CN110542755A - kit for detecting Selenecar valley virus by using immune labeling method and detection method thereof - Google Patents
kit for detecting Selenecar valley virus by using immune labeling method and detection method thereof Download PDFInfo
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- CN110542755A CN110542755A CN201810526601.4A CN201810526601A CN110542755A CN 110542755 A CN110542755 A CN 110542755A CN 201810526601 A CN201810526601 A CN 201810526601A CN 110542755 A CN110542755 A CN 110542755A
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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
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/533—Production of labelled immunochemicals with fluorescent label
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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
- G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
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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
Abstract
The invention relates to a kit for detecting Sernica Valley Virus (SVV) by an immune labeling method and a detection method thereof. The kit is prepared by using a fluorescent dye or peroxidase to mark an IgG antibody and using the principle of detecting the Seneca valley virus by an immunofluorescence method or an immunoperoxidase method. The kit can quickly and accurately detect SVV virus, is applied to quick diagnosis in a laboratory, can accurately position pathogen, indicates the infection degree of each tissue organ, has important significance for researching the pathogenesis of SVV, can be flexibly selected according to actual production, and the sensitivity, stability and specificity of the prepared kit can meet the detection requirement, thereby being suitable for batch production to deal with sudden epidemic situation.
Description
Technical Field
The invention relates to the technical field of biological detection, in particular to a kit for detecting Sernica valley virus by an immune labeling method and a detection method thereof.
background
Seneca Valley Virus (SVV) is an animal infectious disease caused by Seneca Valley virus A (SVA) of the family MicroRNA viruses, mainly infected pigs and pigs at different ages are susceptible to the infectious diseases, but the pathogenicity of different strains is obviously different, and the caused clinical symptoms are different. The early isolates mostly cause subclinical infection, the isolates in recent years usually cause the clinical symptoms of foot-and-mouth disease, blisters and ulcers appear on the nose, mouth and hoof of sick pigs, and the fatality rate of newborn piglets reaches 30-70%. At present, the disease is mainly prevalent in countries such as canada, brazil and the united states; SVV epidemic situation occurs in certain pig farms in Guangdong and Hubei provinces of China in sequence since 2015.
SVV can cause vesicular lesions similar to foot-and-mouth disease, swine vesicular disease, vesicular stomatitis, swine water herpes and the like, and if accurate differential diagnosis cannot be timely made, improper prevention and control measures can be taken, and the breeding cost is increased. Corresponding laboratory detection methods such as an electron microscope, immunohistochemistry, RT-PCR, virus serum antibody neutralization tests, competitive ELISA, fluorescent quantitative PCR dye methods and the like are developed for SVV at home and abroad at present, but the methods are all complex in operation and are not suitable for being widely applied to daily detection of SVV. SVV is also isolated for the first time in 2016 in China. The research establishes the kit for detecting the Seneca valley virus by using the immune labeling method and the detection method thereof, is suitable for batch production, can quickly and accurately detect the SVV virus, is applied to quick diagnosis in a laboratory, can accurately position the pathogen to indicate the infection degree of each tissue organ, and has important significance for researching the pathogenesis of SVV.
disclosure of Invention
the invention aims to provide a kit for detecting Sernica valley virus by an immune labeling method and a detection method thereof, the kit can quickly and accurately detect SVV virus, is applied to quick diagnosis in a laboratory, can identify and accurately locate pathogen, indicate the infection degree of each tissue organ, and has important significance for researching the pathogenesis of SVV.
firstly, the invention provides a kit for detecting Sernica valley virus by an immune labeling method, wherein the kit comprises cells for detection, a detection antibody and an IgG antibody marked by a marker; the cells for detection are PK-15 cells; the detection antibody is an anti-SVV monoclonal antibody; the marker is a fluorescent dye or peroxidase.
the detection antibody is preferably a mouse anti-SVV monoclonal antibody, and the marker-labeled IgG antibody is preferably a marker-labeled goat anti-mouse IgG antibody.
the fluorescent dye is preferably fluorescein isothiocyanate, and the peroxidase is preferably horseradish peroxidase.
The kit of the invention also comprises the following reagents: sample diluent, washing liquid, stationary liquid, positive control and negative control, wherein:
The sample diluent is serum-free MEM solution;
the washing solution is phosphate buffer solution, and the pH value is 7.2-7.6;
the stationary liquid is 80 percent cold acetone with the temperature of-20 ℃;
The negative control was PK-15 cells not inoculated with SVV.
as a detection kit, cells, a detection antibody and an enzyme-labeled antibody are core components thereof, and a basic antigen-antibody binding reaction can be realized as long as the three components are present. As for the auxiliary components, such as sample diluent, washing solution, enzyme substrate solution, stop solution, positive control and negative control, can be assembled in a detection kit with the above three components or provided separately, so that the detection kit of the present invention may or may not include these auxiliary components, and the present invention preferably includes these auxiliary components for convenient use.
in the present invention, the detection antibody may be a monoclonal antibody against SVV derived from a mammal such as mouse, rat, rabbit, etc., and preferably a mouse monoclonal antibody against SVV.
in the present invention, the source of the secondary antibody is not particularly limited, and is specifically determined by the source of the monoclonal antibody used. Preferred markers of the invention are goat anti-mouse antibodies.
In the present invention, the label in the secondary antibody of the label can be a fluorescent dye, and can also be a peroxidase, wherein the fluorescent dye of the present invention is preferably fluorescein isothiocyanate, and the peroxidase is preferably horseradish peroxidase.
when the marker is horseradish peroxidase, peroxidase substrate developing solution can be optionally added into the kit.
the invention also provides a detection method of the kit for detecting the Seneca valley virus by using an immune labeling method, which comprises the following steps:
S1, inoculating and fixing the virus to be detected;
s2, antibody incubation and virus detection.
the kit prepared by the invention is used for detecting SVV antigen, and comprises the following specific steps:
s1, inoculating and fixing the virus to be detected
(1) Subculture of cells: digesting the full monolayer PK-15 cells by using pancreatin, adding a cell nutrient solution to prepare a subculture cell suspension, paving a 96-hole cell culture plate at the density of 2-4 multiplied by 105/ml, culturing the 100 mu L cell culture plate in each hole under the condition of 5% CO2 at 37 ℃, and paving the cells at the bottom of the hole after 24 hours; wherein the cell nutrient solution is MEM culture medium containing 10% newborn calf serum;
(2) virus inoculation: diluting the virus to be detected by a proper multiple, inoculating the diluted virus to the PK-15 cell monolayer cultured in the step (1), adding serum-free MEM solution to 200 mu L per well, and culturing for 24 h; meanwhile, PK-15 cells without SVV inoculation are used as negative control;
(3) washing: taking out the cell culture plate from the incubator, discarding the maintenance liquid of the 96-well cell culture plate, and washing with PBS for 2-3 times, each time for 4-6 min;
(4) fixing: fixing with 100 μ L-pre-cooled fixing solution at-20 deg.C for 10-30min, discarding acetone solution, washing with PBS for 2-3 times, each time for 4-6 min; wherein, the fixing solution is preferably acetone with the concentration of 60-80%, and the fixing is carried out in a low-temperature environment of 2-8 ℃.
S2, antibody incubation and virus detection
(1) primary antibody incubation: adding SVV monoclonal antibody diluted with PBS, incubating at 37 deg.C for 45-90min, discarding primary antibody, washing with PBS for 2-3 times, each for 4-6 min;
(2) and (3) secondary antibody incubation: diluting FITC/peroxidase-labeled goat anti-mouse IgG secondary antibody with PBS (phosphate buffer solution), incubating at 37 ℃ for 45-90min with 50 μ L of PBS per well, discarding the secondary antibody, and washing with PBS for 2-3 times, 4-6min each time;
(3) and (3) detection: the 96-well plate was placed on an inverted microscope and observed in a dark room, and the results were judged.
The dilution multiple of the SVV monoclonal antibody is 50-400 times, and the dilution multiple of the goat anti-mouse IgG secondary antibody is 50-800 times.
The invention can also carry out fluorescent dye or peroxidase labeling on the SVV primary antibody, detect SVV virus by adopting a direct immunofluorescence method or a direct immunoperoxidase method, and the S2 comprises the following specific steps:
(1) Adding SVV antibody labeled with marker diluted with PBS, incubating at 37 deg.C for 45-90min with 50 μ L of each well, discarding antibody, and washing with PBS 3-5 times;
(2) The 96-well plate was placed on an inverted microscope and observed in a dark room, and the results were judged.
the invention has the following observation and judgment results: and (5) observing the color change condition under a microscope, and judging the result. The immunofluorescence determination criteria were: negative control and blank control have no specific green fluorescence, and positive cells have specific bright green fluorescence in cytoplasm. The determination standard of the immunoperoxidase method is as follows: negative control and blank control have no specific red staining, and positive cells have specific red staining in cytoplasm.
the invention has the beneficial effects that: the kit can quickly and accurately detect SVV virus, is applied to quick diagnosis in a laboratory, can identify and accurately locate pathogen, indicates the infection degree of each tissue organ, and has important significance for researching the pathogenesis of SVV; the method for preparing the kit can be flexibly selected according to actual production, and the prepared kit can meet the detection requirement and is suitable for batch production so as to cope with sudden epidemic situations.
drawings
FIG. 1 is a negative control chart of the test in example 1;
FIG. 2 is a positive control in example 1;
FIG. 3 is a negative control chart of the test in example 2;
FIG. 4 is a positive control in example 2;
FIG. 5 is a negative control chart of the test in example 3;
FIG. 6 is a positive control in example 3;
FIG. 7 is a negative control chart of the test in example 4;
FIG. 8 is a positive control in example 4.
The specific implementation mode is as follows:
The various biological materials described in the examples are obtained by way of experimental acquisition for the purposes of this disclosure and should not be construed as limiting the source of the biological material of the invention. In fact, the sources of the biological materials used are wide and any biological material that can be obtained without violating the law and ethics can be used instead as suggested in the examples.
the embodiments are provided in order to provide detailed embodiments and specific procedures, which will help understanding of the present invention, but the scope of the present invention is not limited to the following embodiments.
example 1: SVV detection kit and detection method (indirect immunofluorescence method)
the SVV detection kit comprises: PK-15 cells, SVV monoclonal antibody, 96-well cell culture plate, FITC fluorescent labeled IgG antibody (goat anti-mouse); the specific detection method comprises the following steps:
1. inoculating and fixing the virus to be detected:
(1) subculture of cells: digesting the full monolayer PK-15 cells by using pancreatin, adding a cell nutrient solution to prepare a subculture cell suspension, paving a 96-hole cell culture plate at the density of 3 multiplied by 105/ml, wherein each hole is 100 mu L, culturing under the condition of 5% CO2 at 37 ℃, and paving the cells at the bottom of the hole after 24 hours;
(2) virus inoculation: diluting the virus to be detected by a proper multiple, inoculating the diluted virus to the PK-15 cell monolayer cultured in the step (1), adding a serum-free cell culture solution to each hole by 200 mu L, and culturing for 24 h; meanwhile, PK-15 cells without SVV inoculation are used as negative control;
(3) Washing: taking out the cell culture plate from the incubator, discarding the maintenance liquid of the 96-well cell culture plate, and washing with PBS for 3 times, 5min each time;
(4) Fixing: each well was fixed with 100. mu.L of 80% acetone pre-cooled at-20 ℃ for 30min, the acetone solution was discarded, and the wells were washed 3 times with PBS for 5min each.
2. Detecting an antibody:
SVV monoclonal antibodies and enzyme-labeled secondary antibodies are commercially available or commercially available as technical outsourcing, and can be prepared by methods conventional in the art, and methods for preparing antibodies to known antigens are well known in the art; the mouse anti-SVV monoclonal antibody used in the invention is self-made in laboratories.
3. Antibody incubation and virus detection
(1) adding SVV monoclonal antibody (primary antibody, made by laboratory) diluted with PBS, incubating at 37 deg.C for 60min, discarding primary antibody, washing with PBS for 3 times, each for 4-6 min;
(2) FITC-labeled goat anti-mouse IgG secondary antibody (secondary antibody, purchased from Sigma) was diluted with PBS, 50. mu.L per well was incubated at 37 ℃ for 60min, the secondary antibody was discarded, and washed 2-3 times with PBS, each for 4-6 min.
preparing other solutions of the kit:
Sample diluent: serum-free MEM solutions;
Washing liquid: a phosphate buffer solution with a pH of 7.2-7.6;
③ fixing liquid: 80 percent cold acetone with the temperature of-20 ℃;
fourthly, negative control: PK-15 cells not inoculated with SVV;
Positive control: SVV virus fluid.
The test negative control is shown in FIG. 1, and the test positive control is shown in FIG. 2.
example 2: SVV detection kit and detection method thereof (indirect immunoperoxidase method)
the SVV detection kit comprises: PK-15 cells, SVV monoclonal antibodies, 96-well cell culture plates and IgG antibodies marked by HRP; the specific detection method comprises the following steps:
1. Inoculation and immobilization of the Virus to be tested
(1) Subculture of cells: digesting the full monolayer PK-15 cells by using pancreatin, adding a cell nutrient solution to prepare a subculture cell suspension, paving a 96-hole cell culture plate at the density of 3 multiplied by 105/ml, wherein each hole is 100 mu L, culturing under the condition of 5% CO2 at 37 ℃, and paving the cells at the bottom of the hole after 24 hours;
(2) Virus inoculation: diluting the virus to be detected by a proper multiple, inoculating the diluted virus to the PK-15 cell monolayer cultured in the step (1), adding a serum-free cell culture solution to each hole by 200 mu L, and culturing for 24 h; meanwhile, PK-15 cells without SVV inoculation are used as negative control;
(3) Washing: taking out the cell culture plate from the incubator, discarding the maintenance liquid of the 96-well cell culture plate, and washing with PBS for 3 times, 5min each time;
(4) Fixing: each well was fixed with 100. mu.L of 80% acetone pre-cooled at-20 ℃ for 30min, the acetone solution was discarded, and the wells were washed 3 times with PBS for 5min each.
2. detecting an antibody:
The SVV monoclonal antibody antibodies and enzyme-labeled secondary antibodies can be obtained by technical outsourcing or are commercially available, and can also be prepared by conventional methods in the art, and methods for preparing antibodies to known antigens are well known in the art; the mouse anti-SVV monoclonal antibody used in the invention is self-made in laboratories.
3. Peroxidase-labeled secondary antibody
(1) Adding 100 μ L of 0.1% hydrogen peroxide solution into each well, and allowing to act at room temperature for 20 min;
(2) Adding 100 times of SVV monoclonal antibody (primary antibody, self-made in laboratory) diluted with PBS, incubating at 37 deg.C for 1h with 50 μ L per well, discarding the primary antibody, and washing with PBS for 3-5 times;
(3) Adding goat anti-mouse IgG (secondary antibody, purchased from Abeam) labeled with HRP diluted 300 times with PBS, incubating at 37 ℃ for 1h, discarding the secondary antibody, and washing 3-5 times with PBS;
(4) Color developing solution (purchased from Vector of USA) is added for developing at room temperature for 15min, the color developing solution is discarded, and PBS is used for washing.
preparing other solutions of the kit:
sample diluent: serum-free MEM solutions;
washing liquid: a phosphate buffer solution with the pH value of 7-2-7.4;
③ fixing liquid: 80 percent cold acetone with the temperature of-20 ℃;
a peroxidase substrate color developing solution;
Negative control: PK-15 cells not inoculated with SVV;
sixthly, positive comparison: SVV virus fluid.
The test negative control is shown in FIG. 3, and the test positive control is shown in FIG. 4.
example 3: SVV detection kit and detection method (direct immunofluorescence method)
the SVV detection kit comprises: PK-15 cells, FITC-labeled SVV monoclonal antibody, and a 96-well cell culture plate; the specific detection method comprises the following steps:
(1) culture of PK-15 cells: PK-15 cells were cultured in a cell culture flask in MEM medium containing 10% bovine serum, and cultured in a 5% CO2 incubator at 37 ℃ for 1 passage over 2 days to grow a monolayer of PK-15 cells. Digesting with pancreatin, adding growth liquid to prepare cell suspension, inoculating the cell suspension into a 96-well plate at the density of 3 multiplied by 105/ml, culturing the cell suspension in a condition of 5% CO2 at 37 ℃ with 100 mu L per well, and paving the cell at the bottom of the well in an adherent manner after 24 hours;
(2) Inoculation of a sample to be detected: inoculating SVV with the PK-15 cells cultured in the step 1, adding serum-free MEM solution to 200 mu L per well, and culturing for 24h, wherein the PK-15 cells which are not inoculated with SVV are used as negative control;
(3) Taking out the cell culture plate from the incubator, removing liquid in culture holes, and washing 3 times by using PBS;
(4) adding 80% of 100 μ L of cold acetone at-20 deg.C into each well, fixing at 4 deg.C for 30min, discarding acetone solution, and washing with PBS for 3 times;
(5) A300-fold dilution of FITC-labeled SVV fluorescent antibody (prepared in the laboratory) in PBS was added thereto, 50. mu.L of the antibody was added to each well, incubated at 37 ℃ for 1 hour, the fluorescent antibody was discarded, and the mixture was washed 3 to 5 times with PBS.
Preparing other solutions of the kit:
sample diluent: serum-free MEM solutions;
washing liquid: a phosphate buffer solution with a pH of 7.2-7.6;
③ fixing liquid: 80 percent cold acetone with the temperature of-20 ℃;
Fourthly, negative control: PK-15 cells not inoculated with SVV;
Positive control: SVV virus fluid.
the test negative control is shown in FIG. 5, and the test positive control is shown in FIG. 6.
example 4: SVV detection kit and detection method thereof (direct immunoperoxidase method)
The SVV detection kit comprises: PK-15 cells, an SVV monoclonal antibody marked by HRP, and a 96-well cell culture plate; the specific detection method comprises the following steps:
(1) culture of PK-15 cells: PK-15 cells were cultured in a cell culture flask in MEM medium containing 10% bovine serum, and cultured in a 5% CO2 incubator at 37 ℃ for 1 passage over 2 days to grow a monolayer of PK-15 cells. Digesting with pancreatin, adding growth liquid to prepare cell suspension, inoculating the cell suspension into a 96-well plate at the density of 3 multiplied by 105/ml, culturing the cell suspension in a condition of 5% CO2 at 37 ℃ with 100 mu L per well, and paving the cell at the bottom of the well in an adherent manner after 24 hours;
(2) inoculation of a sample to be detected: inoculating SVV with the PK-15 cells cultured in the step 1, adding serum-free MEM solution to 200 mu L per well, and culturing for 24h, wherein the PK-15 cells which are not inoculated with SVV are used as negative control;
(3) Taking out the cell culture plate from the incubator, removing liquid in culture holes, and washing 3 times by using PBS;
(4) adding 80% of 100 μ L of cold acetone at-20 deg.C into each well, fixing at 4 deg.C for 30min, discarding acetone solution, and washing with PBS for 3 times;
(5) Adding 100 μ L of 0.1% hydrogen peroxide solution into each well, and allowing to act at room temperature for 20 min;
(6) A300-fold dilution of HRP-labeled SVV monoclonal antibody (self-made in the laboratory) in PBS was added, 50. mu.L of each well was incubated at 37 ℃ for 1 hour, the antibody was discarded, and the mixture was washed 3 to 5 times with PBS.
Preparing other solutions of the kit:
Sample diluent: serum-free MEM solutions;
Washing liquid: a phosphate buffer solution with a pH of 7.2-7.6;
③ fixing liquid: 80 percent cold acetone with the temperature of-20 ℃;
a peroxidase substrate color developing solution;
negative control: PK-15 cells not inoculated with SVV;
sixthly, positive comparison: SVV virus fluid.
The test negative control is shown in FIG. 7, and the test positive control is shown in FIG. 8.
example 5: specificity test
the above-mentioned kits of examples 1-4 were used to detect positive samples of porcine pseudorabies virus (PRV), Classical Swine Fever Virus (CSFV), Foot and Mouth Disease Virus (FMDV), Porcine Circovirus (PCV), and Sernica Valley Virus (SVV), and the results are shown in Table 1.
TABLE 1 results of specificity test
from the table above, it can be seen that the four kits and the detection methods are negative for detection results of positive samples of porcine pseudorabies virus (PRV), Classical Swine Fever Virus (CSFV), Foot and Mouth Disease Virus (FMDV) and Porcine Circovirus (PCV), and positive for detection results of positive samples of Seikaga Valley Virus (SVV), indicating that the specificity is good, and no cross reaction exists with the porcine pseudorabies virus (PRV), the Classical Swine Fever Virus (CSFV), the Foot and Mouth Disease Virus (FMDV) and the Porcine Circovirus (PCV).
example 6: sensitivity and stability test
1. and (3) sensitivity test: SVV cell cytotoxicity of known TCID50 was serially diluted 10-fold, 1TCID50, 10TCID50, 100TCID50, 103TCID50, 104TCID50 and 105TCID50 cytotoxics were inoculated to a full monolayer of PK-15 cells, and each dilution was inoculated to 4 wells, cultured for 24 hours, and tested using the above-mentioned kits of examples 1 to 4, and the results were as follows:
TABLE 2 results of sensitivity test
As can be seen from the above table, the SVV indirect immunofluorescence kit and the indirect immunoperoxidase kit can detect the SVV antigen of 10TCID50 at the lowest, and the SVV direct immunofluorescence kit and the direct immunoperoxidase kit can detect the SVV antigen of 100TCID50 at the lowest, which indicates that the sensitivity of the indirect immunolabeling kit is slightly better than that of the direct immunolabeling kit, but both meet the detection requirements.
2. and (3) stability test: the same sample was tested 3 times at different times using the kits of examples 1-4 above, 10 samples each time, and the stability of the results was verified. The test results are given in the following table:
TABLE 3 stability test results
as can be seen from the above table, the repetition rate of the 3 detection results of the kit of embodiments 1-4 is above 85%, and the stability of the kit results is good.
Claims (10)
1. a kit for detecting Seikagaku virus by an immune labeling method is characterized by comprising cells for detection, a detection antibody and an IgG antibody labeled by a marker; the cells for detection are PK-15 cells; the detection antibody is an anti-SVV monoclonal antibody; the marker is a fluorescent dye or peroxidase.
2. The kit for detecting Securitacavirus by an immunological labeling method according to claim 1, wherein the detection antibody is preferably a mouse anti-SVV monoclonal antibody, and the marker-labeled IgG antibody is preferably a marker-labeled goat anti-mouse IgG antibody.
3. the kit for detecting Seneca valley virus by using the immunolabeling method of claim 1, wherein the fluorescent dye is preferably fluorescein isothiocyanate, and the peroxidase is preferably horseradish peroxidase.
4. The kit for detecting Seikagavuv virus according to any one of claims 1 to 3, which further comprises the following reagents: sample diluent, washing solution, stationary liquid and negative control;
The sample diluent is serum-free MEM solution;
the washing solution is phosphate buffer solution, and the pH value is 7.2-7.6;
the stationary liquid is 80 percent cold acetone with the temperature of-20 ℃;
The negative control was PK-15 cells not inoculated with SVV.
5. the method for detecting the Selenka valley virus kit according to claim 1 to 4 by using the immuno-labeling method, comprising the steps of:
S1, inoculating and fixing the virus to be detected;
S2, antibody incubation and virus detection.
6. the method for detecting Seneca valley virus using the kit for immune labeling according to claim 5, wherein the step S1 specifically comprises:
(1) Subculture of cells: digesting the full monolayer PK-15 cells by using pancreatin, adding a cell nutrient solution to prepare a subculture cell suspension, paving a 96-hole cell culture plate at the density of 2-4 multiplied by 105/ml, culturing the 100 mu L cell culture plate in each hole under the condition of 5% CO2 at 37 ℃, and paving the cells at the bottom of the hole after 24 hours; wherein the cell nutrient solution is MEM culture medium containing 10% newborn calf serum;
(2) virus inoculation: diluting the virus to be detected, inoculating the diluted virus to the PK-15 cell monolayer cultured in the step (1), adding serum-free MEM solution to 200 mu L per well, and culturing for 24 h; meanwhile, PK-15 cells without SVV inoculation are used as negative control;
(3) Washing: taking out the cell culture plate from the incubator, discarding the maintenance liquid of the 96-well cell culture plate, and washing with PBS for 2-3 times, each time for 4-6 min;
(4) fixing: fixing with 100 μ L-pre-cooled fixing solution at-20 deg.C for 10-30min, discarding the fixing solution, washing with PBS for 2-3 times, each time for 4-6 min; wherein, the fixing solution is preferably acetone with the concentration of 60-80%, and the fixing is carried out in a low-temperature environment of 2-8 ℃.
7. The method for detecting Seneca valley virus using the kit for immune labeling according to claim 5, wherein the step S2 specifically comprises:
(1) Primary antibody incubation: adding SVV monoclonal antibody diluted with PBS, incubating at 37 deg.C for 45-90min, discarding primary antibody, washing with PBS for 2-3 times, each for 4-6 min;
(2) and (3) secondary antibody incubation: diluting fluorescent dye/peroxidase-labeled goat anti-mouse IgG secondary antibody with PBS (phosphate buffer solution), incubating at 37 ℃ for 45-90min with 50 mu L of each well, discarding the secondary antibody, and washing with PBS for 2-3 times, 4-6min each time;
(3) and (3) detection: the 96-well plate was placed on a fluorescence inverted microscope and observed in a dark room, and the results were judged.
8. The method for detecting Seneca valley virus using the kit for immune labeling according to claim 5, wherein the step S2 specifically comprises:
(1) adding fluorescent dye/peroxidase-labeled SVV antibody diluted by PBS, incubating at 37 deg.C for 45-90min with 50 μ L of each well, discarding antibody, and washing with PBS for 3-5 times;
(2) The 96-well plate was placed on an inverted microscope and observed in a dark room, and the results were judged.
9. The method for detecting the seneca valley virus kit by using the immune labeling method as claimed in claims 8-9, wherein the dilution factor of the SVV monoclonal antibody is 50-400 times, and the dilution factor of the goat anti-mouse IgG secondary antibody is 50-800 times.
10. the method of detecting a seneca valley virus by using an immunological labeling method according to claim 5 to 9, wherein the observation and judgment of the result are as follows: the resultant was observed under a microscope to determine the color development.
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| CN111286491A (en) * | 2020-02-03 | 2020-06-16 | 河南省动物疫病预防控制中心 | Porcine Seneca virus nucleic acid standard substance and application thereof |
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