Preferably, the dilution ratio of the enzyme-labeled secondary antibody is 1:
(20,000-50,000).
The present invention provides a preparation method for the visual rapid
detection kit, including a preparation method of the coated ELISA plate.
The preparation method for the coated ELISA plate includes the following steps:
1) an antiserum dilution is obtained by diluting the0-type foot-and-mouth
disease virus antiserum;
2) the antiserum dilution is added into each well of the ELISA plate to incubate
for 12 h for a first time at 37°C;
3) the ELISA plate which is incubated for the first time is washed for 3-4 times,
blocked by a blocking solution, and incubated for 20 min for a second time at 37°C;
4) the ELISA plate which is incubated for the second time is washed for 3-4
times, and dried in air to obtain the coated ELISA plate.
Preferably, the antiserum dilution is added to each well in the ELISA plate in a
volume of 100 pl.
Preferably, the blocking solution is a bovine serum albumin aqueous solution
with a mass concentration of 0.1% to 0. 5 %; and the blocking liquid is added in an
amount of 100 l/well.
A visual rapid detection kit for an0-type foot-and-mouth disease virus provided
in the present invention includes a coated ELISA plate, a washing solution, a sample
diluent, a substrate color developing solution, an enzyme-labeled secondary antibody,
a stop solution, a positive standard substance and a negative standard substance, where 0-type foot-and-mouth disease virus antiserum is coated in the coated ELISA plate; and the dilution ratio of the0-type foot-and-mouth disease virus antiserum is 1:
(1,000-3,000). The kit provided in the present invention has the advantages of being
efficient and good in repeatability, can realize the visual detection at the room
temperature, does not depend on experimental large-scale detection instruments, has
the characteristics of being simple, convenient and rapid in operation and is suitable
for detection in the environment outside a laboratory, thereby providing a reliable
technical means for the rapid detection of the 0-type foot-and-mouth disease virus. At
the same time, the visual rapid detection kit provided in the present invention is low
in cost, and is suitable for popularization under various detection conditions.
FIG. 1 shows the detection results of test samples, where 1 is positive control
color developing results; 2 and 3 are positive sample color developing results; 4, 5, 6
and 7 are negative sample color developing results; and 8 is negative control color
developing results.
A visual rapid detection kit for 0-type foot-and-mouth disease virus provided in
the present invention includes a coated ELISA plate, a washing solution, a sample
diluent, a substrate color developing solution, an enzyme-labeled secondary antibody,
a stop solution, a positive standard substance and a negative standard substance,
where 0-type foot-and-mouth disease virus antiserum is coated in the coated ELISA
plate; and the dilution ratio of the0-type foot-and-mouth disease virus antiserum is 1:
(1,000-3,000).
The kit provided in the present invention includes the coated ELISA plate. In the
present invention, the ELISA plate in the coated ELISA plate is preferably a transparent ELISA plate. The specification of the ELISA plate is preferably a 96-well plate. The source of the ELISA plate in the present invention is not particularly limited, and a commercial ELISA plate well known in the art can be used.
In the present invention, the preparation method for the 0-type foot-and-mouth
disease virus antiserum preferably includes the following steps: the animals are
immunized with 0.1-0.5 mg of the 0-type foot-and-mouth disease virus 146s for the
first time; after 14 days, the animals are immunized with 0.1-0.5 mg of the 0-type
foot-and-mouth disease virus 146s for the second time; and after 28 days, animal
serum is collected to obtain the 0-type foot-and-mouth disease virus antiserum. The
animals are preferably rabbits.
The source of the 0-type foot-and-mouth disease virus 146s in the present
invention is not particularly limited, and the 0-type foot-and-mouth disease virus
146s can be purchased by a conventional purchase route in the art. When the animals
are immunized with the 0-type foot-and-mouth disease virus 146s for the first time
and the second time, the dilution concentration is 1,000-5,000.
The kit provided in the present invention includes the washing solution. In the
present invention, the washing solution is a 1 mol/L phosphate buffer solution
containing Tween-20 with a volume concentration of 0.01% to 0.05%. The pH value
of the washing solution is 7.2. The preparation method of the washing solution in the
present invention is not particularly limited, and a washing solution preparation
scheme well known in the art can be adopted. The source of the Tween-20 in the
present invention is not particularly limited, and a Tween-20 and a phosphate product
well known in the art can be adopted.
The kit provided in the present invention includes the sample diluent. In the
present invention, the sample diluent is a 1 mol/L phosphate buffer solution containing skim milk powder with a mass concentration of 20-50 g/L and Tween-20 with a volume concentration of 0.01% to 0.05%. The preparation method of the sample diluent in the present invention is not particularly limited, and a sample diluent preparation scheme well known in the art can be adopted. The source of the skim milk powder, the Tween-20 and the phosphate in the present invention is not particularly limited, and a skim milk powder, Tween-20 and phosphate products well known in the art can be adopted.
The kit provided in the present invention includes the substrate color developing
solution. In the present invention, the substrate color developing solution is a 1 mol/L
tetramethylbenzidine solution containing hydrogen peroxide with a volume
concentration of 1% to 5%. The preparation method of the substrate color developing
solution in the present invention is not particularly limited, and a substrate color
developing solution preparation scheme well known in the art can be adopted. To
guarantee the color developing effect, the substrate color developing solution is
preferably prepared when needed. The source of the substrate color developing
solution in the present invention is not particularly limited, and hydrogen peroxide
and tetramethylbenzidine products well known in the art can be adopted.
The kit provided in the present invention includes the stop solution. In the
present invention, the stop solution is preferably an aqueous solution of sodium lauryl
sulfate with a mass concentration of 1% to 5%. The source of the sodium lauryl
sulfate in the present invention is not particularly limited, and a sodium lauryl sulfate
product well known in the art can be adopted.
The kit provided in the present invention includes the enzyme-labeled secondary
antibody. In the present invention, the dilution ratio of the enzyme-labeled secondary
antibody is preferably 1: (20,000-50,000), more preferably 1:30,000. Enzyme of the enzyme-labeled secondary antibody is horseradish peroxidase. The source of the enzyme-labeled secondary antibody in the present invention is not particularly limited, and may be obtained by a commercially available purchase route well known in the art. In an embodiment of the present invention, the enzyme-labeled secondary antibody is purchased from the Sigma company.
The kit provided in the present invention includes the positive standard substance.
The positive standard substance is the 0-type foot-and-mouth disease virus. The
positive standard substrate is from the Foot and Mouth Disease Reference Laboratory
of Lanzhou Veterinary Research Institute.
The kit provided in the present invention includes the negative standard
substance. The negative standard substance is a 1 mol/L phosphate buffer solution.
The present invention provides a preparation method for the visual rapid
detection kit, preferably, the including a preparation method of the coated ELISA
plate.
The preparation method for the coated ELISA plate preferably includes the
following steps:
1) an antiserum dilution is obtained by diluting the0-type foot-and-mouth
disease virus antiserum;
2) the antiserum dilution is added into each well of the ELISA plate to
incubate for 12 h at 37°C;
3) the ELISA plate which is incubated for the first time is washed for 3-4
times, blocked by a blocking solution, and incubated for 20 min for a second time
at 37°C;
4) the ELISA plate which is incubated for the second time is washed for 3-4
times, and dried in air to obtain the coated ELISA plate.
The antiserum dilution is obtained by diluting the0-type foot-and-mouth disease
virus antiserum in the present invention. The solution for dilution is a coating solution.
The coating solution is a 0.05 mol/L carbonate buffer solution. A preparation method
for the .05 mol/L carbonate buffer solution preferably includes: weighing 1.59 g of
Na2CO3 and 2.93 g of NaHCO3, adding distilled water to 1000 mL, and mixing to
obtain the coating solution. The pH value of the coating solution is preferably 9.6.
The dilution ratio is preferably 1:1,000. The dilution method is not particularly
limited in the present invention, and a scheme well known in the art can be adopted.
After being diluted, the antiserum dilution is added into each well of the ELISA
plate to incubate for 12 h for a first time at 37C. In the present invention, the
antiserum dilution is added to each well in the ELISA plate in a volume of 50 l
preferably. Standing is preferable during the first-time incubation, so that antibodies
in the antiserum are stably coated onto inner walls of wells in the ELISA plate.
After being incubated for the first time, the ELISA plate which is incubated for
the first time is washed for 3-4 times, blocked by a blocking solution, and incubated
for 20 min for a second time at 37C.
The washing method in the present invention is not particularly limited, and a
washing scheme well known in the art can be adopted. The solution for washing is
preferably the washing solution in the kit in the present invention. The washing is
beneficial for removing free or unstably-coated antibodies in the antiserum.
In the present invention, the blocking solution is preferably a bovine serum
albumin aqueous solution with a concentration of 0.1%-0.5%. The preparation
method of the blocking solution in the present invention is not particularly limited,
and a blocking solution preparation scheme well known in the art can be adopted. The
blocking dilution is added in a volume of 50 1/well preferably. The effect of the blocking is to coat the inner wall not coated with antibody in the ELISA plate with the bovine serum albumin to prevent other antibodies from binding to the inner wall during subsequent detection, resulting in inaccurate detection results.
After being incubated for the second time, the ELISA plate which is incubated
for the second time is washed for 3-4 times, and dried in air to obtain the coated
ELISA plate. Air-drying is preferably indoor natural air drying.
The visual rapid detection kit for the 0-type foot-and-mouth disease virus
provided in the present invention has the effects of being high in detection speed,
convenient in operation and visual in detection results. Based on this, the present
invention further provides application of the visual rapid detection kit in diagnosis of
foot-and-mouth disease.
In the present invention, the diagnosis method for the foot-and-mouth disease
preferably includes the following steps:
a. a test sample is diluted with the sample diluent, and the diluted sample is
added to each well of the coated ELISA plate, and the negative standard substance
and the positive standard substance are respectively added into two wells as a control
group, and incubated at 20 to 27°C for 1 h;
b. the incubated ELISA plate is washed once with the washing solution, and
incubated for 35-55 min for a second time at 20-27°C by adding the enzyme-labeled
secondary antibody;
c. the ELISA plate which is incubated for the second time is washed twice with
the washing solution, then added with the substrate color developing solution to
develop color for 15-25 min away from light, and is added with the stop solution; and
color of the solution is observed through naked eyes, and the solution is positive if it
is blue.
In the present invention, the diluted sample, the washing solution, the substrate
color developing solution or the stop solution are added in a volume of 100 1 /well
preferably.
Preferably, the dilution ratio of the test sample is preferably 40. The color
developing time away from the light is preferably 20 min. The second-time incubation
time is preferably 40 min.
The detection method based on the kit provided in the present invention does not
depend on large-scale detection instruments in the laboratory, and the detection
results which are accurate and reliable can be obtained within detection time of 2 h.
The visual rapid detection kit for 0-type foot-and-mouth disease virus and the
preparation method thereof provided in the present invention are described in detail
below with reference to the embodiments, but are not to be construed as limiting the
scope of the present invention.
Embodiment 1:
1. preparation of a sample diluent, a washing solution and a stop solution
A coating solution was a 0.05 mol/L carbonate buffer solution. A preparation
method was as follows: 1.59 g of Na2CO3 and 2.93 g of NaHCO3 were weighed,
distilled water was added to 1,000 mL to obtain the coating solution with a pH value
of 9.6.
The sample diluent is a washing solution containing 50 g/L skim milk powder.
The washing solution is a 0.01 mol/L phosphate buffer solution containing
Tween-20 with a volume concentration of 0.05%, and the pH value of the washing
solution was 7.2. A preparation method was as follows: 8.5 g of NaCl, 0.356 g of
NaH2PO4-2H20 and 2.772 g of NaH2PO4-12H20were mixed, and dissolved with
distilled water and made up to 1,000 mL, and 0.5 mL of Tween-20 was added into the solution to obtain the washing solution.
The stop solution was an SDS (Sodium Dodecyl Sulfate) aqueous solution with a
mass concentration of 1%.
2. determination of optimal dilution of test sample
The test sample was diluted in a ratio of 1:40. The enzyme-labeled secondary
antibody was diluted in a ratio of 1:20,000.
3. preparation method of antiserum
The animals were immunized with 0.1 mg of the0-type foot-and-mouth disease
virus 146s for the first time; after 14 days, the animals were immunized with 0.1 mg
of the 0-type foot-and-mouth disease virus 146s for the second time; and after 28
days, serum was collected. Rabbit serum was used to dilute the coated ELISA plate in
a ratio of 1:1000.
4. preparation method of coated ELISA plate:
1) an antiserum dilution was obtained by diluting the 0-type foot-and-mouth
disease virus antiserum;
2) 100 1 of antiserum dilution was added into each well of the ELISA plate to
incubate for a first time for 12h at 37°C;
3) the ELISA plate which was incubated for the first time was washed for 3-4
times, blocked by a 100u1/well blocking solution, and incubated for 20 min for a
second time at 37°C;
4) the ELISA plate which was incubated for the second time was washed for 3-4
times, and dried in air to obtain the coated ELISA plate.
5. the specific detection method was as follows:
According to the optimal operating conditions determined in the above items, the
determined operating procedure is as follows: the coated and blocked ELISA plate strips were taken out, and the test samples were diluted in a ratio of 1:40, and were added into each well of the ELISA plate, 100 L per well. Positive and negative standard substances of the 0-type foot-and-mouth disease virus were added into two wells and acted for 1 h at the room temperature; liquid in the wells was discarded, and each well was washed with the washing solution for three times and was shaken to remove the liquid in the well; 100 L of enzyme-labeled secondary antibody was added into each well and acted for 1 h at the room temperature; liquid in the well was discarded, and each well was washed with the washing solution for three times and was shaken to remove the liquid in the well; after 100 L of of the substrate color developing solution was added into each well, color developing away from light was performed for 20 min at the room temperature, and 100 L of the SDS stop solution was added; and the color of the solution was observed with naked types, and the solution was positive if it was blue.
FIG. 1 showed the detection results of test samples, where 1 was positive control
color developing results; 2 and 3 were positive sample color developing results; 4, 5,
6 and 7 were negative sample color developing results; and 8 was negative control
color developing results. It could be known from FIG. 1 that the kit provided in the
present invention could achieve a qualitative detection purpose by observing the
detected results with naked eyes to judge whether the test samples had the 0-type
foot-and-mouth disease virus.
Embodiment 2:
Specificity test of the kit provided in the present invention
The kit provided in the present invention was used to repressively detect a
known swine fever virus (No. 4), a porcine circovirus (No. 5), a porcine parvovirus
(No. 6), and a pseudorabies virus (No. 7), a blue ear virus (No. 8) and 0-type foot- and-mouth disease virus samples (No. 1-3) according to the detection method in
Embodiment 1.
The result judgment method: the negative color was colorless, and the positive
color was blue. The kit provided in the present invention showed only blue for the 0
type foot-and-mouth disease virus sample, and showed negative for the other five
viruses. It was indicated that the kit provided in the present invention had a higher
specificity.
It could be known from the embodiment that the kit for detecting the 0-type
foot-and-mouth disease virus in the present invention had the advantages of being
efficient, good in sensitive specificity and good in repeatability. The detection method
was simple and rapid in operation, was low in cost, could be used for visual detection
at the room temperature, and was suitable for being popularized in clinical application,
thereby providing a reliable technical means for the rapid detection of the 0-type
foot-and-mouth disease virus.
The above is only a preferred embodiment of the present invention. It should be
noted that, for those of ordinary skill in the art, without departing from the principle
of the present invention, several improvements and retouches, which are regarded as
the protection scope of the present invention, further may be made.
Claims (10)
1. A visual rapid detection kit for an0-type foot-and-mouth disease virus,
comprising a coated ELISA plate, a washing solution, a sample diluent, a substrate
color developing solution, an enzyme-labeled secondary antibody, a stop solution, a
positive standard substance and a negative standard substance, wherein 0-type foot
and-mouth disease virus antiserum is coated in the coated ELISA plate; and the
dilution ratio of the 0-type foot-and-mouth disease virus antiserum is 1: (1,000-3,000).
2. The visual rapid detection kit according to claim 1, wherein a preparation
method for the 0-type foot-and-mouth disease virus antiserum comprises the
following steps that:
the animals are immunized with 0.1-0.5 mg of the0-type foot-and-mouth
disease virus 146s for the first time; after 14 days, the animals are immunized with
0.1-0.5 mg of the 0-type foot-and-mouth disease virus 146s for the second time; and
after 28 days, animal serum is collected to obtain the0-type foot-and-mouth disease
virus antiserum.
3. The visual rapid detection kit according to claim 1, wherein the washing
solution is a 1 mol/L phosphate buffer solution containing Tween-20 with a volume
concentration of 0.01% to 0.05%, and the pH value of the washing solution is 7.2.
4. The visual rapid detection kit according to claim 1, wherein the sample diluent
is a 1 mol/L phosphate buffer solution containing skim milk powder with a mass
concentration of 20-50 g/L and Tween-20 with a volume concentration of 0.01% to
0.05%.
5. The visual rapid detection kit according to claim 1, wherein the substrate color
developing solution is a 1 mol/L tetramethylbenzidine solution containing hydrogen
peroxide with a volume concentration of 1% to 5%.
6. The visual rapid detection kit according to claim 1, wherein the stop solution
is an aqueous solution of sodium lauryl sulfate with a mass concentration of 1% to 5%.
7. The visual rapid detection kit according to any one of claims 1 to 6, wherein a
dilution ratio of the enzyme-labeled secondary antibody is 1: (20,000-50,000).
8. A preparation method for the visual rapid detection kit according to any one of
claims 1 to 7, comprising a preparation method for the coated ELISA plate,
wherein preparation method for the coated ELISA plate comprises the following
steps that:
1) an antiserum dilution is obtained by diluting the0-type foot-and-mouth
disease virus antiserum;
2) the antiserum dilution is added into each well of the ELISA plate to incubate
for 12 h at 37°C;
3) the ELISA plate which is incubated for the first time is washed for 3-4 times,
blocked by a blocking solution, and incubated for 20 min for a second time at 37°C;
4) the ELISA plate which is incubated for the second time is washed for 3-4
times, and dried in air to obtain the coated ELISA plate.
9. The preparation method according to claim 8, wherein the antiserum dilution
is added to each well in the ELISA plate in a volume of 100 l.
10. The preparation method according to claim 8 or 9, wherein the blocking
solution is a bovine serum albumin aqueous solution with a mass concentration of 0.1%
to 0.5%; and the blocking liquid is added in an amount of 100 l/ well.
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| Application Number | Priority Date | Filing Date | Title |
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| AU2021105824A AU2021105824A4 (en) | 2021-08-18 | 2021-08-18 | Visual Rapid Detection Kit for O-type Foot-and-Mouth Disease Virus and Preparation Method Thereof |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| AU2021105824A AU2021105824A4 (en) | 2021-08-18 | 2021-08-18 | Visual Rapid Detection Kit for O-type Foot-and-Mouth Disease Virus and Preparation Method Thereof |
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ID=78177103
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| AU2021105824A Ceased AU2021105824A4 (en) | 2021-08-18 | 2021-08-18 | Visual Rapid Detection Kit for O-type Foot-and-Mouth Disease Virus and Preparation Method Thereof |
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| AU (1) | AU2021105824A4 (en) |
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