CN113295868A - Kit capable of rapidly detecting tilapia streptococcus agalactiae and preparation method thereof - Google Patents
Kit capable of rapidly detecting tilapia streptococcus agalactiae and preparation method thereof Download PDFInfo
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- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
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Abstract
The invention belongs to the technical field of preparation of kits, and discloses a kit capable of rapidly detecting tilapia streptococcus agalactiae and a preparation method thereof, wherein the kit capable of rapidly detecting tilapia streptococcus agalactiae comprises: the kit comprises a kit shell and kit internal components, wherein the kit shell comprises an upper kit shell and a lower kit shell, and a sample dripping area and a result display area are arranged on the surface of the upper kit shell; the internal components of the kit comprise a bottom plate and a colloidal gold immunochromatographic test strip; the colloidal gold immunochromatographic test strip is provided with a quality control line and a detection line. The kit prepared by the method for detecting the tilapia streptococcus agalactiae antibody established by the tilapia monoclonal antibody and the colloidal gold technology is simple to detect and operate, does not need inspectors with professional knowledge, can be detected on site, can be used by culturists without special knowledge during use, can be used only by simple detection training, and strives for precious time for early control of epidemic diseases.
Description
Technical Field
The invention belongs to the technical field of preparation of kits, and particularly relates to a kit capable of rapidly detecting tilapia streptococcus agalactiae and a preparation method thereof.
Background
At present, streptococcus agalactiae is an important pathogen which is common to people and livestock and harms various animals, and at present, streptococcus agalactiae mainly harms tilapia breeding industry in China. According to the statistics of the world Food and Agriculture Organization (FAO), the breeding amount of the tilapia in China in 2008 reaches 706585 tons, which accounts for 50 percent of the total world production, and the tilapia becomes a genuine waterfowl. According to statistics, after 1980, the streptococcal infection in freshwater fish and marine fish is reported to outbreak in several countries, and the fish streptococcal infection begins to be widely popular worldwide. To date, the endemic area of the disease has covered freshwater and marine fish in europe, africa, australia, asia, and 15 countries in north and south africa, 6 continents. The tilapia industry is seriously injured due to the fact that the tilapia streptococcus agalactiae has to greatly reduce the tilapia culture amount.
The streptococcus agalactiae disease is serious in danger and extremely high in infectivity, and currently, no commercial vaccine exists for prevention and control of the streptococcus iniae disease in China, and only strict production management and antibiotic treatment can be relied on, so that establishment of a quick and effective early disease diagnosis method and monitoring of epidemic situations are key technical problems to be solved at present. However, the method for detecting the tilapia streptococcus agalactiae in the prior art is complex in operation, long in time and inconvenient for large-scale detection.
Through the above analysis, the problems and defects of the prior art are as follows: in the prior art, the method for detecting the tilapia streptococcus agalactiae is complex in operation, long in time and inconvenient for large-scale detection.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a kit capable of quickly detecting tilapia streptococcus agalactiae and a preparation method thereof.
The preparation method of the kit capable of rapidly detecting the tilapia streptococcus agalactiae comprises the following steps:
preparing an upper shell and a lower shell of the kit by using polycarbonate as a main material, wherein the lower shell is a semi-sealed shell with an opening at the upper end, and the lower shell is a shell with an opening at the lower end and two openings at the upper end;
step two, adhering a bottom plate to the lower end position in the lower shell of the kit, and marking the area corresponding to the sample dripping area and the result display area on the bottom plate; the sample dripping area and the result display area correspond to the two openings of the upper shell;
selecting a polyester film, and cutting the selected polyester film into long strips with the specification of 8-10 mm wide and 4-5 cm long; taking the cut strip as a reaction test strip, and trimming the edge of the reaction test strip;
step four, immunizing tilapia for 3-5 times, and collecting whole blood; naturally separating out serum at room temperature, centrifuging, and collecting supernatant to obtain serum; diluting by using PBS (phosphate buffer solution) for 2-3 times, centrifuging, separating, purifying and concentrating serum IgM protein to obtain tilapia serum IgM;
emulsifying the purified tilapia IgM protein and a Freund's complete adjuvant with the same volume, injecting the tilapia IgM protein and the Freund's complete adjuvant into the subcutaneous of the mouse at multiple points, immunizing the mouse, repeating the immunization for 2-3 times, and then performing the enhanced immunization on the direct abdominal cavity and vein of the mouse by using the tilapia IgM protein again;
taking the mouse cells after 2-5 days of boosting immunity, preheating to 30-40 ℃, adding a cell fusion agent and a DMEM medium preheated to 35-40 ℃, centrifuging, and placing in a solid medium for cell culture fusion; when the cells grow to the bottom of the hole by half, screening to obtain a tilapia IgM (immunoglobulin M) resisting monoclonal antibody cell strain;
selecting an NC membrane as a carrier of a quality control line and a detection line on the colloidal gold immunochromatography test strip, wherein the quality control line is a C line, and the detection line is a T line; accurately cutting the NC membrane to obtain a colloidal gold immunochromatographic test strip matrix;
step eight, coating the anti-tilapia IgM monoclonal antibody cell strain obtained in the step six onto a detection line of the colloidal gold immunochromatographic test strip matrix, and coating a goat anti-mouse immunoglobulin antibody onto a quality control line of the colloidal gold immunochromatographic test strip matrix;
step nine, preparing a colloidal gold solution, soaking the antibody-coated colloidal gold immunochromatographic test strip substrate in the colloidal gold solution for 2-5 min, diluting the antibody-coated colloidal gold immunochromatographic test strip substrate by using a phosphate buffer solution, then printing a film, and drying to obtain the colloidal gold immunochromatographic test strip;
and step ten, placing the colloidal gold immunochromatographic test strip in a shell to obtain the kit capable of quickly detecting the tilapia streptococcus agalactiae.
Further, in the sixth step, the centrifugation method comprises: and (3) placing the mixture containing the mouse cells, the cell fusion agent and the DMEM medium in a centrifuge with the speed of 800-1200 rpm for centrifugation for 15-20 min.
Further, in the eighth step, the coating concentration of the anti-tilapia IgM monoclonal antibody cell strain is 0.5-0.8 mg/mL.
Further, in the eighth step, the coating concentration of the goat anti-mouse immunoglobulin antibody is 1.0-2.0 mg/mL.
Further, in step ten, the placing the colloidal gold immunochromatographic test strip in the casing comprises:
(1) determining the relative position of the sample dripping area and the result display area, and placing the colloidal gold immunochromatographic test strip according to the determined relative position;
(2) the placed colloidal gold immunochromatographic test strip corresponds to the sample dripping area and the result display area of the bottom plate and covers the bottom plate;
(3) pressing the joint of the bottom plate and the colloidal gold immunochromatographic test strip to be smooth, and preparing sealing glue;
(4) and connecting the upper shell with the lower shell, using sealing glue to wipe the joint, and solidifying to obtain the kit capable of quickly detecting the tilapia streptococcus agalactiae.
Further, the placing of the colloidal gold immunochromatographic test strip according to the determined relative position comprises: placing the colloidal gold immunochromatographic test strip according to the sequence that the sample dripping area is on the left, the result display area is on the right, the detection line in the result display area is on the left, and the quality control line is on the right; or the colloidal gold immunochromatographic test strip is placed according to the sequence that the sample dripping area is on the right, the result display area is on the left, the quality control line in the result display area is on the left, and the detection line is on the right.
The invention also aims to provide a kit for rapidly detecting tilapia streptococcus agalactiae, which is prepared by applying the preparation method of the kit for rapidly detecting tilapia streptococcus agalactiae, and the kit for rapidly detecting tilapia streptococcus agalactiae comprises:
the kit comprises a kit shell and kit internal components, wherein the kit shell comprises an upper kit shell and a lower kit shell, and a sample dripping area and a result display area are arranged on the surface of the upper kit shell.
Further, the sample dripping area and the result display area are hollow areas.
Further, the sample dripping area is a circular hole-shaped area, and the result display area is a square area.
Further, the kit internal components comprise a bottom plate and a colloidal gold immunochromatographic test strip; the colloidal gold immunochromatographic test strip is provided with a quality control line and a detection line.
By combining all the technical schemes, the invention has the advantages and positive effects that: the kit for rapidly detecting the tilapia streptococcus agalactiae provided by the invention is a detection method of the tilapia streptococcus agalactiae antibody established by using a tilapia monoclonal antibody and a colloidal gold technology, the prepared kit is simple in detection operation, does not need a tester with professional knowledge, can be used for on-site detection, and can be used by a farmer without special knowledge during use and only needs simple detection training. Meanwhile, the kit provided by the invention can directly convert the complex, long and professional detection process of a laboratory into simple field detection for detection, thereby striving for precious time for early control of epidemic diseases.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings needed to be used in the embodiments of the present application will be briefly described below, and it is obvious that the drawings described below are only some embodiments of the present application, and it is obvious for those skilled in the art that other drawings can be obtained from the drawings without creative efforts.
FIG. 1 is a schematic diagram of a kit for rapidly detecting tilapia streptococcus agalactiae provided by an embodiment of the invention;
in the figure: 1. a cartridge housing; 2. a reagent cartridge internal assembly; 3. a sample dripping zone; 4. and a result display area.
FIG. 2 is a schematic view of the internal components of the kit provided by an embodiment of the present invention;
in the figure: 5. a base plate; 6. a colloidal gold immunochromatographic test strip; 7. a quality control line; and 8, detecting lines.
FIG. 3 is a flowchart of a method for preparing a kit for rapidly detecting tilapia streptococcus agalactiae according to an embodiment of the present invention.
FIG. 4 is a flow chart of a method for preparing monoclonal antibodies against tilapia Streptococcus agalactiae by using obtained tilapia non-head tissues according to an embodiment of the present invention.
Fig. 5 is a flowchart of a method for placing a colloidal gold immunochromatographic test strip in a housing according to an embodiment of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Aiming at the problems in the prior art, the invention provides a kit capable of rapidly detecting tilapia streptococcus agalactiae and a preparation method thereof, and the invention is described in detail with reference to the accompanying drawings.
As shown in fig. 1, the kit for rapidly detecting tilapia streptococcus agalactiae comprises: the kit comprises a kit shell 1 and a kit internal component 2, wherein the kit shell 1 comprises an upper kit shell and a lower kit shell, and a sample dripping area 3 and a result display area 4 are arranged on the surface of the upper kit shell;
the sample dripping area 3 and the result display area 4 are hollow areas;
the sample dripping area 3 is a circular hole-shaped area, and the result display area 4 is a square area.
As shown in fig. 2, the reagent cartridge internal component provided in the embodiment of the present invention includes a bottom plate 5, a colloidal gold immunochromatographic test strip 6; the colloidal gold immunochromatographic test strip 6 is provided with a quality control line 7 and a detection line 8.
As shown in fig. 3, the preparation method of the kit for rapidly detecting tilapia streptococcus agalactiae provided by the embodiment of the invention comprises the following steps:
s101, preparing an upper shell and a lower shell of the kit by using polycarbonate as a main material, wherein the lower shell is a semi-sealed shell with an opening at the upper end, and the lower shell is a shell with an opening at the lower end and two openings at the upper end;
s102, adhering a bottom plate to the lower end position in the lower shell of the kit, and marking the area, corresponding to the sample dripping area and the result display area, on the bottom plate; the sample dripping area and the result display area correspond to the two openings of the upper shell;
s103, selecting a polyester film, and cutting the selected polyester film into long strips with the specification of 8-10 mm wide and 4-5 cm long; taking the cut strip as a reaction test strip, and trimming the edge of the reaction test strip;
s104, separating, purifying and concentrating tilapia serum IgM protein to obtain tilapia serum IgM; emulsifying the purified tilapia IgM protein and an equivalent volume of Freund's complete adjuvant, and then carrying out mouse immunization; fusing the mouse cells to obtain a tilapia IgM (immunoglobulin M) resistant monoclonal antibody cell strain;
s105, selecting an NC membrane as a carrier of a quality control line and a detection line on the colloidal gold immunochromatographic test strip, wherein the quality control line is a C line, and the detection line is a T line; accurately cutting the NC membrane to obtain a colloidal gold immunochromatographic test strip matrix;
s106, coating the anti-tilapia IgM monoclonal antibody cell strain obtained in S104 on a detection line of the colloidal gold immunochromatographic test strip matrix, and coating a goat anti-mouse immunoglobulin antibody on a quality control line of the colloidal gold immunochromatographic test strip matrix;
s107, preparing a colloidal gold solution, soaking the antibody-coated colloidal gold immunochromatographic test strip substrate in the colloidal gold solution for 2-5 min, diluting the antibody-coated colloidal gold immunochromatographic test strip substrate by using a phosphate buffer solution, then printing a film, and drying to obtain the colloidal gold immunochromatographic test strip;
s108, placing the colloidal gold immunochromatographic test strip in a shell to obtain the kit capable of rapidly detecting tilapia streptococcus agalactiae.
As shown in fig. 4, in step S104, obtaining anti-tilapia IgM monoclonal antibody cell lines includes:
s201, immunizing tilapia for 3-5 times, and collecting whole blood; naturally separating out serum at room temperature, centrifuging, and collecting supernatant to obtain serum; diluting by using PBS (phosphate buffer solution) for 2-3 times, centrifuging, separating, purifying and concentrating serum IgM protein to obtain tilapia serum IgM;
s202, emulsifying the purified tilapia IgM protein and an equivalent volume of Freund 'S complete adjuvant, injecting the emulsified tilapia IgM protein and the Freund' S complete adjuvant at multiple points under the skin of the mouse, immunizing the mouse, repeating the immunization for 2-3 times, and then performing the enhanced immunization on the direct abdominal cavity and vein of the mouse by using the tilapia IgM protein again;
s203, preheating the mouse cells after 2-5 days of boosting immunity to 30-40 ℃, adding a cell fusion agent and a DMEM culture medium preheated to 35-40 ℃, centrifuging, and placing the mixture in a solid culture medium for cell culture fusion; and when the cells grow to the bottom of the hole by half, screening to obtain the anti-tilapia IgM monoclonal antibody cell strain.
The tilapia IgM antibody monoclonal antibody cell strain provided by the embodiment of the invention is a monoclonal antibody cell strain 2C10, and is named after classification: hybridoma cells, deposited at the chinese type culture collection on 21/3/2016, address: in the Wuhan university school of eight paths 299 # in Wuchang area of Wuhan city, Hubei province, the preservation number is CCTCC NO: C201644.
in step S201 provided in the embodiment of the present invention, the centrifugation method includes: and (3) placing the mixture containing the mouse cells, the cell fusion agent and the DMEM medium in a centrifuge with the speed of 800-1200 rpm for centrifugation for 15-20 min.
In step S106 provided by the embodiment of the invention, the coating concentration of the anti-tilapia IgM monoclonal antibody cell strain is 0.5-0.8 mg/mL.
In step S106 provided by the embodiment of the invention, the coating concentration of the goat anti-mouse immunoglobulin antibody is 1.0-2.0 mg/mL.
As shown in fig. 5, in step S108, the step of placing the colloidal gold immunochromatographic strip in the casing includes:
s301, determining the relative position of the sample dripping area and the result display area, and placing the colloidal gold immunochromatographic test strip according to the determined relative position;
s302, enabling the placed colloidal gold immunochromatographic test strip to correspond to a sample dripping area and a result display area of the bottom plate, and covering the sample dripping area and the result display area on the bottom plate;
s303, compacting and flattening the joint of the bottom plate and the colloidal gold immunochromatographic test strip, and preparing sealing glue;
s304, connecting the upper shell with the lower shell, using sealing glue to wipe the joint, and solidifying to obtain the kit capable of quickly detecting the tilapia streptococcus agalactiae.
The placement of the colloidal gold immunochromatographic test strip according to the determined relative position provided by the embodiment of the invention comprises the following steps: placing the colloidal gold immunochromatographic test strip according to the sequence that the sample dripping area is on the left, the result display area is on the right, the detection line in the result display area is on the left, and the quality control line is on the right; or the colloidal gold immunochromatographic test strip is placed according to the sequence that the sample dripping area is on the right, the result display area is on the left, the quality control line in the result display area is on the left, and the detection line is on the right.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention, and the scope of the present invention is not limited thereto, and any modification, equivalent replacement, and improvement made by those skilled in the art within the technical scope of the present invention disclosed herein, which is within the spirit and principle of the present invention, should be covered by the present invention.
Claims (10)
1. A preparation method of a kit capable of rapidly detecting tilapia streptococcus agalactiae is characterized by comprising the following steps:
preparing an upper shell and a lower shell of the kit by using polycarbonate as a main material, wherein the lower shell is a semi-sealed shell with an opening at the upper end, and the lower shell is a shell with an opening at the lower end and two openings at the upper end;
step two, adhering a bottom plate to the lower end position in the lower shell of the kit, and marking the area corresponding to the sample dripping area and the result display area on the bottom plate; the sample dripping area and the result display area correspond to the two openings of the upper shell;
selecting a polyester film, and cutting the selected polyester film into long strips with the specification of 8-10 mm wide and 4-5 cm long; taking the cut strip as a reaction test strip, and trimming the edge of the reaction test strip;
step four, immunizing tilapia for 3-5 times, and collecting whole blood; naturally separating out serum at room temperature, centrifuging, and collecting supernatant to obtain serum; diluting by using PBS (phosphate buffer solution) for 2-3 times, centrifuging, separating, purifying and concentrating serum IgM protein to obtain tilapia serum IgM;
emulsifying the purified tilapia IgM protein and a Freund's complete adjuvant with the same volume, injecting the tilapia IgM protein and the Freund's complete adjuvant into the subcutaneous of the mouse at multiple points, immunizing the mouse, repeating the immunization for 2-3 times, and then performing the enhanced immunization on the direct abdominal cavity and vein of the mouse by using the tilapia IgM protein again;
taking the mouse cells after 2-5 days of boosting immunity, preheating to 30-40 ℃, adding a cell fusion agent and a DMEM medium preheated to 35-40 ℃, centrifuging, and placing in a solid medium for cell culture fusion; when the cells grow to the bottom of the hole by half, screening to obtain a tilapia IgM (immunoglobulin M) resisting monoclonal antibody cell strain;
selecting an NC membrane as a carrier of a quality control line and a detection line on the colloidal gold immunochromatography test strip, wherein the quality control line is a C line, and the detection line is a T line; accurately cutting the NC membrane to obtain a colloidal gold immunochromatographic test strip matrix;
step eight, coating the anti-tilapia IgM monoclonal antibody cell strain obtained in the step six onto a detection line of the colloidal gold immunochromatographic test strip matrix, and coating a goat anti-mouse immunoglobulin antibody onto a quality control line of the colloidal gold immunochromatographic test strip matrix;
step nine, preparing a colloidal gold solution, soaking the antibody-coated colloidal gold immunochromatographic test strip substrate in the colloidal gold solution for 2-5 min, diluting the antibody-coated colloidal gold immunochromatographic test strip substrate by using a phosphate buffer solution, then printing a film, and drying to obtain the colloidal gold immunochromatographic test strip;
and step ten, placing the colloidal gold immunochromatographic test strip in a shell to obtain the kit capable of quickly detecting the tilapia streptococcus agalactiae.
2. The method for preparing the kit for rapidly detecting tilapia streptococcus agalactiae according to claim 1, wherein in the sixth step, the centrifugation method comprises the following steps: and (3) placing the mixture containing the mouse cells, the cell fusion agent and the DMEM medium in a centrifuge with the speed of 800-1200 rpm for centrifugation for 15-20 min.
3. The method for preparing a kit for rapidly detecting tilapia streptococcus agalactiae according to claim 1, wherein in the eighth step, the coating concentration of the anti-tilapia IgM monoclonal antibody cell strain is 0.5-0.8 mg/mL.
4. The method for preparing the kit for rapidly detecting tilapia streptococcus agalactiae according to claim 1, wherein in the eighth step, the coating concentration of the goat anti-mouse immunoglobulin antibody is 1.0-2.0 mg/mL.
5. The method for preparing a kit for rapidly detecting tilapia streptococcus agalactiae according to claim 1, wherein in the tenth step, the colloidal gold immunochromatographic test strip is placed in a shell and comprises the following steps:
(1) determining the relative position of the sample dripping area and the result display area, and placing the colloidal gold immunochromatographic test strip according to the determined relative position;
(2) the placed colloidal gold immunochromatographic test strip corresponds to the sample dripping area and the result display area of the bottom plate and covers the bottom plate;
(3) pressing the joint of the bottom plate and the colloidal gold immunochromatographic test strip to be smooth, and preparing sealing glue;
(4) and connecting the upper shell with the lower shell, using sealing glue to wipe the joint, and solidifying to obtain the kit capable of quickly detecting the tilapia streptococcus agalactiae.
6. The method for preparing the kit for rapidly detecting tilapia streptococcus agalactiae according to claim 5, wherein the placing of the colloidal gold immunochromatographic test strip according to the determined relative position comprises: placing the colloidal gold immunochromatographic test strip according to the sequence that the sample dripping area is on the left, the result display area is on the right, the detection line in the result display area is on the left, and the quality control line is on the right; or the colloidal gold immunochromatographic test strip is placed according to the sequence that the sample dripping area is on the right, the result display area is on the left, the quality control line in the result display area is on the left, and the detection line is on the right.
7. The kit for rapidly detecting tilapia streptococcus agalactiae, which is prepared by the preparation method of the kit for rapidly detecting tilapia streptococcus agalactiae according to any one of claims 1 to 6, is characterized by comprising the following steps:
the kit comprises a kit shell and kit internal components, wherein the kit shell comprises an upper kit shell and a lower kit shell, and a sample dripping area and a result display area are arranged on the surface of the upper kit shell.
8. The kit for rapidly detecting tilapia streptococcus agalactiae according to claim 7, wherein the sample dripping area and the result display area are hollow areas.
9. The kit for rapidly detecting tilapia streptococcus agalactiae according to claim 7, wherein the sample dripping zone is a circular-hole-shaped zone, and the result display zone is a square-shaped zone.
10. The kit for rapidly detecting tilapia streptococcus agalactiae according to claim 7, wherein the kit internal components comprise a bottom plate, a colloidal gold immunochromatographic test strip; the colloidal gold immunochromatographic test strip is provided with a quality control line and a detection line.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
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| CN106834235A (en) * | 2016-12-26 | 2017-06-13 | 广西大学 | Anti- Tilapia mossambica IgM monoclonal antibody cell line and its screening technique and application |
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| CN107942060A (en) * | 2017-11-16 | 2018-04-20 | 广西大学 | A kind of method using immune colloid gold percolation detection Tilapia mossambica Streptococcusagalactiae antibody |
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