CN113759118A - Avian influenza virus infection and vaccine immunity differential diagnosis detection card and preparation method thereof - Google Patents
Avian influenza virus infection and vaccine immunity differential diagnosis detection card and preparation method thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of biological detection, and discloses an avian influenza virus infection and vaccine immunity differential diagnosis detection card and a preparation method thereof, wherein the avian influenza virus infection and vaccine immunity differential diagnosis detection card comprises a shell and a sample diluent which is matched with the shell for use, a test strip is assembled in the shell, the test strip comprises a PVC bottom plate, a sample pad, a marking pad, a coating film and a water absorption pad are adhered on the PVC bottom plate, and the avian influenza virus infection and vaccine immunity differential diagnosis detection card is characterized in that: the marking pad is a glass cellulose membrane and is coated with a coupling marker of a mouse anti-chicken IgG monoclonal antibody and latex microspheres; the coating film is a nitrocellulose film, and is coated with a detection line coated with the purified avian influenza NS1 protein and a quality control line coated with the purified recombinant streptococcus G protein; the invention can identify and diagnose the antibodies generated by avian influenza virus infection and vaccine immunization, has good specificity and convenience, and can well identify avian influenza infected chicken flocks.
Description
Technical Field
The invention belongs to the technical field of biological detection, and particularly relates to an avian influenza virus infection and vaccine immunity differential diagnosis detection card and a preparation method thereof.
Background
Avian Influenza (AI) is a highly contagious infectious disease of birds caused by influenza a virus of orthomyxoviridae, HAs 16 HA subtypes and 10 NA subtypes, and is called as fowl plague in folks in the past, and is determined as an infectious disease a by the international veterinary health authority, and is one of devastating diseases in poultry farming.
At present, one of the methods for effectively controlling avian influenza is vaccination, one index for effectively evaluating the immune effect is the antibody level after detection of immunity, but the antibody is also induced to be generated in the later stage of wild virus infection; in addition, because the avian influenza has numerous serosubtypes and the cross protection among the subtypes is very low, the phenomenon that an immunized poultry group is continuously infected with heterotypic strains exists, and great trouble is brought to the differential diagnosis of whether the disease is wild virus infection or vaccine immunity, so that the effective distinction of antibodies generated by natural infection and antibodies generated by immunization is very important.
The chicken flock has a large amount of NS1 protein in the nucleus of early cell infected by avian influenza virus, and NS1 protein appears in cytoplasm in the late stage of infection, which can stimulate the body to produce NS1 nonstructural protein antibody, but the protein is not found in mature virus particles, and a large amount of NS1 protein usually appears in wild infected chicken due to the reproduction and replication of virus, thereby inducing the antibody production.
After the vaccine is applied for immunization, NS1 protein antibodies or extremely low-content NS1 protein antibodies do not appear in chicken bodies, so the NS1 protein and the antibodies thereof can be used as an important marker for avian influenza virus infected organisms.
At present, the traditional serological experimental methods, such as hemagglutination and blood inhibition experiment (HA-HI) and agar diffusion experiment (AGP), can only detect the antibody level after immunization, and can not distinguish natural infected chicken flocks from immune inoculated chicken flocks; although there are reports of enzyme-linked immunosorbent assay (ELISA) methods for identifying avian influenza infection and vaccine immunity, the ELISA methods are cumbersome to operate, long in time consumption, require precise instruments and equipment, have high requirements on operators, and have limitations in practical application.
The gold colloid method is the most common method in immunochromatography, but the sintering of the gold colloid is easy to cause the phenomenon of nonuniform particles, so that the product quality is unstable and the uniformity is poor.
Disclosure of Invention
The invention aims to solve the main technical problem of providing a differential diagnosis detection card for avian influenza virus infection and vaccine immunization and a preparation method thereof, wherein the differential diagnosis detection card can distinguish avian influenza virus infected chicken groups from vaccine immune chicken groups, and can solve the problems of unstable product quality and poor uniformity caused by the phenomenon of uneven particles easily generated by firing colloidal gold in the preparation process of the traditional colloidal gold test strip.
In order to solve the technical problems, the invention provides the following technical scheme:
avian influenza virus infects and bacterin immunity differential diagnosis detects card, including shell and the supporting sample diluent that uses, is equipped with the test paper strip in the shell, and the test paper strip includes the PVC bottom plate, and it has sample pad, mark pad, envelope membrane and the pad that absorbs water to paste on the PVC bottom plate, its characterized in that: the marking pad is a glass cellulose membrane and is coated with a coupling marker of a mouse anti-chicken IgG monoclonal antibody and latex microspheres; the coating film is a nitrocellulose film, and is coated with a detection line coated with the purified avian influenza NS1 protein and a quality control line coated with the purified recombinant streptococcus G protein.
The following is a further optimization of the above technical solution of the present invention:
the sample diluent comprises a base solution, wherein 1 percent of sucrose and 0.05 percent of NaN are added into the base solution in percentage by mass3Tween-20 with the volume percentage of 0.3 percent is also added into the base fluid;
the base solution was PBS solution, the concentration of PBS solution was 0.01mol/L, and the pH of PBS solution was 7.2.
Further optimization: the shell is provided with an observation window and a sample adding hole, after the test strip is assembled in the shell, the coating film is positioned at the position of the observation window, the sample pad is positioned at the position of the sample adding hole, the shell is provided with marks, namely C and T, the mark C corresponds to the quality control line, and the mark T corresponds to the detection line.
The invention also discloses a preparation method of the avian influenza virus infection and vaccine immunity differential diagnosis detection card, which comprises the following steps:
s1, treatment of latex microspheres: adding 0.1% by mass of latex microspheres into a latex microsphere cleaning solution, centrifuging for 15 minutes at the temperature of 2-8 ℃, wherein the centrifugation speed is 13000 r/min, discarding the supernatant, adding a cleaning solution with the same volume as that of the latex microsphere cleaning solution into the precipitate for resuspension, and repeatedly cleaning for 1 time, wherein NHS with the concentration of 20mg/mL is firstly added into the suspended latex microsphere solution, EDC with the concentration of 20mg/mL is then added, and the volume ratio of NHS to the suspended latex microsphere solution is 1: 20; the volume ratio of EDC to the suspended latex microsphere solution is 1: 100; then oscillating for 15 minutes at an oscillation speed of 140-150 r/min, centrifuging for 15 minutes at the temperature of 2-8 ℃ after oscillation is finished, wherein the centrifugation speed is 13000 r/min, discarding supernatant, adding latex microsphere preservative fluid with the volume of the original latex microsphere cleaning fluid for resuspension after precipitation is finished, and the average diameter of the latex microspheres is 100 nm;
s2, protein labeling: adding a mouse anti-chicken IgG monoclonal antibody into the treated latex microspheres according to 1/10 of the mass of the latex microspheres, oscillating for 1 hour at room temperature at the oscillation speed of 140-150 r/min, adding sealing liquid with the volume of 1/10 volume of original latex microsphere cleaning liquid after oscillation is finished, and oscillating for 30 minutes at room temperature at the oscillation speed of 140-150 r/min; after the oscillation is finished, centrifuging for 15 minutes at the temperature of 2-8 ℃, wherein the centrifugation speed is 13000 r/min, discarding the supernatant, precipitating, and adding a redissolution with the volume 2 times that of the original emulsion microsphere cleaning solution for redissolving;
s3, preparation of a marking pad: and (3) paving the protein solution redissolved in the step S2 on a glass cellulose membrane, and drying at the room temperature of 37 ℃ for 2 hours to obtain the marking pad.
The following is a further optimization of the above technical solution of the present invention:
the preparation method also comprises the following steps:
s4, preparation of coating film: selecting a nitrocellulose membrane as a coating membrane, respectively diluting the purified NS1 protein and the recombinant streptococcus G protein by using a membrane scribing solution until the concentrations are 1mg/mL, obtaining a purified NS1 protein diluent and a recombinant streptococcus G protein diluent, and using the purified NS1 protein diluent for scribing a detection line; the recombinant streptococcus G protein diluent is used for marking a quality control line; a coated film was obtained by scribing on a nitrocellulose membrane at a concentration of 1. mu.L/cm using a three-dimensional scribing goldput apparatus, and then drying at 37 ℃ for 2 hours.
Further optimization: the preparation method also comprises the following steps:
s5, preparing a sample diluent: the formulation of the sample diluent comprises: 1 percent of sucrose and 0.05 percent of NaN are added into the base liquid in percentage by mass3Tween-20 with the volume percentage of 0.3 percent is also added into the base fluid;
the base solution is PBS solution, the concentration of the PBS solution is 0.01mol/L, and the pH value of the PBS solution is 7.2;
s6, assembling of the diagnosis detection card: sequentially sticking the coating film, the marking pad, the sample pad and the water absorption pad on a PVC (polyvinyl chloride) base plate to prepare a test strip, and putting the test strip into a shell to obtain the avian influenza virus infection and vaccine immunity differential diagnosis test card;
s7, packaging: the assembled avian influenza virus infection and vaccine immunity differential diagnosis detection card and the drying agent are put into an aluminum foil bag for sealing and label pasting, and then the sample diluent pipe is put into an outer packing box.
Further optimization: the formula of the latex microsphere cleaning solution comprises: MES solution with pH value of 6.1 and concentration of 25mmol/L and Tween-20 in 0.05 vol%.
Further optimization: the formula of the latex microsphere preservative fluid is as follows: MES solution with pH value of 6.1 and concentration of 25mmol/L, to which NaN3 with mass percent of 0.05% was added.
Further optimization: the formula of the confining liquid comprises water, wherein 1.0 mass percent of BSA is added into the water;
the formula of the compound solution comprises: the pH value is 6.5, the concentration is 0.01mol/L PBS buffer solution, the PBS buffer solution is added with the following components by mass percent: 10% sucrose, 1% BSA, 0.05% PEG 20000 and 0.05% NaN3Tween-20 was also added to the PBS buffer in an amount of 0.5% by volume.
Further optimization: the formula of the scribing liquid comprises: PBS buffer with pH 7.2 and concentration 0.01 mol/L.
By adopting the technical scheme, the invention has the following beneficial effects:
1. the invention has the characteristics of low cost, higher sensitivity, convenience, rapidness, no need of equipment and instruments and no need of professional personnel.
2. The antibody generated by the avian influenza virus infection and vaccine immunization can be identified and diagnosed, and the method has good specificity and convenience and can well identify avian influenza infected chicken flocks.
3. The avian influenza virus infection and vaccine immunity differential diagnosis detection card provided by the invention adopts the latex microspheres to replace colloidal gold labeled mouse anti-chicken IgG monoclonal antibody for the first time in the preparation process, so that the preparation process repeatability of the avian influenza virus infection and vaccine immunity differential diagnosis detection card is better, the product is more stable, and the adaptability is stronger.
The invention is further illustrated with reference to the following figures and examples.
Drawings
FIG. 1 is a schematic view of the overall structure of embodiment 1 of the present invention;
FIG. 2 is a schematic structural diagram of a diagnostic test card in embodiment 1 of the present invention;
FIG. 3 is a graph showing the determination of a strong positive result when the diagnostic test card is used in example 1 of the present invention;
FIG. 4 is a diagram showing the judgment of a weak positive result of the diagnostic test card in use in example 1 of the present invention;
FIG. 5 is a graph showing the negative result judgment of the diagnostic test card in use in example 1 of the present invention;
FIG. 6 is a graph showing the determination of the invalid result only displayed by the detection lines when the diagnostic test card is in use in embodiment 1 of the present invention;
FIG. 7 is a graph showing the determination of the invalid result of the diagnostic test card in use in embodiment 1 of the present invention;
FIG. 8 shows the results of pH measurements of different labels in example 2 of the present invention;
FIG. 9 shows the results of detection of different amounts of protein markers in example 3 of the present invention;
FIG. 10 shows the results of measurements of different sealant formulations in example 4 of the present invention;
FIG. 11 shows the selection result of the optimal reconstituted solution in example 5 of the present invention;
FIG. 12 shows the results of different recipes for different scrims in example 6 of the present invention;
FIG. 13 shows the results of detection of sensitivity in example 7 of the present invention;
FIG. 14 shows the results of the specificity detection in example 8 of the present invention;
FIG. 15 shows the results of detection of 30 sera from non-immunized avian influenza vaccine with avian influenza in example 9 of the present invention;
FIG. 16 shows the results of 20 sera immunized with avian influenza vaccine in example 9 of the present invention.
In the figure: 1-sample pad; 2-PVC base plate; 3-a marker pad; 4-detection line; 5-quality control line; 6-coating film; 7-absorbent pad; 8-a housing; 81-observation window; 82-addition well.
Detailed Description
Example 1: referring to fig. 1-2, the avian influenza virus infection and vaccine immunodifferential diagnosis test card includes a housing 8 and a sample diluent, wherein the housing 8 is equipped with a test strip.
The test strip comprises a PVC base plate 2, wherein a sample pad 1, a marking pad 3, a coating film 6 and a water absorption pad 7 are sequentially adhered to the PVC base plate 2 from one side of the PVC base plate 2 to the other side.
The coating film 6 is a nitrocellulose film.
In this example, a nitrocellulose membrane model CN140 manufactured by Sartorius corporation was used as the coating film 6.
The coating film 6 is coated with a detection line 4 and a quality control line 5, the detection line 4 is coated at a position close to the marking pad 3, and the quality control line 5 is coated at a position close to the absorbent pad 7.
The detection line 4 is coated with purified avian influenza NS1 protein, and the quality control line 5 is coated with purified recombinant streptococcus G protein.
The marking pad 3 is a glass cellulose membrane and is coated with a coupling marker of a mouse anti-chicken IgG monoclonal antibody and latex microspheres.
In this embodiment, the marking pad 3 is a glass cellulose film model 8964, manufactured by Shanghai Jiening Biotech, Inc., and available directly from the market.
In this example, the avian influenza NS1 protein, recombinant streptococcal G protein, and murine anti-chicken IgG monoclonal antibody were all produced by the "Loyang Bai Otto center for laboratory materials" and are all commercially available.
An observation window 81 is arranged on the shell 8 and close to the coating film 6 of the test strip.
After the strip is assembled in the housing 8, the envelope 6 is positioned at the position of the observation window 81.
The two sides of the observation window 81 on the shell 8 are respectively provided with marks C and T, the mark C corresponds to the quality control line 5, and the mark T corresponds to the detection line 4.
The shell 8 is provided with a sample adding hole 82 at a position close to the sample pad 1 of the test strip.
After the strip is assembled in the housing 8, the sample pad 1 is positioned at the location of the loading aperture 82.
The sample diluent comprises a base solution, wherein 1 percent of sucrose and 0.05 percent of NaN are added into the base solution in percentage by mass3Tween-20 with the volume percentage of 0.3 percent is also added into the base fluid;
the base solution is PBS solution, the concentration of the PBS solution is 0.01mol/L, and the pH value of the PBS solution is 7.2.
The invention also discloses a preparation method of the avian influenza virus infection and vaccine immunity differential diagnosis detection card, which comprises the following steps:
s1, treatment of latex microspheres: adding 0.1 mass percent of latex microspheres into a latex microsphere cleaning solution, performing a centrifugation process at the ambient temperature of 2-8 ℃, centrifuging by using a centrifuge at the centrifugation speed of 13000 r/min for 15 minutes, discarding the supernatant, adding a cleaning solution with the same volume as that of the latex microsphere cleaning solution into the precipitate, and repeatedly cleaning for 1 time.
Adding NHS (N-hydroxysuccinimide) solution with the concentration of 20mg/mL into the suspended latex microsphere solution, and then adding EDC (1-ethyl- (3-dimethylaminopropyl) diimine hydrochloride) solution with the concentration of 20mg/mL, wherein the volume ratio of NHS to the suspended latex microsphere solution is 1: 20; the volume ratio of EDC to the suspended latex microsphere solution was 1: 100.
And then oscillating at room temperature, wherein the oscillation speed is 140-150 r/min, and the oscillation time is 15 minutes.
And after the oscillation is finished, performing a centrifugation process at the ambient temperature of 2-8 ℃, centrifuging by using a centrifuge at the centrifugation speed of 13000 r/min for 15 minutes, discarding the supernatant, and adding the latex microsphere preservation solution with the volume of the original latex microsphere cleaning solution for resuspension after the precipitation is finished.
In step S1, the formula of the latex microsphere cleaning solution includes: MES solution (2- (N-morpholine) ethanesulfonic acid solution) with pH value of 6.1 and concentration of 25mmol/L, Tween-20 with volume percentage of 0.05% is added into the MES solution.
In step S1, the formula of the latex microsphere preservation solution is as follows: MES solution with pH value of 6.1 and concentration of 25mmol/L and NaN in 0.05 wt%3。
In step S1, the average diameter of the latex microspheres is 100 nm.
S2, protein labeling: adding a mouse anti-chicken IgG monoclonal antibody into the treated latex microspheres according to 1/10 of the mass of the latex microspheres, performing primary oscillation at room temperature, wherein the primary oscillation speed is 140-150 r/min, the primary oscillation time is 1 hour, adding a sealing liquid with the volume of 1/10 volume of the original latex microsphere cleaning liquid after the primary oscillation is completed, and then performing secondary oscillation at room temperature, wherein the secondary oscillation speed is 140-150 r/min and the secondary oscillation speed is 30 minutes.
And after the secondary oscillation is finished, performing a centrifugation process at the ambient temperature of 2-8 ℃, centrifuging by using a centrifuge at the centrifugation speed of 13000 r/min for 15 minutes, discarding the supernatant, and precipitating and adding a redissolution with 2 times of volume of the original emulsion microsphere cleaning solution for redissolution.
In the step S2, the mass ratio of the latex microspheres to the mouse-anti-chicken monoclonal antibody for labeling is 10: 1.
In step S2, the formulation of the blocking solution includes water, and BSA (bovine serum albumin) is added to the water in a mass percentage of 1.0%.
In the step S2, the volume ratio of the latex microsphere cleaning solution to the confining liquid is 10: 1.
In step S2, the formula of the reconstituted solution includes: the pH value is 6.5, the concentration is 0.01mol/L PBS buffer solution, the PBS buffer solution is added with the following components by mass percent: 10% sucrose, 1% BSA, 0.05% PEG 20000 and 0.05% NaN3Tween-20 was also added to the PBS buffer in an amount of 0.5% by volume.
In the step S2, the volume ratio of the latex microsphere cleaning solution to the reconstituted solution is 1: 2.
S3, preparation of a marking pad: and (3) paving the protein solution redissolved in the step S2 on the glass cellulose membrane 8964 to obtain the marking pad 3, and then drying the marking pad 3 at the room temperature of 37 ℃ for 2 hours, wherein the marking pad 3 is sealed in a tin foil bag and stored at the normal temperature for later use.
S4, preparation of coating film: selecting a nitrocellulose membrane as a coating membrane 6, and diluting the purified NS1 protein and the recombinant streptococcal G protein with the scribing solution respectively to obtain a purified NS1 protein diluent and a recombinant streptococcal G protein diluent, wherein the concentrations of the purified NS1 protein diluent and the recombinant streptococcal G protein diluent are both 1 mg/mL.
The purified NS1 protein diluent is used for scribing a detection line 4 (T line); the recombinant streptococcus G protein diluent is used for marking a quality control line 5 (a C line); and (3) sequentially scribing the purified NS1 protein diluent and the recombinant streptococcus G protein diluent on a nitrocellulose membrane by using a three-dimensional scribing and metal spraying instrument at the concentration of 1 mu L/cm to obtain a detection line 4 (T line) and a quality control line 5 (C line).
And (3) after the marking is finished, performing a drying process, wherein the drying temperature is 37 ℃, the time is 2 hours, obtaining a coating film 6 after the drying is finished, sealing the coating film 6 in a tin foil bag, and storing at normal temperature for later use.
In the step S4, the model of the three-dimensional film-scribing metal spraying instrument is HM 3030.
In step S4, the formulation of the scribing solution includes: PBS buffer with pH 7.2 and concentration 0.01 mol/L.
The purified NS1 protein diluent is used for marking a detection line 4, so that the detection line 4 is coated with the purified avian influenza NS1 protein.
The recombinant streptococcal G protein diluent is used for marking a quality control line 5, so that the quality control line 5 is coated with purified recombinant streptococcal G protein.
S5, preparing a sample diluent: the formula of the sample diluent comprises base liquid, wherein 1 percent of sucrose and 0.05 percent of NaN are added into the base liquid in percentage by mass3Tween-20 with the volume percentage of 0.3 percent is also added into the base fluid.
The base solution is PBS solution, the concentration of the PBS solution is 0.01mol/L, and the pH value of the PBS solution is 7.2.
After the diluent is prepared, the diluent is filled into a diluent tube.
In this embodiment, the specific formulation of the sample diluent is: 100mL of PBS solution with the pH value of 7.2 and the concentration of 0.01mol/L, 1g of sucrose, 0.3mL of Tween-20 and 0.05g of NaN are added into the PBS solution3。
S6, assembling the test strip: the coating film 6, the marking pad 3, the sample pad 1 and the water absorption pad 7 are sequentially stuck on the PVC base plate 2, then a strip cutting machine is adopted to cut strips to obtain test strips, and the test strips are put into a shell 8 to obtain the avian influenza virus infection and vaccine immunity differential diagnosis test card.
S7, packaging: the assembled avian influenza virus infection and vaccine immunity differential diagnosis detection card and the drying agent are put into an aluminum foil bag for sealing and label pasting, and then the sample diluent pipe is put into an outer packing box.
The invention also discloses a use method of the avian influenza virus infection and vaccine immunity differential diagnosis detection card, which comprises the following specific steps:
(1) collecting serum on chicken body, placing the serum into a disposable dropper, dripping 2-3 drops of serum into a diluent tube by using the disposable dropper, stirring uniformly, and taking the mixed solution for detection.
In the step (1): if the sample can not be detected immediately, the sample should be refrigerated in the dark, and the sample should be refrigerated for more than 24 hours; the sample was allowed to return to room temperature before re-testing.
(2) And (3) recovering the unopened avian influenza virus infection and vaccine immunization differential diagnosis detection card and the detection sample to room temperature.
(3) The avian influenza virus infection and vaccine immunity differential diagnosis detection card is horizontally placed, and 4-5 drops of detection liquid without air bubbles are slowly and dropwise added into the sample adding hole 82 of the avian influenza virus infection and vaccine immunity differential diagnosis detection card by a dropper.
(5) After the sample liquid is added, the red liquid flows out from the edge of the observation window 81 near the sample addition hole 82 and flows in the other direction.
(6) The result is judged in 5-10 minutes, and the result after 10 minutes is only used as reference.
And (5) judging a result: as shown in fig. 3-7, positive: the quality control line 5 shows a red color band, and the detection line 4 also shows a red color band, which is judged to be positive regardless of the color depth.
Negative: the quality control line 5 shows a red color band, and the detection line 4 shows no red color band, and the result is judged to be negative.
And (4) invalidation: the quality control line 5 does not display a red color band, and the avian influenza virus infection and vaccine immunity differential diagnosis detection card is judged to be invalid whether the detection line 4 displays the red color band or not.
Example 2: in example 1 above, determination of the optimal protein labeling pH: in the process of marking the proteins of the latex microspheres, MES with pH values of 7.0, 7.5 and 8.0 is respectively adopted for marking, and the influences of different pH values on the specificity and the sensitivity of the avian influenza virus infection and vaccine immunodiagnosis detection card are compared, so that the pH value of the optimal protein marker is determined, and the experimental structure is shown in the following table and figure 8.
The following table shows the experimental effect of different labeled pH values:
in the above table, "+" indicates positive; "-" indicates negative.
As can be seen from the table and the figure 8, the sensitivity of the detection line of the avian influenza virus infection and vaccine immunodifferential diagnosis detection card is better when the pH value is 7.5, the color development of the detection line is better, the sensitivity is poorer when the pH value is 7.0 and the pH value is 8.0, and false negative appears, so that the pH value of the selective marker is 7.5 according to the experimental result.
Example 3: in example 1 above, the optimal amount of protein marker was chosen: in the process of marking the protein of the latex microsphere, the mouse anti-chicken IgG monoclonal antibodies with the mass ratio of the marked protein to the latex microsphere of 1:15, 1:10 and 1:5 are respectively adopted for marking, and the experimental structure is shown in the following table and figure 9.
The following table shows the experimental effect of different protein labeling amounts:
in the above table, "+" indicates positive; "-" indicates negative.
As can be seen from the table and FIG. 9, when the mass ratio of the marker protein to the latex microspheres is 1:15, the sensitivity is poor, and the color development of the detection line is weak; the sensitivity and specificity are better at 1:10, and the developing effect of the avian influenza virus infection and vaccine immunity differential diagnosis detection card at 1:5 is not obviously changed at 1:10, so that the mass ratio of the selected marker protein to the latex microspheres is 1:10 finally according to the experimental result.
Example 4: in example 1 above, the selection of the optimal sealant formulation: the latex microspheres labeled with protein were blocked with 0.5%, 1.0%, and 1.5% BSA as blocking solutions, and the experimental structures are shown in the following table and FIG. 10.
The following table: the experimental effects of different sealant liquid formulas are as follows:
in the above table, "+" indicates positive; "-" indicates negative.
As can be seen from the above table and FIG. 10, when the final concentration of BSA is 0.5%, the specificity is poor and false positive appears; the sensitivity and specificity are better when the BSA final concentration is 1%; the sensitivity is relatively poor when the BSA final concentration is 1.5%, and the detection line is relatively weak in color development, so that the BSA final concentration is 1.0% according to the experimental result.
Example 5: in example 1 above, selection of the optimal reconstituted solution: different redissolving solutions are adopted for redissolving, the formula of the redissolving solution is shown in the following table, and the optimal formula of the redissolving solution is determined according to the influence of the different formulas of the redissolving solutions on the specificity and sensitivity of the avian influenza virus infection and vaccine immunity differential diagnosis detection card.
The following table shows the formulations of the different complex solutions:
the different re-solution formulas have the effects on the specificity and the sensitivity of the detection card for the avian influenza virus infection and the vaccine immunity differential diagnosis, and the experimental structure is shown in the table and the figure 11.
The following table: experimental results for different double solutions:
in the above table, "+" indicates positive; "-" indicates negative.
As can be seen from the above table and fig. 11, no false positive occurred in any of the 4 groups of reconstituted solutions from the aspect of specificity; from the aspect of sensitivity, the redissolved solution 2 has the highest sensitivity, and the detection line has the best color development, so the redissolved solution 2 is finally selected as the final redissolved solution of the product.
Example 6: preparation of coating film: selecting a nitrocellulose membrane as a coating film 6, respectively diluting purified NS1 protein and recombinant streptococcus G protein with a film scribing solution until the concentrations are both 1mg/mL, respectively scribing a detection line 4 (T line) and a quality control line 5 (C line), sequentially scribing the detection line 4 (T line) and the quality control line 5 (C line) on the nitrocellulose membrane at the concentration of 1 muL/cm by adopting an XYZ three-dimensional film scribing and gold spraying instrument, drying the coating film for 2 hours at 37 ℃ to obtain the coating film 6, sealing the coating film 6 in a tin foil bag, and storing at normal temperature for later use.
(1) Selecting an optimal formula of the film-scribing liquid: adopting different membrane-scribing liquid formulas (shown in a table below), diluting NS1 protein to 1.0mg/mL for detecting the lineation of a line 4 (T line), diluting recombinant streptococcus G protein to 1.0mg/mL for detecting a quality control line (C line), and determining the optimal membrane-scribing liquid formula according to the influence of different membrane-scribing liquid formulas on the detection specificity and sensitivity.
The following table shows the formulations of 3 different scribes:
the effect of different formulation of the streaking solutions on the specificity and sensitivity of the assay, the experimental structure is shown in the following table and FIG. 12.
The following table: experimental results for different streaking solutions:
in the above table: "+" indicates positive; "-" indicates negative.
As can be seen from the table and FIG. 12, the sensitivity and specificity of the PBS buffer system in the 3 buffer systems to be selected are better than those of the PBST buffer system and the TRIS buffer system, wherein the sensitivity of the TRIS buffer system is poorer, and the color development of the detection line is weaker; the PBST buffer system has poor specificity and false positive appears, so PBS (0.01 mol/L) with the pH value of 7.2 is finally selected as the optimal solution formula of the scribing solution.
Example 7: and (3) sensitivity detection:
sequentially carrying out the following steps on the avian influenza virus serum sample: 1:2, 1:4 and 1:8.. 1:512 times, and then the avian influenza virus infection and vaccine immunity differential diagnosis detection card is adopted for detection, and the detection structure is shown in the following table and figure 13.
The following table: results of sensitivity measurement:
in the above table: "+" indicates positive avian influenza virus antibody and "-" indicates negative avian influenza virus antibody.
As can be seen from the above table and FIG. 13, the serum samples were all positive at 1: 128-fold dilution.
Example 8: and (3) specificity test:
the avian influenza virus infection and Vaccine immunity differential diagnosis detection card is used for detecting chicken serum (H9 Vaccine) of immune avian influenza, infectious bronchitis virus positive serum (IBV), infectious bursal disease virus positive serum (IBDV), newcastle disease virus positive serum (NDV) and escherichia coli culture solution (E.coli), and the detection structure is shown in the following table and figure 14.
The following table: and (3) specific detection results:
in the above table: "-" indicates that the avian influenza virus antibody was negative.
As can be seen from the above table and fig. 14, the avian influenza virus infection and vaccine immunization differential diagnosis detection card pair of the present invention: the detection structures of H9 Vaccine, IBV, IBDV, NDV and E.coli are all negative, so the avian influenza virus infection and Vaccine immunity differential diagnosis detection card can meet the requirement of specificity.
Example 9: batch test:
30 parts of serum (serial numbers: S1-S30) collected from chicken farms which have not been immunized with avian influenza vaccines and have avian influenza and 20 parts of serum (serial numbers: V1-V20) collected from chicken farms which have been immunized with avian influenza vaccines are detected by adopting the avian influenza virus infection and vaccine immunization differential diagnosis detection card, and the detection structure is shown in the following two tables and figures 15-16.
The following table: the detection results of 30 parts of serum collected from a chicken farm which has not been immunized with avian influenza vaccine and has avian influenza:
in the above table: + "indicates positive avian influenza antibody.
The following table: the detection results of 20 parts of serum collected from the chicken farm immunized with the avian influenza vaccine are as follows:
in the above table: "+" indicates positive avian influenza antibody and "-" indicates negative avian influenza antibody.
As can be seen from the above table and fig. 14, the detection results of 30 sera collected from chicken farms that had not been immunized with avian influenza vaccine and had developed avian influenza were all positive, i.e., the positive detection rate was 100% (30/30), and the positive detection rate of 20 sera collected from chicken farms that had been immunized with avian influenza vaccine was 20% (4/20).
It will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in the embodiments described above without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims.
Claims (10)
1. The utility model provides an avian influenza virus infects and bacterin immunity differential diagnosis detects card, includes shell (8) and the supporting sample diluent that uses, is equipped with the test paper strip in shell (8), and the test paper strip includes PVC bottom plate (2), and it has sample pad (1), mark pad (3), envelope membrane (6) and absorbent pad (7) to paste on PVC bottom plate (2), its characterized in that: the marking pad (3) is a glass cellulose membrane and is coated with a coupling marker of a mouse anti-chicken IgG monoclonal antibody and latex microspheres; the coating film (6) is a nitrocellulose film, and a detection line (4) coated with the purified avian influenza NS1 protein and a quality control line (5) coated with the purified recombinant streptococcus G protein are coated on the coating film (6).
2. The differential diagnosis detection card for avian influenza virus infection and vaccine immunization according to claim 1, which is characterized in that: the sample diluent comprises a base solution, wherein 1 percent of sucrose and 0.05 percent of NaN are added into the base solution in percentage by mass3Tween-20 with the volume percentage of 0.3 percent is also added into the base fluid;
the base solution was PBS solution, the concentration of PBS solution was 0.01mol/L, and the pH of PBS solution was 7.2.
3. The differential diagnosis detection card for avian influenza virus infection and vaccine immunization according to claim 2, which is characterized in that: an observation window (81) and a sample adding hole (82) are formed in the shell (8), after the test strip is assembled in the shell (8), the coating film (6) is located at the position of the observation window (81), the sample pad (1) is located at the position of the sample adding hole (82), marks C and T are arranged on the shell (8), the mark C corresponds to the quality control line (5), and the mark T corresponds to the detection line (4).
4. The method for preparing the avian influenza virus infection and vaccine immunization differential diagnosis detection card as claimed in any one of claims 1 to 3, which is characterized in that: the method comprises the following steps:
s1, treatment of latex microspheres: adding 0.1% by mass of latex microspheres into a latex microsphere cleaning solution, centrifuging for 15 minutes at the temperature of 2-8 ℃, wherein the centrifugation speed is 13000 r/min, discarding the supernatant, adding a cleaning solution with the same volume as that of the latex microsphere cleaning solution into the precipitate for resuspension, and repeating the cleaning for 1 time, wherein NHS with the concentration of 20mg/mL is firstly added into the suspended latex microsphere solution, EDC with the concentration of 20mg/mL is then added, and the volume ratio of NHS to the suspended latex microsphere solution is 1: 20; the volume ratio of EDC to the suspended latex microsphere solution is 1: 100; then oscillating for 15 minutes at an oscillation speed of 140-150 r/min, centrifuging for 15 minutes at the temperature of 2-8 ℃ after oscillation is finished, wherein the centrifugation speed is 13000 r/min, discarding supernatant, adding latex microsphere preservative fluid with the volume of the original latex microsphere cleaning fluid for resuspension after precipitation is finished, and the average diameter of the latex microspheres is 100 nm;
s2, protein labeling: adding a mouse anti-chicken IgG monoclonal antibody into the treated latex microspheres according to 1/10 of the mass of the latex microspheres, oscillating for 1 hour at room temperature at the oscillation speed of 140-150 r/min, adding sealing liquid with the volume of 1/10 volume of original latex microsphere cleaning liquid after oscillation is finished, and oscillating for 30 minutes at room temperature at the oscillation speed of 140-150 r/min; after the oscillation is finished, centrifuging for 15 minutes at the temperature of 2-8 ℃, wherein the centrifugation speed is 13000 r/min, discarding the supernatant, precipitating, and adding a redissolution with the volume 2 times that of the original emulsion microsphere cleaning solution for redissolving;
s3, preparation of a marking pad: and (3) paving the protein solution redissolved in the step S2 on a glass cellulose membrane, and drying at the room temperature of 37 ℃ for 2 hours to obtain the marking pad (3).
5. The method for preparing the avian influenza virus infection and vaccine immunity differential diagnosis detection card according to claim 4, is characterized in that: the preparation method also comprises the following steps:
s4, preparation of coating film: selecting a nitrocellulose membrane as a coating membrane (6), respectively diluting the purified NS1 protein and the recombinant streptococcal G protein by using a membrane scribing solution until the concentrations are 1mg/mL to obtain a purified NS1 protein diluent and a recombinant streptococcal G protein diluent, wherein the purified NS1 protein diluent is used for scribing a detection line (4); the recombinant streptococcus G protein diluent is used for marking a quality control line (5); the coated film (6) was obtained by streaking on a nitrocellulose membrane at a concentration of 1. mu.L/cm using a three-dimensional streaking gold spraying instrument, followed by drying at 37 ℃ for 2 hours.
6. The method for preparing the avian influenza virus infection and vaccine immunity differential diagnosis detection card according to claim 5, is characterized in that: the preparation method also comprises the following steps:
s5, preparing a sample diluent: the formula of the sample diluent comprises base liquid, wherein 1 percent of sucrose and 0.05 percent of NaN are added into the base liquid in percentage by mass3Tween-20 with the volume percentage of 0.3 percent is also added into the base fluid;
the base solution is PBS solution, the concentration of the PBS solution is 0.01mol/L, and the pH value of the PBS solution is 7.2;
s6, assembling of the diagnosis detection card: sequentially sticking the coating film (6), the marking pad (3), the sample pad (1) and the water absorption pad (7) on the PVC base plate (2) to prepare a test strip, and putting the test strip into a shell (8) to obtain the avian influenza virus infection and vaccine immunity differential diagnosis test card;
s7, packaging: the assembled avian influenza virus infection and vaccine immunity differential diagnosis detection card and the drying agent are put into an aluminum foil bag for sealing and label pasting, and then the sample diluent pipe is put into an outer packing box.
7. The method for preparing the avian influenza virus infection and vaccine immunity differential diagnosis detection card according to claim 6, which is characterized in that: the formula of the latex microsphere cleaning solution comprises: MES solution with pH value of 6.1 and concentration of 25mmol/L and Tween-20 in 0.05 vol%.
8. The method for preparing the avian influenza virus infection and vaccine immunity differential diagnosis detection card according to claim 7, is characterized in that: the formula of the latex microsphere preservative fluid is as follows: MES solution with pH of 6.1 and concentration of 25mmol/LAdding 0.05 percent of NaN in percentage by mass into the solution3。
9. The method for preparing the avian influenza virus infection and vaccine immunity differential diagnosis detection card according to claim 8, which is characterized in that: the formula of the confining liquid comprises water, wherein 1.0 mass percent of BSA is added into the water;
the formula of the compound solution comprises: the pH value is 6.5, the concentration is 0.01mol/L PBS buffer solution, the PBS buffer solution is added with the following components by mass percent: 10% sucrose, 1% BSA, 0.05% PEG 20000 and 0.05% NaN3Tween-20 was also added to the PBS buffer in an amount of 0.5% by volume.
10. The method for preparing the avian influenza virus infection and vaccine immunity differential diagnosis detection card according to claim 9, is characterized in that: the formula of the scribing liquid comprises: PBS buffer with pH 7.2 and concentration 0.01 mol/L.
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