Kit and detection method for detecting vomitoxin

Abstract

The present invention belongs to the technical field of mycotoxin detection, and particularly relates to a kit and a detection method for detecting vomitoxin. The kit includes an antigen, a primary antibody, an enzyme-labeled secondary antibody, a chromogenic regulator, a phosphate buffer solution, and a detection reagent; the antigen is bovine serum albumin conjugated with vomitoxin; the primary antibody is a monoclonal antibody against vomitoxin; the enzyme-labeled secondary antibody is a goat anti-mouse IgG secondary antibody labeled with horseradish peroxidase; the chromogenic regulator is ethylenediaminetetraacetic acid or its sodium salt, or vitamin C; the detection reagent includes a TMB chromogenic solution. The present invention uses ethylenediaminetetraacetic acid or its sodium salt as a chromogenic regulator to change the chemical microenvironment around the chromogenic substance, enhance the optical properties of the chromogenic substance, and thereby reduce IC 50 , and improve the detection sensitivity.

Description

Technical Field

[0001] The present invention belongs to the technical field of mycotoxin detection, and particularly relates to a kit and a detection method for detecting vomitoxin. Background Art

[0002] Vomitoxin is a toxic secondary metabolite produced by fungi of the genus Fusarium, and its chemical structural formula is as Figure 1 shown, named deoxynivalenol, with the English name 4-Deoxynivalenol and the English abbreviation DON. Prior art research has found that vomitoxin is difficult to destroy its toxicity during processing. Therefore, it has certain harmful effects on humans and animals. After being poisoned by vomitoxin, symptoms such as diarrhea, vomiting, loss of appetite, slow reaction, gastroenteritis, immunosuppression, and blood diseases will occur. Therefore, in order to ensure food safety, the content of vomitoxin in food is strictly restricted.

[0003] The prior art has established an enzyme-linked immunosorbent assay for detecting deoxynivalenol and detected actual samples, and used the indirect competitive ELISA method to determine DON. However, the lower the IC 50 of the indirect competitive ELISA method, the higher the sensitivity. The prior art lacks an indirect competitive ELISA method for reducing IC 50 . Summary of the Invention

[0004] In order to solve the above technical problems, the present invention provides a kit and a detection method for detecting vomitoxin, which are used to reduce the IC 50 of the indirect competitive ELISA method.

[0005] The purpose of the present invention is to provide a kit for detecting vomitoxin, and the kit includes an antigen, a primary antibody, an enzyme-labeled secondary antibody, a color development regulator, a phosphate buffer solution, and a detection reagent;

[0006] The antigen is bovine serum albumin conjugated with vomitoxin;

[0007] The primary antibody is a monoclonal antibody against vomitoxin;

[0008] The enzyme-labeled secondary antibody is a horseradish peroxidase-labeled goat anti-mouse IgG secondary antibody;

[0009] The color development regulator is ethylenediaminetetraacetic acid or its sodium salt, or vitamin C;

[0010] The detection reagent includes a TMB color development solution.

[0011] The present invention uses an indirect competitive ELISA method to detect vomitoxin. Antigen is coated on the microplate wells. The vomitoxin in the sample to be detected and the pre-coated antigen on the wells compete for the vomitoxin antibody. After adding the enzyme-labeled secondary antibody, TMB is used for color development, and then the content of vomitoxin is calculated according to the standard curve. The lower the IC 50 of the indirect competitive ELISA method, the higher the sensitivity. The IC 50 is the key to determining the detection sensitivity. Since the IC 50 is calculated based on the standard curve established from the absorbance values after color development, the response of the absorbance values also determines the detection sensitivity. The present invention uses ethylenediaminetetraacetic acid or its sodium salt or vitamin C as a color development regulator to change the chemical microenvironment around the color-developing substance, enhance the optical properties of the color-developing substance, and then adjust the color development result to reduce the IC 50 .

[0012] Preferably, the sodium salt of ethylenediaminetetraacetic acid is sodium ethylenediaminetetraacetate.

[0013] Preferably, for the kit for detecting vomitoxin, the phosphate buffer solution is a 0.01 mol / L phosphate buffer solution with the pH adjusted to 7.4.

[0014] The color development regulator is dissolved in the phosphate buffer solution to form a color development regulator solution, which is then used to detect vomitoxin. The concentration of the color development regulator solution is 0.5 g / L to 1 g / L. Preferably, the concentration of the color development regulator is 0.5 g / L to 0.7 g / L. The concentration of the color development regulator cannot be too high, otherwise it will have the side effect of interfering with the absorbance and instead reduce the detection sensitivity. The concentration of the color development regulator cannot be too low either, otherwise the IC 50 cannot be reduced.

[0015] The detection reagent includes a coating solution, a blocking solution, a washing solution, and a termination solution for performing an enzyme-linked immunosorbent assay.

[0016] Preferably, the coating solution is a carbonate buffer solution, and the termination solution is an H2SO4 solution.

[0017] The color development regulator is ethylenediaminetetraacetic acid.

[0018] The present invention provides a method for detecting vomitoxin using the above kit, including:

[0019] Preparing a reaction solution using an antigen, a primary antibody, an enzyme-labeled secondary antibody, a sample to be detected, and a detection reagent. A color development regulator solution is added to the reaction solution to adjust the absorbance value, and the absorbance value is detected;

[0020] Detecting the absorbance value of the vomitoxin standard under the same operating conditions as above, and plotting a standard curve;

[0021] Quantitatively analyze the content of vomitoxin in the sample to be detected by using a standard curve.

[0022] Preferably, the method is an enzyme-linked immunosorbent assay (ELISA), including:

[0023] Take an enzyme-labeled plate coated with an antigen, block it, add a primary antibody and the sample to be detected, incubate in a light-proof environment at 37 °C, wash the plate, add an enzyme-labeled secondary antibody, incubate in a light-proof environment at 37 °C, wash the plate, add a chromogenic solution and a chromogenic regulator solution, mix well, incubate in a light-proof environment at 37 °C for 15 min to 20 min; add a stop solution, mix well, and detect the absorbance value of each well at 450 nm with an enzyme-labeled instrument; quantitatively analyze the content of vomitoxin in the sample to be detected by using a standard curve.

[0024] Preferably, the volume ratio of the chromogenic solution to the chromogenic regulator solution is 4:1.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] The present invention uses ethylenediaminetetraacetic acid or its sodium salt as a chromogenic regulator to change the chemical microenvironment around the chromogenic substance, enhance the optical properties of the chromogenic substance, and thus reduce IC 50 , and improve the detection sensitivity. Description of the Drawings

[0027] Figure 1 is the structural formula of vomitoxin.

[0028] Figure 2 is the standard curve of Example 1 and the blank control.

[0029] Figure 3 is the standard curve of Example 2.

[0030] Figure 4 is the standard curve of Example 3.

[0031] Figure 5 is the standard curve of Example 4.

[0032] Figure 6 is the standard curve of Example 5.

[0033] Figure 7 is the standard curve of Example 6.

[0034] Figure 8 is the standard curve of Example 7. Detailed Embodiments

[0035] In order to enable those skilled in the art to better understand and implement the technical solutions of the present invention, the present invention will be further described below with reference to specific embodiments and drawings.

[0036] In the description of the present invention, unless otherwise specified, the reagents used are commercially available, and the methods used are conventional techniques in the art.

[0037] In the experiments of the present invention, the preparation methods of the main drugs are shown in Table 1.

[0038] Table 1 Preparation of main drugs for ELISA

[0039] The present invention provides a kit for detecting vomitoxin, which includes an antigen, a primary antibody, an enzyme-labeled secondary antibody, a color development regulator, a phosphate buffer solution, and a detection reagent;

[0040] The antigen is bovine serum albumin conjugated with vomitoxin;

[0041] The primary antibody is a monoclonal antibody against vomitoxin;

[0042] The enzyme-labeled secondary antibody is a goat anti-mouse IgG secondary antibody labeled with horseradish peroxidase;

[0043] The color development regulator is ethylenediaminetetraacetic acid or its sodium salt, or vitamin C;

[0044] The detection reagent includes a TMB color development solution.

[0045] The method for detecting vomitoxin includes the following examples. The information of CBS, antigen, primary antibody, enzyme-labeled secondary antibody, PBS, TMB color development solution, sample dilution solution, blocking solution, PBST, and termination solution described in the following examples is shown in Table 1.

[0046] Example 1

[0047] The indirect competitive ELISA method is used to detect vomitoxin, and the detection steps include:

[0048] (1) Sample treatment

[0049] The corn kernels are ground into powder. Weigh 2 g of the powder and dissolve it in 10 mL of a 50% methanol aqueous solution by volume. Mix it evenly in a vortex mixer, and then place it in a high-speed centrifuge at 4000 r / min and centrifuge at 4 °C for 10 min. Take the supernatant as the sample to be detected.

[0050] The sample to be detected is diluted with the sample dilution solution to obtain diluted samples to be detected at different multiples. When the concentration of vomitoxin in the sample to be detected is too high and exceeds the concentration range set by the standard curve, it is necessary to prepare a diluted sample to be detected and re-measure until the detected concentration range of vomitoxin is within the concentration range set by the standard curve. Making the concentration of the target substance in the sample to be detected meet the set value of the standard curve by dilution belongs to the common knowledge in the art.

[0051] (2) Coating and blocking

[0052] Dilute the antigen with CBS to 0.12 mg / mL to obtain the antigen dilution, and set it aside. Add the antigen dilution to a 96-well ELISA plate, 100 μL per well, place it in a refrigerator at 4 °C for 12 h, wash it 3 times with PBST, 3 min each time, and then pat dry the ELISA plate. Add 100 μL of blocking solution to each well to block, place it in an incubator at 37 °C for 1 h, take it out, wash the ELISA plate 3 times with PBST, and pat dry each time. Vacuum dry at -20 °C for 3 h. Coating and blocking are completed.

[0053] (3) Add the test sample dilution or vomitoxin standard to each well, 50 μL per well; add the primary antibody in Table 1, 50 μL per well; place it in an incubator at 37 °C for 1 h, then take it out, rinse it 3 times with PBST, and pat dry. Among them, the concentration of the primary antibody is 1.05 μg / mL.

[0054] Add the enzyme-labeled secondary antibody, 100 μL per well, incubate in an incubator at 37 °C at room temperature for 1 h; wash the plate 3 times with PBST, and pat dry.

[0055] (4) Add 80 μL of freshly prepared TMB chromogenic solution mixed in equal volume at 1:1 to each well, and add 20 μL of chromogenic regulator solution to each well, mix well, wrap it with aluminum foil and place it in an incubator at 37 °C for 10 min for color development.

[0056] Add 50 μL of stop solution per well, mix well, set the microplate reader to detect at 450 nm, and measure the absorbance value of each well; quantitatively analyze the content of vomitoxin in the test sample using the standard curve.

[0057] Among them, the chromogenic regulator is dissolved in the phosphate buffer solution in Table 1 to form a chromogenic regulator solution, and then it is used to detect vomitoxin. The concentration of the chromogenic regulator solution is 0.5 g / L.

[0058] Among them, when making the standard curve, prepare vomitoxin standard solutions with different concentrations. Set the microplate reader to detect at 450 nm and measure the absorbance value.

[0059] Among them, the detection method for the blank control is as follows:

[0060] (1) Sample treatment

[0061] Crush the corn kernels into powder, weigh 2 g of the powder and dissolve it in 10 mL of 50% methanol aqueous solution by volume fraction, mix well in a vortex mixer, place it in a high-speed centrifuge at 4000 r / min, centrifuge at 4 °C for 10 min, take the supernatant as the test sample.

[0062] (2) Coating and blocking

[0063] Dilute the antigen with CBS to 0.12 mg / mL to obtain an antigen dilution solution for standby. Add the antigen dilution solution to a 96-well enzyme-linked immunosorbent assay (ELISA) plate, 100 μL per well, and place it in a refrigerator at 4 °C for 12 h. Wash it 3 times with PBST, 3 min each time, and then pat the ELISA plate dry. Add 100 μL of blocking solution to each well for blocking, and incubate it in an incubator at 37 °C for 1 h. Take it out, wash the ELISA plate 3 times with PBST, and pat it dry each time. Vacuum dry it at -20 °C for 3 h. Coating and blocking are completed.

[0064] (3) Add the sample dilution solution to be tested or the vomitoxin standard to each well, 50 μL per well; add the primary antibody in Table 1, 50 μL per well; after incubating it in an incubator at 37 °C for 1 h, take it out and rinse it 3 times with PBST, and pat it dry. Among them, the concentration of the primary antibody is 1.05 μg / mL.

[0065] Add the enzyme-labeled secondary antibody, 100 μL per well, and incubate it in an incubator at 37 °C for 1 h at room temperature; wash the plate 3 times with PBST and pat it dry.

[0066] (4) Add 100 μL of freshly prepared TMB chromogenic solution mixed in equal volume at a ratio of 1:1 to each well, mix well, wrap it with aluminum foil, and place it in an incubator at 37 °C for 10 min for color development.

[0067] Add 50 μL of stop solution to each well, mix well, set the microplate reader to detect at 450 nm, and measure the absorbance value of each well; quantitatively analyze the content of vomitoxin in the sample to be tested using the standard curve.

[0068] The difference between the blank control and Example 1 is that the chromogenic regulator solution is not added. Since the difference between different examples is the chromogenic regulator solution, the same blank control and standard curve are used for all examples.

[0069] With logC DON (the logarithm of the vomitoxin concentration) as the abscissa and the ratio of the absorbance value B of the well with the vomitoxin standard to the absorbance value B0 of the well without the vomitoxin standard as the ordinate, as Figure 2 shown.

[0070] IC 50 refers to the concentration of vomitoxin in the sample to be tested corresponding to B / B0 = 50%, and the lower the IC 50 , the higher the sensitivity of the detection method.

[0071] The half-maximal inhibitory concentration IC 50 of the method in this example is 1.79 pg / mL, which is lower than the IC 50It was significantly reduced to 166.38 pg / mL, indicating that the detection accuracy of the method of the present invention is high. To verify the stability of the detection method, a spike recovery test was carried out, independently repeated 15 times, and the standard deviation was 1.1%.

[0072] Table 2 Spike Recovery Test

[0073]

[0074] Example 2

[0075] The indirect competitive ELISA method was used to detect deoxynivalenol, and the detection steps included:

[0076] (1) Sample treatment

[0077] The corn kernels were ground into powder, 2 g of the powder was weighed and dissolved in 10 mL of a 50% methanol aqueous solution by volume. After mixing evenly in a vortex mixer, it was placed in a high-speed centrifuge at 4000 r / min and centrifuged at 4 °C for 10 min. The supernatant was taken as the sample to be tested.

[0078] The sample to be tested was diluted with the sample diluent to obtain the sample diluents to be tested with different multiples. When the concentration of deoxynivalenol in the sample to be tested was too high and exceeded the concentration setting range of the standard curve, it was necessary to prepare the sample diluent to be tested and re-determine until the concentration range of the detected deoxynivalenol was within the concentration setting range of the standard curve. Making the concentration of the target substance in the sample to be tested meet the set value of the standard curve by dilution belongs to the common general knowledge in the art.

[0079] (2) Coating and blocking

[0080] The antigen was diluted to 0.12 mg / mL with CBS to obtain the antigen diluent for standby. The antigen diluent was added to a 96-well enzyme-linked immunosorbent assay (ELISA) plate, 100 μL per well, and placed in a refrigerator at 4 °C for 12 h. It was washed 3 times with PBST, 3 min each time, and then the ELISA plate was patted dry. 100 μL of the blocking solution was added to each well for blocking, and it was incubated in an incubator at 37 °C for 1 h. After taking it out, the ELISA plate was washed 3 times with PBST, and patted dry each time. It was vacuum dried at -20 °C for 3 h. Coating and blocking were completed.

[0081] (3) Add 50 μL of the sample diluent to be tested or the deoxynivalenol standard product to each well; add 50 μL of the primary antibody in Table 1 to each well; after incubating in an incubator at 37 °C for 1 h, take it out and wash it 3 times with PBST and pat it dry. The concentration of the primary antibody was 1.05 μg / mL.

[0082] Add 100 μL of the enzyme-labeled secondary antibody per well and incubate it in an incubator at 37 °C for 1 h at room temperature; wash the plate 3 times with PBST and pat it dry.

[0083] (4) Add 80 μL of freshly prepared TMB chromogenic solution mixed in equal volume at 1:1 to each well, and add 20 μL of chromogenic regulator solution to each well. Mix well, wrap with aluminum foil and place in an incubator at 37 °C for 10 min for color development.

[0084] Add 50 μL / well of stop solution, mix well, set the microplate reader to detect at 450 nm, and measure the absorbance value of each well; use the standard curve to quantitatively analyze the content of vomitoxin in the sample to be tested.

[0085] Among them, the chromogenic regulator is dissolved in phosphate buffer to form a chromogenic regulator solution, which is then used to detect vomitoxin. The concentration of the chromogenic regulator solution is 0.6 g / L.

[0086] Among them, when making the standard curve, prepare standard solutions of vomitoxin at different concentrations. Set the microplate reader to detect at 450 nm and measure the absorbance value.

[0087] Take logC DON (the logarithm of the vomitoxin concentration) as the abscissa, and the ratio of the absorbance value B of the well with the addition of the vomitoxin standard to the absorbance value B0 of the well without the addition of the vomitoxin standard as the ordinate, as Figure 3 shown.

[0088] IC 50 refers to the concentration of vomitoxin in the sample to be tested corresponding to B / B0 = 50%. The lower the IC 50 , the higher the sensitivity of the detection method.

[0089] The half inhibitory concentration IC 50 of the method in this example is 1.59 pg / mL, which is significantly lower than the blank control IC 50 without the chromogenic regulator being 166.38 ng / mL, indicating that the detection limit of the method of the present invention is low.

[0090] Example 3

[0091] The indirect competitive ELISA method is used to detect vomitoxin, and the detection steps include:

[0092] (1) Sample treatment

[0093] The corn kernels are ground into powder, weigh 2 g of the powder and dissolve it in 10 mL of 50% methanol aqueous solution by volume. Mix well in a vortex mixer, place it in a high-speed centrifuge at 4000 r / min, centrifuge at 4 °C for 10 min, and take the supernatant as the sample to be tested.

[0094] The test sample is diluted with a sample diluent to obtain test sample diluents with different dilution multiples. When the concentration of vomitoxin in the test sample is too high and exceeds the concentration setting range of the standard curve, it is necessary to prepare a test sample diluent and re-measure until the detected concentration range of vomitoxin is within the concentration setting range of the standard curve. Making the concentration of the target substance in the test sample meet the set value of the standard curve by dilution belongs to the common general knowledge in the art.

[0095] (2) Coating and blocking

[0096] The antigen is diluted to 0.12 mg / mL with CBS to obtain an antigen diluent for standby. Add the antigen diluent to a 96-well enzyme-linked immunosorbent assay (ELISA) plate, 100 μL per well, and place it in a refrigerator at 4°C for 12 h. Wash it 3 times with PBST, 3 min each time, and then pat the ELISA plate dry. Add 100 μL of blocking solution to each well for blocking, place it in an incubator at 37°C for 1 h, take it out, wash the ELISA plate 3 times with PBST, and pat it dry each time. Vacuum dry it at -20°C for 3 h. Coating and blocking are completed.

[0097] (3) Add the test sample diluent or the vomitoxin standard to each well, 50 μL per well; add the primary antibody in Table 1, 50 μL per well; after incubating in an incubator at 37°C for 1 h, take it out, rinse it 3 times with PBST, and pat it dry. The concentration of the primary antibody is 1.05 μg / mL.

[0098] Add the enzyme-labeled secondary antibody, 100 μL per well, and incubate it in an incubator at 37°C for 1 h at room temperature; wash the plate 3 times with PBST and pat it dry.

[0099] (4) Add 80 μL of freshly prepared TMB chromogenic solution mixed in equal volume at a ratio of 1:1 to each well, add 20 μL of chromogenic regulator solution to each well, mix well, wrap it with aluminum foil, and place it in an incubator at 37°C for 10 min for color development.

[0100] Add 50 μL of stop solution to each well, mix well, set the microplate reader to detect at 450 nm, and measure the absorbance value of each well; quantitatively analyze the content of vomitoxin in the test sample using the standard curve.

[0101] Among them, the chromogenic regulator is dissolved in phosphate buffer to form a chromogenic regulator solution, which is then used to detect vomitoxin, and the concentration of the chromogenic regulator solution is 0.7 g / L.

[0102] Among them, when preparing the standard curve, prepare vomitoxin standard solutions with different concentrations. Set the microplate reader to detect at 450 nm and measure the absorbance value.

[0103] With logC DON(Logarithm of vomitoxin concentration) is used as the abscissa, and the ratio of the absorbance value B of the vomitoxin standard added to the absorbance value B0 of the vomitoxin standard not added is used as the ordinate, as Figure 4 shown.

[0104] IC 50 refers to the concentration of vomitoxin in the test sample corresponding to B / B0 = 50%, and the lower the IC 50 , the higher the sensitivity of the detection method.

[0105] The half inhibitory concentration IC 50 of the method in this example is 2.44 pg / mL, which is significantly lower than the blank control IC 50 of 166.38 ng / mL without the chromogenic regulator.

[0106] Example 4

[0107] Detecting vomitoxin by indirect competitive ELISA method, the detection steps include:

[0108] (1) Sample treatment

[0109] The corn kernels are ground into powder, 2 g of the powder is weighed and dissolved in 10 mL of 50% methanol aqueous solution by volume. After mixing evenly in a vortex mixer, it is placed in a high-speed centrifuge at 4000 r / min and centrifuged at 4°C for 10 min. The supernatant is taken as the test sample.

[0110] The test sample is diluted with the sample diluent to obtain test sample diluents with different multiples. When the concentration of vomitoxin in the test sample is too high and exceeds the concentration setting range of the standard curve, it is necessary to prepare a test sample diluent and re-measure until the detected concentration range of vomitoxin is within the concentration setting range of the standard curve. Making the concentration of the target substance in the test sample meet the standard curve setting value by dilution belongs to the common knowledge in the art.

[0111] (2) Coating and blocking

[0112] The antigen is diluted to 0.12 mg / mL with CBS to obtain an antigen diluent for standby. Add the antigen diluent to a 96-well enzyme-linked immunosorbent assay (ELISA) plate, 100 μL per well, and place it in a refrigerator at 4°C for 12 h. Wash it 3 times with PBST, 3 min each time, and then pat the ELISA plate dry. Add 100 μL of blocking solution to each well to block, place it in an incubator at 37°C for 1 h, take it out, wash the ELISA plate 3 times with PBST, and pat it dry each time. Vacuum dry at -20°C for 3 h. Coating and blocking are completed.

[0113] (3) Add the sample dilution to be tested or the vomitoxin standard to each well, 50 μL per well; add the primary antibody in Table 1, 50 μL per well; incubate in an incubator at 37 °C for 1 h, then take it out, rinse it 3 times with PBST, and pat it dry. The concentration of the primary antibody is 1.05 μg / mL.

[0114] Add the enzyme-labeled secondary antibody, 100 μL per well, and incubate in an incubator at 37 °C at room temperature for 1 h; wash the plate 3 times with PBST and pat it dry.

[0115] (4) Add 80 μL of freshly prepared TMB chromogenic solution mixed in equal volume at a ratio of 1:1 to each well, and add 20 μL of chromogenic regulator solution to each well, mix well, wrap it with aluminum foil and place it in an incubator at 37 °C for 10 min for color development.

[0116] Add 50 μL of stop solution to each well, mix well, set the microplate reader to detect at 450 nm, and measure the absorbance value of each well; use the standard curve to quantitatively analyze the content of vomitoxin in the sample to be tested.

[0117] Among them, the chromogenic regulator is dissolved in phosphate buffer to form a chromogenic regulator solution, and then used to detect vomitoxin. The concentration of the chromogenic regulator solution is 0.8 g / L.

[0118] Among them, when making the standard curve, prepare vomitoxin standard solutions with different concentrations. Set the microplate reader to detect at 450 nm and measure the absorbance value.

[0119] Take logC DON (the logarithm of the vomitoxin concentration) as the abscissa, and the ratio of the absorbance value B of the well with the vomitoxin standard added to the absorbance value B0 of the well without the vomitoxin standard added as the ordinate, as Figure 5 shown.

[0120] IC 50 refers to the concentration of vomitoxin in the sample to be tested corresponding to B / B0 = 50%, and the lower the IC 50 , the higher the sensitivity of the detection method.

[0121] The half-inhibitory concentration IC 50 of the method in this example is 8.47 pg / mL, which is significantly lower than the blank control IC 50 without the chromogenic regulator being 166.38 ng / mL, indicating that the detection limit of the method of the present invention is low.

[0122] Example 5

[0123] Detect vomitoxin by the indirect competitive ELISA method. The detection steps include:

[0124] (1) Sample treatment

[0125] The corn kernels are ground into powder. Weigh 2 g of the powder and dissolve it in 10 mL of a 50% methanol aqueous solution by volume. Mix well in a vortex mixer, then place it in a high-speed centrifuge at 4000 r / min and centrifuge at 4 °C for 10 min. Take the supernatant as the sample to be tested.

[0126] The sample to be tested is diluted with the sample diluent to obtain diluted samples to be tested at different multiples. When the concentration of vomitoxin in the sample to be tested is too high and exceeds the concentration setting range of the standard curve, it is necessary to prepare a diluted sample to be tested and re-measure until the concentration range of the detected vomitoxin is within the concentration setting range of the standard curve. Making the concentration of the target substance in the sample to be tested meet the set value of the standard curve by dilution is common knowledge in the art.

[0127] (2) Coating and blocking

[0128] The antigen is diluted to 0.12 mg / mL with CBS to obtain an antigen diluent for standby. Add the antigen diluent to a 96-well enzyme-linked immunosorbent assay (ELISA) plate, 100 μL per well, and place it in a refrigerator at 4 °C for 12 h. Wash it 3 times with PBST, 3 min each time, and then pat the ELISA plate dry. Add 100 μL of the blocking solution to each well to block, place it in an incubator at 37 °C for 1 h, take it out, wash the ELISA plate 3 times with PBST, and pat it dry each time. Vacuum dry at -20 °C for 3 h. Coating and blocking are completed.

[0129] (3) Add 50 μL of the sample to be tested or the vomitoxin standard to each well; add 50 μL of the primary antibody in Table 1 to each well; after incubating in an incubator at 37 °C for 1 h, take it out, rinse it 3 times with PBST, and pat it dry. The concentration of the primary antibody is 1.05 μg / mL.

[0130] Add 100 μL of the enzyme-labeled secondary antibody per well and incubate in an incubator at 37 °C for 1 h at room temperature; wash the plate 3 times with PBST and pat it dry.

[0131] (4) Add 80 μL of freshly prepared TMB chromogenic solution mixed in equal volume ratio of 1:1 to each well, and add 20 μL of the chromogenic regulator solution to each well. Mix well, wrap it with aluminum foil and place it in an incubator at 37 °C for 10 min for color development.

[0132] Add 50 μL of the stop solution per well, mix well, set the microplate reader to detect at 450 nm, and measure the absorbance value of each well; quantitatively analyze the content of vomitoxin in the sample to be tested using the standard curve.

[0133] Among them, the chromogenic regulator is dissolved in phosphate buffer to form a chromogenic regulator solution, which is then used to detect vomitoxin. The concentration of the chromogenic regulator solution is 0.9 g / L.

[0134] Among them, when preparing the standard curve, prepare standard solutions of different concentrations of vomitoxin. Set the microplate reader to detect at 450 nm and measure the absorbance value.

[0135] Using logC DON (the logarithm of the vomitoxin concentration) as the abscissa, and the ratio of the absorbance value B of the sample added with the vomitoxin standard to the absorbance value B0 of the sample without adding the vomitoxin standard as the ordinate, as Figure 6 shown.

[0136] IC 50 refers to the concentration of vomitoxin in the sample to be detected corresponding to B / B0 = 50%. The lower the IC 50 , the higher the sensitivity of the detection method.

[0137] The half-maximal inhibitory concentration IC 50 of the method in this example is 6.10 pg / mL, which is significantly lower than the blank control IC 50 without adding the chromogenic regulator, which is 166.38 pg / mL, indicating that the detection limit of the method of the present invention is low.

[0138] It should be noted that in Examples 1 to 5, the chromogenic regulator is ethylenediaminetetraacetic acid.

[0139] Example 6

[0140] The indirect competitive ELISA method is used to detect vomitoxin. The detection steps include:

[0141] (1) Sample treatment

[0142] The corn kernels are ground into powder. Weigh 2 g of the powder and dissolve it in 10 mL of a 50% (v / v) methanol aqueous solution. Mix it evenly in a vortex mixer, and then place it in a high-speed centrifuge at 4000 r / min and centrifuge at 4 °C for 10 min. Take the supernatant as the sample to be detected.

[0143] The sample to be detected is diluted with the sample diluent to obtain different-fold dilutions of the sample to be detected. When the concentration of vomitoxin in the sample to be detected is too high and exceeds the concentration setting range of the standard curve, it is necessary to prepare a diluted sample to be detected and re-measure until the detected concentration range of vomitoxin is within the concentration setting range of the standard curve. Making the concentration of the target substance in the sample to be detected meet the standard curve setting value by dilution belongs to the common general knowledge in the art.

[0144] (2) Coating and blocking

[0145] Dilute the antigen with CBS to 0.12 mg / mL to obtain the antigen dilution, which is reserved. Add the antigen dilution to a 96-well ELISA plate, 100 μL per well, place it in a refrigerator at 4 °C for 12 h, wash it 3 times with PBST, 3 min each time, and then pat the ELISA plate dry. Add 100 μL of blocking solution to each well to block, place it in an incubator at 37 °C for 1 h, take it out, wash the ELISA plate 3 times with PBST, and pat it dry each time. Vacuum dry at -20 °C for 3 h. Coating and blocking are completed.

[0146] (3) Add the test sample dilution or vomitoxin standard to each well, 50 μL per well; add the primary antibody in Table 1, 50 μL per well; place it in an incubator at 37 °C for 1 h, then take it out, rinse it 3 times with PBST, and pat it dry. The concentration of the primary antibody is 1.05 μg / mL.

[0147] Add the enzyme-labeled secondary antibody, 100 μL per well, incubate in an incubator at 37 °C at room temperature for 1 h; wash the plate 3 times with PBST and pat it dry.

[0148] (4) Add 80 μL of freshly prepared TMB chromogenic solution mixed in equal volume at 1:1 to each well, add 20 μL of chromogenic regulator solution to each well, mix well, wrap it with aluminum foil and place it in an incubator at 37 °C for 10 min for color development.

[0149] Add 50 μL of stop solution to each well, mix well, set the microplate reader to detect at 450 nm, and measure the absorbance value of each well; quantitatively analyze the content of vomitoxin in the test sample using the standard curve.

[0150] Among them, the chromogenic regulator is dissolved in phosphate buffer to form a chromogenic regulator solution, which is then used to detect vomitoxin. The concentration of the chromogenic regulator solution is 0.5 g / L. The chromogenic regulator is sodium ethylenediaminetetraacetate.

[0151] Among them, when making the standard curve, prepare vomitoxin standard solutions with different concentrations. Set the microplate reader to detect at 450 nm and measure the absorbance value.

[0152] Using logC DON (the logarithm of the vomitoxin concentration) as the abscissa, and the ratio of the absorbance value B of the well with the vomitoxin standard added to the absorbance value B0 of the well without the vomitoxin standard added as the ordinate, as Figure 7 shown.

[0153] IC 50 refers to the concentration of vomitoxin in the test sample corresponding to B / B0 = 50%, and the lower the IC 50 , the higher the sensitivity of the detection method.

[0154] The half-maximal inhibitory concentration IC of the method in this example 50was 3.17 pg / mL, significantly lower than the blank control IC without the color development regulator 50 which was 166.38 ng / mL, indicating that the detection limit of the method of the present invention is low.

[0155] Example 7

[0156] The indirect competitive ELISA method was used to detect vomitoxin, and the detection steps included:

[0157] (1) Sample treatment

[0158] The corn kernels were ground into powder, 2 g of the powder was weighed and dissolved in 10 mL of a 50% methanol aqueous solution by volume. After mixing evenly in a vortex mixer, it was placed in a high-speed centrifuge at 4000 r / min and centrifuged at 4°C for 10 min. The supernatant was taken as the sample to be tested.

[0159] The sample to be tested was diluted with the sample diluent to obtain diluted samples to be tested at different multiples. When the concentration of vomitoxin in the sample to be tested was too high and exceeded the concentration setting range of the standard curve, it was necessary to prepare a diluted sample to be tested and re-measure until the concentration range of the detected vomitoxin was within the concentration setting range of the standard curve. Diluting to make the concentration of the target substance in the sample to be tested meet the set value of the standard curve belongs to the common knowledge in the art.

[0160] (2) Coating and blocking

[0161] The antigen was diluted to 0.12 mg / mL with CBS to obtain an antigen diluent for standby. The antigen diluent was added to a 96-well enzyme-linked immunosorbent assay (ELISA) plate, 100 μL per well, and placed in a refrigerator at 4°C for 12 h. It was washed 3 times with PBST, 3 min each time, and then the ELISA plate was patted dry. 100 μL of the blocking solution was added to each well for blocking, and it was incubated in an incubator at 37°C for 1 h. Then it was taken out, and the ELISA plate was washed 3 times with PBST, and patted dry each time. It was vacuum dried at -20°C for 3 h. Coating and blocking were completed.

[0162] (3) Add 50 μL of the diluted sample to be tested or the vomitoxin standard product to each well; add 50 μL of the primary antibody in Table 1 to each well; after incubating in an incubator at 37°C for 1 h, take it out and wash it 3 times with PBST and pat it dry. The concentration of the primary antibody was 1.05 μg / mL.

[0163] Add 100 μL of the enzyme-labeled secondary antibody per well and incubate in an incubator at 37°C for 1 h at room temperature; wash the plate 3 times with PBST and pat it dry.

[0164] (4) Add 80 μL of the freshly prepared TMB chromogenic solution mixed in equal volume at a ratio of 1:1 to each well, add 20 μL of the chromogenic regulator solution to each well, mix well, wrap it with aluminum foil and place it in an incubator at 37°C for 10 min for color development.

[0165] Add 50 μL / well of the stop solution, mix well, set the microplate reader to detect at 450 nm, and measure the absorbance value of each well; quantitatively analyze the content of deoxynivalenol in the sample to be detected using the standard curve.

[0166] Among them, the chromogenic regulator is dissolved in the phosphate buffer solution to form a chromogenic regulator solution, and then it is used to detect deoxynivalenol. The concentration of the chromogenic regulator solution is 0.5 g / L. The chromogenic regulator is vitamin C.

[0167] Among them, when preparing the standard curve, prepare standard solutions of deoxynivalenol with different concentrations. Set the microplate reader to detect at 450 nm and measure the absorbance value.

[0168] Taking logC DON (the logarithm of the deoxynivalenol concentration) as the abscissa, and the ratio of the absorbance value B of the standard solution with added deoxynivalenol to the absorbance value B0 of the standard solution without added deoxynivalenol as the ordinate, as Figure 8 shown.

[0169] IC 50 refers to the concentration of deoxynivalenol in the sample to be detected corresponding to B / B0 = 50%. The lower the IC 50 , the higher the sensitivity of the detection method.

[0170] The half-inhibitory concentration IC 50 of the method in this example is 11.51 pg / mL, which is significantly lower than the IC 50 of 166.38 ng / mL of the blank control without the chromogenic regulator, indicating that the detection limit of the method of the present invention is low.

[0171] It should be noted that when the present invention involves a numerical range, it should be understood that any value between the two endpoints of each numerical range and the two endpoints can be selected. Since the steps and methods adopted are the same as those in the examples, in order to prevent repetition, the present invention describes the preferred embodiments. Although the preferred embodiments of the present invention have been described, those skilled in the art can make additional changes and modifications to these embodiments once they know the basic creative concept. Therefore, the appended claims are intended to be interpreted to include the preferred embodiments and all changes and modifications falling within the scope of the present invention.

[0172] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalent technologies, the present invention is also intended to include these changes and modifications.

Claims

1. A kit for detecting vomitoxin, characterized in that, Including antigen, primary antibody, enzyme-labeled secondary antibody, color regulator, phosphate buffer, and detection reagent; The antigen is bovine serum albumin coupled to vomitoxin; The primary antibody is a monoclonal antibody against vomitoxin; The enzyme-labeled secondary antibody is a goat anti-mouse IgG secondary antibody labeled with horseradish peroxidase; The color development regulator is sodium ethylenediaminetetraacetate; The detection reagent consists of TMB colorimetric solution, coating solution, blocking solution, washing solution, sample diluent and stop solution, wherein the coating solution is carbonate buffer and the stop solution is H2SO4 solution; The TMB chromogenic solution is composed of Solution A and Solution B. The formula of Solution A is as follows: 13.6 g of CH3COONa, 1.6 g of C6H8O7·H2O, 0.3 mL of 30% H2O2, and after dissolution, it is made up to 1000 mL; the formula of Solution B is as follows: 10 H 12 0.2 g of FeN2NaO8·3H2O, 50 mL of glycerol, 0.15 g of TMB·2HCl is made up to 1000 mL with deionized water; Solution A and Solution B are mixed in a volume ratio of 1:1 and used immediately after preparation; The color regulator is dissolved in a phosphate buffer to form a color regulator solution, the concentration of the color regulator solution is 0.5 g / L to 0.6 g / L, and the volume ratio of the color developing solution to the color regulator solution is 4:

1.

2. The kit for detecting vomitoxin according to claim 1, characterized in that, The phosphate buffer is 0.01 mol / L phosphate buffer with a pH of 7.

4.

3. The kit for detecting vomitoxin according to claim 2, wherein, The carbonate buffer solution has the following formula: 0.75 g Na2CO3, 1.46 g NaHCO3, and the volume is adjusted to 500 mL with deionized water, and the pH value is adjusted to 9.

6.

4. A method for detecting vomitoxin using the kit according to claim 1, characterized in that, include: A reaction solution is prepared using antigen, primary antibody, enzyme-labeled secondary antibody, sample to be tested and detection reagent, a color regulator solution is added to the reaction solution, and the absorbance value is detected after the reaction; The absorbance value of the vomitoxin standard was detected under the same operating conditions as above, and a standard curve was drawn; The standard curve was used to quantitatively analyze the content of DON in the sample.

5. The method for detecting vomitoxin by the kit according to claim 4, wherein include: Take the ELISA plate coated with the antigen, block it, add the primary antibody and the sample to be tested, incubate at 37°C in a dark environment, wash the plate, add the enzyme-labeled secondary antibody, incubate at 37°C in a dark environment, wash the plate, add the color developing solution and the color developing regulator solution, mix well, and incubate at 37°C in a dark environment for 15min~20min; add the stop solution, mix well, and detect the absorbance value of each well at 450nm on the ELISA instrument; use the standard curve to quantitatively analyze the content of vomitoxin in the sample to be tested.

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