AU2021105787A4 - Visual Rapid Detection Kit for Swine Fever Virus Antibody and Application Thereof - Google Patents
Visual Rapid Detection Kit for Swine Fever Virus Antibody and Application Thereof Download PDFInfo
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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
- G01N2333/01—DNA viruses
-
- 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
- G01N2469/20—Detection of antibodies in sample from host which are directed against antigens from microorganisms
-
- 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 invention relates to a visual rapid detection kit for swine fever virus antibodies, belonging
to the biological detection technical filed. The kit provided by the invention comprises a coated
ELISA plate, a washing solution, a sample diluent, a substrate color developing solution, an
ELISA secondary antibody, a stop solution, a standard positive serum and a standard negative
serum, wherein the coated ELISA plate takes classical swine fever virus E2 as a coating
antigen, and the coating concentration of the coating antigen is 1.5-3[ g/ml. The kit provided
by the invention can be used for detecting antibodies of classical swine fever virus, and the
result is judged by color change, and has the advantages of high efficiency, high sensitivity,
strong specificity and good repeatability. The kit provided by the invention has the advantages
of simple and convenient operation, rapidness and low cost, can carry out visual detection at
room temperature (20-25°C), is suitable for clinical popularization and application, and
provides a reliable technical means for rapid detection of antibodies against classical swine
fever virus.
Description
Visual Rapid Detection Kit for Swine Fever Virus Antibody and Application
Thereof
The invention belongs to the technical field of biological detection, and particularly
relates to a visual swine fever virus antibodies quick detection kit and an application
thereof.
Classical swine fever is a highly infectious disease, and it is one of the major infectious
diseases threatening the pig industry. It is characterized by acute, septic changes,
bleeding, necrosis and parenchymal organ infarction, which have occurred in most
provinces of China. Swine fever can occur all the year round, especially in rainy seasons
in spring and summer. It is characterized by strong infectivity and rapid epidemic.
Susceptible animals die easily under the condition of weak resistance, which will cause
huge economic losses to farmers and seriously hinder the development of aquaculture.
The pathogen of classical swine fever is the classical swine fever virus of plague virus,
belonging to Trichoderma viride. Mainly through direct contact or by contact with
contaminated media. Digestive tract, nasal mucosa and broken skin are all infection
routes. At present, the methods of diagnosis of classical swine fever virus antibody and
evaluation of vaccine immune effect are mostly carried out in laboratory, which are not
suitable for grass-roots detection. Therefore, it is necessary to establish a more sensitive,
fast and simple visual detection method.
In view of the problems existing in the background technology, the purpose of the present
invention is to provide a visual rapid detection kit for classical swine fever virus
antibody, which comprises a coated ELISA plate, a washing solution, a sample diluent, a
substrate chromogenic solution, an ELISA secondary antibody, a stop solution, a standard
positive serum and a standard negative serum, wherein the coated ELISA plate takes
classical swine fever virus protein E2 as a coating antigen, and the coating concentration
of the coating antigen is 1.5-3 g/ml
Preferably, the coated ELISA plate is prepared by sealing the coated antigen with a
sealing solution, wherein the sealing solution is PBST buffer solution containing 40-60
g/L skimmed milk powder, and the sealing time is 15-25 minutes; The coat enzyme label
plate is stored at 2-6°C
Preferably, the cleaning solution is phosphate buffer solution containing Tween -20 with
a volume concentration of 0. 04% - 0.06%, and the pH value is 7 - 7.4.
Preferably, the sample diluent is phosphate buffer solution containing 40-60 g/L skim
milk powder.
Preferably, the substrate color developing solution is TMB solution.
Preferably, the dilution multiple of the enzyme-labeled secondary antibody is 1: (15000
25000).
Preferably, the ending solution is 0.8-1.2% SDS or 1-3 mol/L sulfuric acid solution.
The invention also provides the application of the visual rapid detection kit in the
detection of the classical swine fever virus antibody after vaccination.
Preferably, the application comprises the following steps:
(1) Taking an isolated sample to be detected, diluting the isolated sample with the sample
diluent, wherein the dilution ratio is 1: (30-50), and obtaining a diluted sample;
(2) Adding the diluted sample to the coated ELISA plate, incubating at 20 ~ 25°C for 30
~ 40 minutes, discarding the reaction solution, washing the plate with washing liquid, and
drying;
(3) Adding the enzyme-labeled second antibody to the enzyme-labeled plate dried in step
(2), incubating at 20-25°C for 30-40 minutes, discarding the reaction solution, washing
the plate with washing liquid, and drying;
(4) Adding the substrate chromogenic solution into the dried enzyme-labeled plate in step
(3), performing chromogenic reaction at 20-25°C for 10-20 minutes, and judging the
detection result.
The invention provides a kit for visual rapid detection of classical swine fever virus
antibody, which comprises a coated ELISA plate, a washing solution, a sample diluent, a
substrate color developing solution, an ELISA secondary antibody, a stop solution, a
standard positive serum and a standard negative serum, wherein the coated ELISA plate
takes classical swine fever virus protein E2 as a coating antigen, and the coating
concentration of the coating antigen is 1.5-3 g/ml. The kit provided by the invention
can be used for detecting antibodies of classical swine fever virus, and the result is judged
by color change, and has the advantages of high efficiency, strong specificity and good
repeatability. The kit provided by the invention has the advantages of simple and
convenient operation, rapidness and low cost, can be visually detected at room
temperature, is suitable for popularization in clinical application, and provides a reliable
technical means for rapid detection of swine fever virus antibodies.
Figure 1 is a graph showing the experimental results of specificity detection described in
example 2 of the present invention.
The invention provides a kit for visual rapid detection of classical swine fever virus
antibody, which comprises a coated ELISA plate, a washing solution, a sample diluent, a
substrate chromogenic solution, an ELISA secondary antibody, a stop solution, a standard
positive serum and a standard negative serum, wherein the coated ELISA plate takes
classical swine fever virus protein E2 as a coating antigen, and the coating concentration
of the coating antigen is 1.5-3 g/ml.
The kit provided by the invention comprises a coated ELISA plate. In the invention, the
coated ELISA plate takes classical swine fever virus as the coating antigen, and can
specifically capture the antibody of classical swine fever virus in the sample to be
detected during detection. In the present invention, the coating antigen is classical swine
fever virus protein E2, and the coating concentration is 1.5-3 g/ml. According to the
invention, the source of the classical swine fever virus protein E2 is not particularly
limited, and conventional commercial products in the field can be used.
In the present invention, a sealing solution is used to seal the coated antigen into the
coated ELISA plate. In the present invention, the blocking solution is preferably PBST
buffer solution containing skim milk powder. The concentration of the skimmed milk
powder in PBST buffer solution is preferably 40-60 g/L, more preferably 50 g/L.. The
sealing time is preferably 15-25 minutes, and more preferably 20minutes. According to
the invention, there is no special restriction on the sources of the PBST buffer solution and skim milk powder, and the conventional commercial products in this field can be used. In an embodiment of the present invention, PBST and skim milk powder are purchased from sigma. After the coated antigen is sealed to the ELISA plate, the invention preferably stores the coated ELISA plate. The preservation temperature is preferably 2-6°C, more preferably 4°C; The preservation is preferably carried out in a sealed bag.
The kit provided by the invention comprises an enzyme-labeled secondary antibody. In
the invention, the enzyme-labeled secondary antibody can be specifically combined with
the classical swine fever virus antibody captured on the enzyme-labeled plate, and can
carry out color reaction with the color developing solution. In the present invention, the
enzyme labeled secondary antibody is purchased from sigma. According to the invention,
the enzyme-labeled secondary antibody is preferably diluted during detection. In the
present invention, the dilution multiple of the enzyme-labeled secondary antibody is
preferably 1: (15000-25000), and more preferably 1: 20000.
The kit provided by the present invention also comprises a washing solution, a sample
diluent, a substrate color developing solution and a stopping solution. In the present
invention, the washing solution is preferably phosphate buffer solution containing Tween
-20. The volume concentration of Tween -20 in phosphate buffer solution is preferably 0.
04% ~ 0.06%, more preferably 0.5%. The pH value of the phosphate buffer is preferably
7-7.4, more preferably 7.2. In the present invention, the sample diluent is preferably
phosphate buffer containing skim milk powder. The concentration of the skim milk
powder in phosphate buffer solution is preferably 40-60 g/L, more preferably 50 g/L. In
the present invention, the substrate color developing solution is preferably TMB solution
(3 ,3 ',5 5 '- tetramethylbenzidine solution) purchased from sigma. In the present
invention, the stopping solution is preferably SDS or sulfuric acid solution. The volume
percentage of SDS is preferably 0. 8% - 1.2%, more preferably 1%. The concentration of
the sulfuric acid solution is preferably 1-3 mol/L, more preferably 2 mol/L.. According to
the invention, the sources of the phosphate buffer solution, tween-20, TMB solution and
SDS solution are not particularly limited, and conventional commercial products in the
field can be used. In an embodiment of the present invention, the phosphate buffer, tween
-20, TMB solution and SDS were all purchased from sigma.
The kit provided by the present invention also preferably comprises standard positive
serum and standard negative serum. The standard positive serum is swine fever virus
positive serum; The standard negative serum is swine fever virus negative pig serum; The
standard positive serum and the standard negative serum can be used for the sample to be
tested in the control test in the detection process. In an embodiment of the invention, the
serum of swine fever virus positive pig and the serum of swine fever virus negative pig
are both provided by Lanzhou Veterinary Research Institute.
The kit provided by the invention can be used for detecting antibodies of classical swine
fever virus, and has the advantages of high efficiency, strong specificity and good
repeatability by judging the result according to color change.
In the present invention, the method for preparing the kit is not particularly limited. The
raw materials of the invention are adopted to prepare each component to obtain solutions
with corresponding concentration.
The invention also provides the application of the visual rapid detection kit in detecting
the antibody of the classical swine fever virus after inoculation. The application
preferably comprises the following steps:
(1) Taking an isolated sample to be detected, diluting the isolated sample with the sample
diluent, wherein the dilution ratio is 1: (30-50), and obtaining a diluted sample;
(2) Adding the diluted sample into the coated ELISA plate, incubating at 20-25°C for 30
minutes, discarding the reaction solution, washing the plate with washing liquid, and
drying;
(3) Adding the enzyme-labeled second antibody to the dried enzyme-labeled plate in step
(2), incubating at 20-25°C for 30-40 minutes, discarding the reaction solution, washing
the plate with washing liquid, and drying;
(4) Adding the substrate chromogenic solution into the dried enzyme-labeled plate in step
(3), performing chromogenic reaction at 20-25°C for 10-20 minutes, and judging the
detection result.
In step (1) of the present invention, the separated sample to be detected is first taken out
and diluted with the sample diluent. In the present invention, the dilution ratio is
preferably 1: (30-50), more preferably 1: 40. A diluted sample are obtained after dilution.
In step (2) of the present invention, the diluted sample is added to the coated ELISA plate
for incubation. In the present invention, the incubation temperature is preferably 20-25°C.
The incubation time is preferably 30-40 minutes and more preferably 35minutes. After
incubation, the reaction solution is discarded, and the washing solution is used to wash
the plate, and the number of times of washing the plate is preferably 2-3. Washing the
board and dry, wherein the drying mode is preferably pat dry with absorbent paper.
In step (3), the enzyme-labeled secondary antibody is added to the dried enzyme-labeled
plate in step (2) for further incubation. In the present invention, the temperature of the
further incubation is preferably 20-25°C., more preferably 25°C. The further incubation
time is preferably 30-40minutes, and more preferably 40 minutes. After further
incubation, the reaction solution is discarded, and the washing solution is used to wash
the plate, and the number of times of washing the plate is preferably 2-3. Washing the
board and dry, wherein the drying mode is preferably pat dry with absorbent paper.
In step (4) of the present invention, the substrate color developing solution is added to the
dried ELISA plate in step (3) for color development. In the present invention, the color
developing temperature is preferably 20-25°C, more preferably 25°C. The time of the
color reaction is preferably 10-20 minutes, more preferably 20 minutes. The detection
result is judged after the color reaction is finished. In the present invention, it is
preferable to judge the detection result by adding SDS termination solution and observing
with naked eyes: blue is positive and colorless is negative.
The kit provided by the invention has the advantages of simple and convenient operation,
rapidness and low cost, can be visually detected at room temperature, is suitable to be
popularized in clinical application, and provides a reliable technical means for rapidly
detecting anti-classical swine fever virus antibodies.
The visual rapid detection kit for classical swine fever virus antibody provided by the
present invention and its application are described in detail in the following examples, but
can not be understood as limiting the scope of protection of the present invention.
Example I
1. Preparation of coated ELISA plate, preparation of sample diluent, washing solution
and stop solution
0.05mol/L carbonate buffer was prepared as coating solution: Na3ABO3 1.59g, NaHCO3
2 .93g, and distilled water was added. The volume is 1000ml, and the pH is 9.6. Classical
swine fever virus antigen E2 was diluted with coating solution, and the dilution degree
was 2 .Otg/mL. Add the diluted CSFV antigen to the ELISA plate, and add 100L
volume to each well. Then, 50g/L skim milk powder PBST was used for 20 minutes to
obtain the coated ELISA plate. Put it in a sealed bag and store at 4°C for later use.
Phosphate buffer containing 50g/L skim milk powder was prepared as sample diluent.
0.01 mol/L phosphate buffer containing 0. 05% tween-20 with ph of 7.2 was prepared as
washing solution: NaCl 8.5g, NaH2PO4 -2H20 0.356 g, NaH2PO4 -12H20 2. 772 g, which
were mixed and dissolved in distilled water to 1000mL.
1% SDS or 2mol/L sulfuric acid solution was prepared as the stopping solution.
2. Sample Testing
(1) Take the serum sample to be detected, and use the sample diluent to dilute the serum
by 1:40 times;
(2) Take out the coated and sealed ELISA strip (coated ELISA plate), and add 100 L of
diluted serum into each hole; At the same time, the control group with standard positive
serum and standard negative serum (foot-and-mouth disease virus A) was set up at room
temperature (20-25°C) incubate for 1 hour, discard the liquid in the wells, wash each well
with washing liquid for 3 times and pat, discarding the liquid in the wells;
(3) Take the enzyme-labeled secondary antibody (purchased from sigma) and use the
sample diluent to dilute the enzyme-labeled secondary antibody by 1:20000 times. Then add the diluted enzyme-labeled secondary antibody to the enzyme-labeled plate, add
100OL to each well, incubate at room temperature (20-25°C) for 40min, discard the liquid
in the reaction well, wash each well with washing liquid for 3 times and pat, and discard
the liquid in the well;
(4) Add 100pL substrate TMB chromogenic solution to each well of the enzyme-labeled
plate, perform chromogenic reaction at room temperature (20-25°C) for 20 minutes in the
dark, and add 100LSDS termination solution; Observe the color with naked eye, blue is
positive, colorless is negative. Or add sulfuric acid stop solution, and read the OD value
of each hole with a microplate reader at 450nm wavelength to judge the result.
Example II
Specific test of the kit: The positive serum samples of known foot-and-mouth disease
virus, Japanese encephalitis virus, porcine circovirus, porcine parvovirus, pseudorabies
virus, blue ear disease virus and classical swine fever virus were detected according to the
method of Example I, and the negative samples were colorless, while the positive
samples were blue.
The test results are shown in Figure 1. Figure 1 shows foot-and-mouth disease virus
(No.1), classical swine fever virus positive serum sample (No.2), japanese encephalitis
virus (No.3), porcine circovirus (No.4), porcine parvovirus (No.5), pseudorabies virus
(No.6), classical swine fever virus positive serum sample (No.7) and blue ear disease
virus (No.8). The results in figure 1 show that the kit provided in example 1 of the present
invention is colorless for positive serum samples of porcine epidemic diarrhea virus, foot
and-mouth disease virus, japanese encephalitis virus, porcine circovirus, porcine
parvovirus, pseudorabies virus and blue for positive serum samples of classical swine fever virus. Therefore, the kit of the invention is used for detecting antibodies of classical swine fever virus, and has the advantages of high efficiency, sensitivity, specificity and repeatability. The method is simple, rapid and low in cost, can be used for visually detection at room temperature, is suitable for clinical application, and provides a reliable technical means for rapid detection of antibodies against classical swine fever virus.
The above are only the preferred embodiment of the present invention, and it should be
pointed out that for ordinary people in the technical field, various improvements and
modifications can be made without departing from the principle of the present invention,
and these improvements and embellishments should also be regarded as the protection
scope of the present invention.
Claims (1)
- THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:1. A kit for visual rapid detection of classical swine fever virus antibody, whichcomprises a coated ELISA plate, a washing solution, a sample diluent, a substrate colordeveloping solution, an ELISA secondary antibody and a stop solution, and ischaracterized in that the coated ELISA plate takes classical swine fever virus protein E2as a coating antigen, and the coating concentration of the coating antigen is 1.5-3 g/ml.2. The visual rapid detection kit according to claim 1, characterized in that the coatedenzyme-linked immunosorbent assay plate is made by sealing the coated antigen with asealing, and the sealing solution is PBST buffer containing 40-60 g/L skim milk powder,and the sealing time is 15-25 minutes; The coat enzyme label plate is stored at 2-6°C.3. The rapid vision detection kit according to claim 1, wherein the washing liquidcomprises 0. 04% - 0.06% volume concentration of Tween -20 phosphate buffer, pH 77.4.4. The visual rapid detection kit of claim 1, wherein the sample diluent comprisesphosphate buffer solution of 40 - 60g/L skim milk powder.5. The rapid vision detection kit according to claim 1, wherein the substrate colordeveloping solution is TMB solution.6. The rapid vision detection kit according to claim 1, characterized in that the dilutionmultiple of the enzyme-labeled secondary antibody is 1: (15000-25000).7. The rapid vision detection kit according to claim 1, characterized in that the stopsolution is 0. 8 -. 1. 2% SDS or 1 - 3 mol/L sulfuric acid solution.8. Application of the visual rapid detection kit according to any one of claims 1-7 indetecting antibodies against classical swine fever virus after vaccination.Figure 1/1Figure 1
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|---|---|---|---|
| AU2021105787A AU2021105787A4 (en) | 2021-08-18 | 2021-08-18 | Visual Rapid Detection Kit for Swine Fever Virus Antibody and Application Thereof |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| AU2021105787A AU2021105787A4 (en) | 2021-08-18 | 2021-08-18 | Visual Rapid Detection Kit for Swine Fever Virus Antibody and Application Thereof |
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| Publication Number | Publication Date |
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| AU2021105787A4 true AU2021105787A4 (en) | 2021-10-21 |
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| AU2021105787A Ceased AU2021105787A4 (en) | 2021-08-18 | 2021-08-18 | Visual Rapid Detection Kit for Swine Fever Virus Antibody and Application Thereof |
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| AU (1) | AU2021105787A4 (en) |
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- 2021-08-18 AU AU2021105787A patent/AU2021105787A4/en not_active Ceased
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