阅读说明：本技术 一种利用免疫标记法检测塞尼卡谷病毒的试剂盒及其检测方法 (kit for detecting Selenecar valley virus by using immune labeling method and detection method thereof ) 是由 孔彩平 张燕红 谢雪岑 史琳凯 齐志涛 路荣 范秀丽 郝鹏 魏学峰 杜宇荣 李雪 于 2018-05-28 设计创作，主要内容包括：本发明涉及一种利用免疫标记法检测塞尼卡谷病毒(SVV)的试剂盒及其检测方法。所述的试剂盒是利用荧光染料或过氧化物酶标记IgG抗体,利用免疫荧光法或免疫过氧化物酶法检测塞尼卡谷病毒的原理制得。本发明试剂盒能够快速、准确检测SVV病毒,应用于实验室快速诊断,而且可对病原进行准确定位,指示各组织器官的感染程度,对研究SVV的致病机理具有重要意义,且制备试剂盒的方法可根据实际生产灵活选择,制成的试剂盒敏感性、稳定性、特异性均能满足检测需要,适合批量生产,以应对突发的疫情。(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.)

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.

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: