A detection kit and method for norostatin

Abstract

This invention discloses a detection kit technology, specifically a detection kit and method for novoarginine, comprising a universal sample processing solution, a novoarginine-specific probe, fluorescently labeled streptavidin, washing solution, chromogenic substrate solution, stop solution, novoarginine standard, and a reaction microplate. The universal sample processing solution is composed of acetonitrile, potassium dihydrogen phosphate buffer, and disodium ethylenediaminetetraacetate in a volume ratio of 3:6:1, with the potassium dihydrogen phosphate buffer concentration being 0.1 mol / L and the disodium ethylenediaminetetraacetate concentration being 0.05 mol / L. The novoarginine-specific probe is a biotin-labeled novoarginine monoclonal antibody at a concentration of 1 mg / mL. The surface of the reaction microplate is coated with novoarginine hapten. This invention, by developing a universal sample processing solution adaptable to different matrix samples, eliminates the need for customized pretreatment procedures, significantly improving the versatility of the kit and solving the cumbersome problem of requiring separate processing for different samples.

Description

Technical Field

[0001] This invention relates to the field of detection kit technology, specifically to a detection kit and detection method for novogene. Background Technology

[0002] Nociceptin is an antibiotic produced by Streptomyces fermentation. Due to its significant inhibitory effect on various Gram-positive bacteria, it is widely used as a bacteriostatic agent in animal husbandry and agriculture, and also has potential applications in the pharmaceutical field. However, the residue level of nociceptin in products directly affects product safety. Excessive residues may lead to bacterial resistance, environmental pollution, and potential harm to human health. Therefore, establishing efficient, accurate, and rapid methods for detecting nociceptin is crucial.

[0003] Currently, the main detection methods for norostatin include high-performance liquid chromatography (HPLC), gas chromatography-mass spectrometry (GC-MS), and enzyme-linked immunosorbent assay (ELISA). While HPLC and GC-MS offer high detection accuracy, they suffer from drawbacks such as expensive equipment, complex procedures, the need for specialized technicians, and long detection cycles (typically 2-4 hours), making them unsuitable for rapid on-site testing or large-scale sample screening. ELISA, although relatively simple to operate, suffers from insufficient antibody specificity, susceptibility to sample matrix interference, and low detection sensitivity (the limit of detection is typically only 10 ng / mL), failing to meet the need for accurate detection of low-residue norostatin.

[0004] Existing detection kits mostly employ a single reaction system, with cumbersome and poorly targeted sample pretreatment steps. Different types of samples (such as feed, serum, soil, and water) require different pretreatment methods, resulting in poor universality. Furthermore, controlling reaction conditions during the detection process is difficult, easily leading to false positive or false negative results, and exhibiting poor stability and repeatability. Therefore, this invention aims to develop a detection kit and method for norostatin. Summary of the Invention

[0005] To solve the above-mentioned technical problems, the present invention provides the following technical solution: A detection kit for novogene includes a universal sample processing solution, a novogene-specific probe, fluorescently labeled streptavidin, washing solution, chromogenic substrate solution, stop solution, novogene standard, and reaction microplate. The universal sample processing solution is composed of acetonitrile, potassium dihydrogen phosphate buffer (pH=6.8), and disodium ethylenediaminetetraacetate (EDTA-2Na) in a volume ratio of 3:6:1. The concentration of the potassium dihydrogen phosphate buffer is 0.1 mol / L, and the concentration of the disodium ethylenediaminetetraacetate is 0.05 mol / L. It is used for the efficient extraction of nifedipine and the removal of impurities from different types of samples. The novocalin-specific probe is a biotin-labeled novocalin monoclonal antibody with a concentration of 1 mg / mL and an antibody purity of ≥95%, used to specifically bind novocalin in the sample. The surface of the reaction microplate is coated with nifedipine hapten for immobilizing antigen-antibody complexes.

[0006] As a preferred embodiment of the detection kit for norogenetic acid described in this invention, the concentration of the fluorescently labeled streptavidin is 0.5 mg / mL, and the fluorescent label is fluorescein isothiocyanate, which is used to bind to the biotin-labeled monoclonal antibody to amplify the detection signal.

[0007] As a preferred embodiment of the detection kit for novogene according to the present invention, the washing solution is a Tris-HCl buffer (pH=7.4) containing 0.05% Tween-20, with a concentration of 0.02 mol / L, used to remove unbound impurities and excess reagents, and reduce non-specific reactions.

[0008] As a preferred embodiment of the detection kit for novogene according to the present invention, wherein: the chromogenic substrate solution is prepared by mixing substrate A solution and substrate B solution in a volume ratio of 1:1, and is used to catalyze the chromogenic reaction; substrate A solution contains 3,3',5,5'-tetramethylbenzidine at a concentration of 0.5 mg / mL; substrate B solution is a citrate buffer solution containing 0.02% hydrogen peroxide, and the pH is 4.0.

[0009] In a preferred embodiment of the detection kit for nifedipine described in this invention, the stop solution is a 2 mol / L sulfuric acid solution used to terminate the colorimetric reaction.

[0010] As a preferred embodiment of the detection kit for novogene according to the present invention, the concentration gradient of the novogene standard is 0 ng / mL, 0.1 ng / mL, 0.5 ng / mL, 1 ng / mL, 5 ng / mL, and 10 ng / mL, which is used to plot a standard curve and realize quantitative detection.

[0011] A detection method for a kit for norepinephrine includes the following specific steps: S1, Sample pretreatment: Collect and preprocess the sample, add general sample processing solution for extraction, centrifuge and take the supernatant, dilute with washing solution to obtain the sample solution to be tested; S2, Kit preparation: Equilibrate the kit components at room temperature, dilute the novogene standard to obtain the standard working solution, and prepare the mixed reaction solution of novogene specific probe and fluorescently labeled streptavidin. S3, immune competitive reaction: Add standard working solution, sample solution to be tested and blank control solution to reaction microplate, then add mixed reaction solution, incubate and wash; S4, Antigen-antibody complex enhancement and residual impurity shielding: Add complex enhancement solution and impurity shielding agent to the washed microwells, incubate briefly, and then wash simply. S5, Color development and signal detection: Add the color development substrate solution and incubate in the dark. Terminate the reaction with the stop solution and read the absorbance value using an ELISA reader. S6, Result Judgment and Quantitative Analysis: Preprocess the detection data, plot the standard curve, substitute the sample absorbance value to calculate the concentration of nifedipine in the original sample and determine the result.

[0012] In a preferred embodiment of the detection method for the detection kit of norepinephrine described in this invention, the specific steps of S1 are as follows: S11, Sample Collection and Preprocessing: Collect the corresponding samples according to the testing requirements. After crushing the feed samples, pass them through an 80-mesh sieve and weigh 1-2g of the sieved sample. Centrifuge the serum samples (3000r / min, 5min) to remove the precipitate and take the supernatant. After air-drying the soil samples, pass them through a 100-mesh sieve and weigh 2-3g of the sieved sample. Take the supernatant directly from the water samples. Place all types of samples into 50mL centrifuge tubes. S12, Extraction: Add 10 mL of general sample processing solution to a centrifuge tube and vortex for 5 min (oscillation frequency 2000 r / min) to fully mix the sample with the processing solution and achieve efficient extraction of nifedipine. S13, Centrifugation: Place the shaken centrifuge tube in a centrifuge and centrifuge at 10000 r / min for 10 min to obtain the supernatant containing benzodiazepines. S14, dilution and purification: Take 1-2 mL of supernatant, add 4-5 mL of washing buffer for dilution, vortex for 1 min to obtain the sample solution to be tested, which will be used for subsequent immune reactions; The specific steps of S2 are as follows: S21, Kit component equilibration: Remove all components in the kit from the refrigerated environment (2-8℃) and place them at room temperature (25±2℃) for equilibration for 30-35 minutes to ensure the activity of each component is stable; S22, Standard dilution: Take the Norfucoagulant standard and dilute each concentration of the standard with washing buffer at a ratio of 1:10 to obtain standard working solutions with concentrations of 0 ng / mL, 0.01 ng / mL, 0.05 ng / mL, 0.1 ng / mL, 0.5 ng / mL, and 1 ng / mL, respectively. S23, Preparation of the mixed solution of probe and fluorescently labeled streptavidin: Take the noradrencin-specific probe and fluorescently labeled streptavidin at a volume ratio of 1:2, add washing solution to dilute 10 times, vortex for 30-40s to prepare a mixed reaction solution.

[0013] In a preferred embodiment of the detection method for the detection kit for norostatin described in this invention, the specific steps of S3 are as follows: S31, Sample addition: Add 100 μL of standard working solution (3 parallel wells for each concentration) and 100 μL of sample solution to be tested (3 parallel wells for each sample) to different wells of the reaction microplate, and add 100 μL of washing solution to blank control wells. S32, Add the mixed reaction solution: Add 50 μL of the probe and fluorescently labeled streptavidin mixture to each microwell, and gently shake the microplate for 1-3 min to fully mix the liquid in the well; S33, Incubation reaction: Place the microplate in a constant temperature incubator and incubate at 38-40℃ for 20-25 min. During this process, the norogenin in the sample competes with the hapten coated on the surface of the microplate for binding to the specific probe, forming an antigen-antibody complex. S34, Washing: After incubation, discard the liquid in the wells, add 200 μL of washing solution to each microwell, let stand for 30-40 seconds and then discard, repeat washing 3-5 times, after the last wash, invert the microplate and use absorbent paper to dry the residual liquid to remove unbound probes and impurities. The specific steps of S4 are as follows: S41, Add complex enhancement solution: Add 30 μL of complex enhancement solution (Tris-HCl buffer containing 2% polyethylene glycol PEG 6000, concentration 0.02 mol / L, pH=7.4) to each washed microwell, and gently shake the microplate for 30-50 s to ensure that the enhancement solution is in full contact with the antigen-antibody complex in the microwell and enhance the stability of complex binding. S42, Add residual impurity shielding agent: Add 20 μL of impurity shielding agent (washing solution containing 1% bovine serum albumin BSA) to each microwell, vortex for 1-3 min, and utilize the specific binding of BSA to the trace amounts of sample matrix impurities (such as protein in feed and humus in soil) remaining in the microwell to avoid interference with the subsequent colorimetric reaction. S43, Short incubation: Place the microplate in an environment of 25-30℃ for 5-10 minutes to ensure that the strengthening liquid and the shielding agent can play their full role; S44, Simple Washing: Add 200 μL of washing solution to each microwell, let stand for 20-30 seconds and then discard. Invert the well to absorb any remaining liquid, removing only excess reinforcing solution and shielding agent, while retaining stable antigen-antibody complexes and bound shielding impurities.

[0014] In a preferred embodiment of the detection method for the detection kit of norepinephrine described in this invention, the specific steps of S5 are as follows: S51, Add colorimetric substrate solution: Add 100μL of colorimetric substrate solution to each microplate, gently shake the microplate for 10-20s, and place it in the dark; S52, colorimetric reaction: Incubate at room temperature (25±2℃) in the dark for 15-17 min. During this process, the enzyme in the fluorescently labeled streptavidin catalyzes the substrate to undergo a colorimetric reaction. The color intensity of the solution is negatively correlated with the concentration of nifedipine. S53, Termination of reaction: Add 50 μL of termination solution to each well, gently shake the microplate for 10-20 s to terminate the colorimetric reaction. At this time, the solution color is stable. S54, Signal detection: Place the microplate into the microplate reader, set the detection wavelength to 450nm, read the absorbance value (OD value) of each microplate, and record the detection data; The specific steps of S6 are as follows: S61, Data preprocessing: Calculate the average absorbance values ​​of parallel wells for each concentration standard and parallel wells for the sample, subtract the absorbance values ​​of the blank control wells, and obtain the corrected absorbance values. S62, Plot the standard curve: Plot the standard working solution concentration as the x-axis and the corrected absorbance value as the y-axis. Use the linear regression method to plot the standard curve and obtain the regression equation y=ax+b, where a is the slope, b is the intercept, and the correlation coefficient R²≥0.99. S63, Sample concentration calculation: Substitute the corrected absorbance value of the sample solution to be tested into the standard curve regression equation to calculate the concentration of nifedipine in the sample solution, and then convert the actual concentration of nifedipine in the original sample according to the dilution factor of S1. S64. Result determination: If the actual concentration of nifedipine in the sample is ≥ the detection limit (0.01 ng / mL), it is determined to be positive; if it is < the detection limit, it is determined to be negative.

[0015] Compared with existing technologies: 1. By developing a universal sample processing solution that is compatible with different matrix samples, there is no need for customized pretreatment processes. This significantly improves the versatility of the kit and solves the cumbersome problem of having to process different samples separately. 2. By designing highly specific probes and signal amplification mechanisms and optimizing the antigen-antibody binding system, the detection sensitivity can be significantly improved, overcoming the limitation of traditional methods in detecting low-residual norogenin. 3. By simplifying sample pretreatment and reaction steps and synergizing the effects of various reagents, it can significantly shorten the detection cycle and meet the needs of rapid on-site testing and large-scale sample screening; 4. By optimizing the reagent kit component ratio and reaction conditions, combined with high-purity specific probes, it is possible to reduce non-specific reactions, lower the probability of false positives and false negatives, and improve detection stability and repeatability; 5. By simplifying the operation process, eliminating the need for expensive and sophisticated equipment, and clearly defining the operation standards for each step, it lowers the testing threshold, allowing testing to be completed without professional technicians, and facilitating its promotion at the grassroots level. Detailed Implementation

[0016] To make the objectives, technical solutions, and advantages of the present invention clearer, the embodiments of the present invention will be described in further detail below. Example

[0017] This invention provides a detection kit and detection method for novogene, including a general sample processing solution, a novogene-specific probe, fluorescently labeled streptavidin, washing solution, chromogenic substrate solution, stop solution, novogene standard, and reaction microplate. The universal sample processing solution is composed of acetonitrile, potassium dihydrogen phosphate buffer (pH=6.8), and disodium ethylenediaminetetraacetate (EDTA-2Na) in a volume ratio of 3:6:1. The concentration of the potassium dihydrogen phosphate buffer is 0.1 mol / L, and the concentration of the disodium ethylenediaminetetraacetate is 0.05 mol / L. It is used for the efficient extraction of nifedipine and the removal of impurities from different types of samples. The novocalin-specific probe is a biotin-labeled novocalin monoclonal antibody with a concentration of 1 mg / mL and an antibody purity of ≥95%, used to specifically bind novocalin in the sample. The surface of the reaction microplate is coated with nifedipine hapten for immobilizing antigen-antibody complexes.

[0018] The concentration of the fluorescently labeled streptavidin is 0.5 mg / mL, and the fluorescent label is fluorescein isothiocyanate, which is used to bind to biotin-labeled monoclonal antibodies to amplify the detection signal.

[0019] The washing solution is a Tris-HCl buffer solution (pH=7.4) containing 0.05% Tween-20, with a concentration of 0.02 mol / L, used to remove unbound impurities and excess reagents, and reduce non-specific reactions.

[0020] The colorimetric substrate solution is prepared by mixing substrate A and substrate B in a volume ratio of 1:1, and is used to catalyze the colorimetric reaction. Substrate A contains 3,3',5,5'-tetramethylbenzidine at a concentration of 0.5 mg / mL. Substrate B is a citrate buffer solution containing 0.02% hydrogen peroxide and has a pH of 4.0.

[0021] The terminating solution is a 2 mol / L sulfuric acid solution, used to terminate the colorimetric reaction.

[0022] The concentration gradient of the noraconazole standard is 0 ng / mL, 0.1 ng / mL, 0.5 ng / mL, 1 ng / mL, 5 ng / mL, and 10 ng / mL, which is used to plot a standard curve and achieve quantitative detection.

[0023] A detection method for a kit for norepinephrine includes the following specific steps: S1, Sample pretreatment: Collect and preprocess the sample, add general sample processing solution for extraction, centrifuge and take the supernatant, dilute with washing solution to obtain the sample solution to be tested; The specific steps of S1 are as follows: S11, Sample Collection and Preprocessing: Collect the corresponding samples according to the testing requirements. After crushing the feed samples, pass them through an 80-mesh sieve and weigh 1g of the sample after sieving. Centrifuge the serum samples (3000r / min, 5min) to remove the precipitate and take the supernatant. After air-drying the soil samples, pass them through a 100-mesh sieve and weigh 2g of the sample after sieving. Take the supernatant directly from the water samples. Place all types of samples into 50mL centrifuge tubes. S12, Extraction: Add 10 mL of general sample processing solution to a centrifuge tube and vortex for 5 min (oscillation frequency 2000 r / min) to fully mix the sample with the processing solution and achieve efficient extraction of nifedipine. S13, Centrifugation: Place the shaken centrifuge tube in a centrifuge and centrifuge at 10000 r / min for 10 min to obtain the supernatant containing benzodiazepines. S14, dilution and purification: Take 1 mL of supernatant, add 4 mL of washing solution for dilution, vortex for 1 min to obtain the sample solution to be tested, which will be used for subsequent immune reactions; S2, Kit preparation: Equilibrate the kit components at room temperature, dilute the novogene standard to obtain the standard working solution, and prepare the mixed reaction solution of novogene specific probe and fluorescently labeled streptavidin. The specific steps of S2 are as follows: S21, Kit component equilibration: Remove all components in the kit from the refrigerated environment (2℃) and place them at room temperature (23℃) for equilibration for 30 minutes to ensure the activity of each component is stable; S22, Standard dilution: Take the Norfucoagulant standard and dilute each concentration of the standard with washing buffer at a ratio of 1:10 to obtain standard working solutions with concentrations of 0 ng / mL, 0.01 ng / mL, 0.05 ng / mL, 0.1 ng / mL, 0.5 ng / mL, and 1 ng / mL, respectively. S23, Preparation of the mixed solution of probe and fluorescently labeled streptavidin: Take the noprigen-specific probe and fluorescently labeled streptavidin at a volume ratio of 1:2, add washing solution to dilute 10 times, vortex for 30s to prepare a mixed reaction solution. S3, immune competitive reaction: Add standard working solution, sample solution to be tested and blank control solution to reaction microplate, then add mixed reaction solution, incubate and wash; The specific steps of S3 are as follows: S31, Sample addition: Add 100 μL of standard working solution (3 parallel wells for each concentration) and 100 μL of sample solution to be tested (3 parallel wells for each sample) to different wells of the reaction microplate, and add 100 μL of washing solution to blank control wells. S32, Add the mixed reaction solution: Add 50 μL of the probe and fluorescently labeled streptavidin mixture to each microwell, and gently shake the microplate for 1 min to fully mix the liquid in the well; S33, Incubation reaction: Place the microplate in a constant temperature incubator and incubate at 38°C for 20 min. During this process, the norogenin in the sample competes with the hapten coated on the surface of the microplate for binding to the specific probe, forming an antigen-antibody complex. S34, Washing: After incubation, discard the liquid in the wells, add 200 μL of washing solution to each microwell, let stand for 30 seconds and then discard, repeat washing 3 times, after the last wash, invert the microplate and use absorbent paper to dry the residual liquid to remove unbound probes and impurities. S4, Antigen-antibody complex enhancement and residual impurity shielding: Add complex enhancement solution and impurity shielding agent to the washed microwells, incubate briefly, and then wash simply. The specific steps of S4 are as follows: S41, Add complex enhancement solution: Add 30 μL of complex enhancement solution (Tris-HCl buffer containing 2% polyethylene glycol PEG 6000, concentration 0.02 mol / L, pH=7.4) to each washed microwell, and gently shake the microplate for 30 s to ensure that the enhancement solution is in full contact with the antigen-antibody complex in the microwell and enhance the stability of complex binding. S42, Add residual impurity shielding agent: Add 20 μL of impurity shielding agent (washing solution containing 1% bovine serum albumin BSA) to each microwell, vortex for 1 min, and utilize the specific binding of BSA to the trace amounts of sample matrix impurities (such as protein in feed and humus in soil) remaining in the microwell to avoid interference with the subsequent colorimetric reaction. S43, Short incubation: Place the microplate in an environment of 25°C for 5 minutes to ensure that the strengthening liquid and the shielding agent play their full role; S44, Simple Washing: Add 200 μL of washing solution to each microwell, let stand for 20 seconds and then discard. Invert the container to absorb the residual liquid, removing only excess enhancement solution and shielding agent, while retaining stable antigen-antibody complexes and bound shielding impurities. S5, Color development and signal detection: Add the color development substrate solution and incubate in the dark. Terminate the reaction with the stop solution and read the absorbance value using an ELISA reader. The specific steps of S5 are as follows: S51, Add colorimetric substrate solution: Add 100 μL of colorimetric substrate solution to each microplate, gently shake the microplate for 10 seconds, and place it in the dark; S52, colorimetric reaction: Incubate at room temperature (23℃) in the dark for 15 min. During this process, the enzyme in the fluorescently labeled streptavidin catalyzes the substrate to produce a colorimetric reaction. The color intensity of the solution is negatively correlated with the concentration of nifedipine. S53, Termination of reaction: Add 50 μL of termination solution to each well, gently shake the microplate for 10 s to terminate the colorimetric reaction, at which point the solution color is stable; S54, Signal detection: Place the microplate into the microplate reader, set the detection wavelength to 450nm, read the absorbance value (OD value) of each microplate, and record the detection data; S6, Result Judgment and Quantitative Analysis: Preprocess the detection data, plot the standard curve, substitute the sample absorbance value to calculate the concentration of nifedipine in the original sample and determine the result; The specific steps of S6 are as follows: S61, Data preprocessing: Calculate the average absorbance values ​​of parallel wells for each concentration standard and parallel wells for the sample, subtract the absorbance values ​​of the blank control wells, and obtain the corrected absorbance values. S62, Plot the standard curve: Plot the standard working solution concentration as the x-axis and the corrected absorbance value as the y-axis. Use the linear regression method to plot the standard curve and obtain the regression equation y=ax+b, where a is the slope, b is the intercept, and the correlation coefficient R²≥0.99. S63, Sample concentration calculation: Substitute the corrected absorbance value of the sample solution to be tested into the standard curve regression equation to calculate the concentration of nifedipine in the sample solution, and then convert the actual concentration of nifedipine in the original sample according to the dilution factor of S1. S64. Result determination: If the actual concentration of nifedipine in the sample is ≥ the detection limit (0.01 ng / mL), it is determined to be positive; if it is < the detection limit, it is determined to be negative. Example

[0024] This invention provides a detection kit and detection method for novogene, including a general sample processing solution, a novogene-specific probe, fluorescently labeled streptavidin, washing solution, chromogenic substrate solution, stop solution, novogene standard, and reaction microplate. The universal sample processing solution is composed of acetonitrile, potassium dihydrogen phosphate buffer (pH=6.8), and disodium ethylenediaminetetraacetate (EDTA-2Na) in a volume ratio of 3:6:1. The concentration of the potassium dihydrogen phosphate buffer is 0.1 mol / L, and the concentration of the disodium ethylenediaminetetraacetate is 0.05 mol / L. It is used for the efficient extraction of nifedipine and the removal of impurities from different types of samples. The novocalin-specific probe is a biotin-labeled novocalin monoclonal antibody with a concentration of 1 mg / mL and an antibody purity of ≥95%, used to specifically bind novocalin in the sample. The surface of the reaction microplate is coated with nifedipine hapten for immobilizing antigen-antibody complexes.

[0025] The concentration of the fluorescently labeled streptavidin is 0.5 mg / mL, and the fluorescent label is fluorescein isothiocyanate, which is used to bind to biotin-labeled monoclonal antibodies to amplify the detection signal.

[0026] The washing solution is a Tris-HCl buffer solution (pH=7.4) containing 0.05% Tween-20, with a concentration of 0.02 mol / L, used to remove unbound impurities and excess reagents, and reduce non-specific reactions.

[0027] The colorimetric substrate solution is prepared by mixing substrate A and substrate B in a volume ratio of 1:1, and is used to catalyze the colorimetric reaction. Substrate A contains 3,3',5,5'-tetramethylbenzidine at a concentration of 0.5 mg / mL. Substrate B is a citrate buffer solution containing 0.02% hydrogen peroxide and has a pH of 4.0.

[0028] The terminating solution is a 2 mol / L sulfuric acid solution, used to terminate the colorimetric reaction.

[0029] The concentration gradient of the noraconazole standard is 0 ng / mL, 0.1 ng / mL, 0.5 ng / mL, 1 ng / mL, 5 ng / mL, and 10 ng / mL, which is used to plot a standard curve and achieve quantitative detection.

[0030] A detection method for a kit for norepinephrine includes the following specific steps: S1, Sample pretreatment: Collect and preprocess the sample, add general sample processing solution for extraction, centrifuge and take the supernatant, dilute with washing solution to obtain the sample solution to be tested; The specific steps of S1 are as follows: S11, Sample Collection and Preprocessing: Collect the corresponding samples according to the testing requirements. After crushing the feed samples, pass them through an 80-mesh sieve and weigh 1.5g of the sieved sample. Centrifuge the serum samples (3000r / min, 5min) to remove the precipitate and take the supernatant. After air-drying the soil samples, pass them through a 100-mesh sieve and weigh 2.5g of the sieved sample. Take the supernatant directly from the water samples. Place all types of samples into 50mL centrifuge tubes. S12, Extraction: Add 10 mL of general sample processing solution to a centrifuge tube and vortex for 5 min (oscillation frequency 2000 r / min) to fully mix the sample with the processing solution and achieve efficient extraction of nifedipine. S13, Centrifugation: Place the shaken centrifuge tube in a centrifuge and centrifuge at 10000 r / min for 10 min to obtain the supernatant containing benzodiazepines. S14, dilution and purification: Take 1-2 mL of supernatant, add 4-5 mL of washing buffer for dilution, vortex for 1 min to obtain the sample solution to be tested, which will be used for subsequent immune reactions; S2, Kit preparation: Equilibrate the kit components at room temperature, dilute the novogene standard to obtain the standard working solution, and prepare the mixed reaction solution of novogene specific probe and fluorescently labeled streptavidin. The specific steps of S2 are as follows: S21, Kit component equilibration: Remove all components in the kit from the refrigerated environment (5℃) and place them at room temperature (25℃) for equilibration for 30 minutes to ensure the activity of each component is stable; S22, Standard dilution: Take the Norfucoagulant standard and dilute each concentration of the standard with washing buffer at a ratio of 1:10 to obtain standard working solutions with concentrations of 0 ng / mL, 0.01 ng / mL, 0.05 ng / mL, 0.1 ng / mL, 0.5 ng / mL, and 1 ng / mL, respectively. S23, Preparation of the mixed solution of probe and fluorescently labeled streptavidin: Take the noprigen-specific probe and fluorescently labeled streptavidin at a volume ratio of 1:2, add washing solution to dilute 10 times, vortex for 35s to prepare a mixed reaction solution; S3, immune competitive reaction: Add standard working solution, sample solution to be tested and blank control solution to reaction microplate, then add mixed reaction solution, incubate and wash; The specific steps of S3 are as follows: S31, Sample addition: Add 100 μL of standard working solution (3 parallel wells for each concentration) and 100 μL of sample solution to be tested (3 parallel wells for each sample) to different wells of the reaction microplate, and add 100 μL of washing solution to blank control wells. S32, Add the mixed reaction solution: Add 50 μL of the probe and fluorescently labeled streptavidin mixture to each microwell, and gently shake the microplate for 2 min to fully mix the liquid in the well; S33, Incubation reaction: Place the microplate in a constant temperature incubator and incubate at 39°C for 22.5 min. During this process, the norogenin in the sample competes with the hapten coated on the surface of the microplate for binding to the specific probe, forming an antigen-antibody complex. S34, Washing: After incubation, discard the liquid in the wells, add 200 μL of washing solution to each microwell, let stand for 35 seconds and then discard, repeat washing 4 times, after the last wash, invert the microplate and use absorbent paper to dry the residual liquid to remove unbound probes and impurities. S4, Antigen-antibody complex enhancement and residual impurity shielding: Add complex enhancement solution and impurity shielding agent to the washed microwells, incubate briefly, and then wash simply. The specific steps of S4 are as follows: S41, Add complex enhancement solution: Add 30 μL of complex enhancement solution (Tris-HCl buffer containing 2% polyethylene glycol PEG 6000, concentration 0.02 mol / L, pH=7.4) to each washed microwell, and gently shake the microplate for 40 s to ensure that the enhancement solution is in full contact with the antigen-antibody complex in the microwell and enhance the stability of complex binding; S42, Add residual impurity shielding agent: Add 20 μL of impurity shielding agent (washing solution containing 1% bovine serum albumin BSA) to each microwell, vortex for 2 min, and utilize the specific binding of BSA to the trace amounts of sample matrix impurities (such as protein in feed and humus in soil) remaining in the microwell to avoid interference with the subsequent colorimetric reaction. S43, Short incubation: Incubate the microplate at 27.5℃ for 7.5 min to ensure that the reinforcing solution and shielding agent work effectively; S44, Simple Washing: Add 200μL of washing solution to each microwell, let stand for 20-30 seconds and then discard. Invert the container to absorb the residual liquid, removing only excess enhancement solution and shielding agent, while retaining stable antigen-antibody complexes and bound shielding impurities. S5, Color development and signal detection: Add the color development substrate solution and incubate in the dark. Terminate the reaction with the stop solution and read the absorbance value using an ELISA reader. The specific steps of S5 are as follows: S51, Add colorimetric substrate solution: Add 100μL of colorimetric substrate solution to each microplate, gently shake the microplate for 10-20s, and place it in the dark; S52, colorimetric reaction: Incubate at room temperature (25℃) in the dark for 16 min. During this process, the enzyme in the fluorescently labeled streptavidin catalyzes the substrate to undergo a colorimetric reaction. The color intensity of the solution is negatively correlated with the concentration of nifedipine. S53, Termination of reaction: Add 50 μL of termination solution to each well, gently shake the microplate for 15 s to terminate the colorimetric reaction, at which point the solution color is stable; S54, Signal detection: Place the microplate into the microplate reader, set the detection wavelength to 450nm, read the absorbance value (OD value) of each microplate, and record the detection data; S6, Result Judgment and Quantitative Analysis: Preprocess the detection data, plot the standard curve, substitute the sample absorbance value to calculate the concentration of nifedipine in the original sample and determine the result; The specific steps of S6 are as follows: S61, Data preprocessing: Calculate the average absorbance values ​​of parallel wells for each concentration standard and parallel wells for the sample, subtract the absorbance values ​​of the blank control wells, and obtain the corrected absorbance values. S62, Plot the standard curve: Plot the standard working solution concentration as the x-axis and the corrected absorbance value as the y-axis. Use the linear regression method to plot the standard curve and obtain the regression equation y=ax+b, where a is the slope, b is the intercept, and the correlation coefficient R²≥0.99. S63, Sample concentration calculation: Substitute the corrected absorbance value of the sample solution to be tested into the standard curve regression equation to calculate the concentration of nifedipine in the sample solution, and then convert the actual concentration of nifedipine in the original sample according to the dilution factor of S1. S64. Result determination: If the actual concentration of nifedipine in the sample is ≥ the detection limit (0.01 ng / mL), it is determined to be positive; if it is < the detection limit, it is determined to be negative. Example

[0031] This invention provides a detection kit and detection method for novogene, including a general sample processing solution, a novogene-specific probe, fluorescently labeled streptavidin, washing solution, chromogenic substrate solution, stop solution, novogene standard, and reaction microplate. The universal sample processing solution is composed of acetonitrile, potassium dihydrogen phosphate buffer (pH=6.8), and disodium ethylenediaminetetraacetate (EDTA-2Na) in a volume ratio of 3:6:1. The concentration of the potassium dihydrogen phosphate buffer is 0.1 mol / L, and the concentration of the disodium ethylenediaminetetraacetate is 0.05 mol / L. It is used for the efficient extraction of nifedipine and the removal of impurities from different types of samples. The novocalin-specific probe is a biotin-labeled novocalin monoclonal antibody with a concentration of 1 mg / mL and an antibody purity of ≥95%, used to specifically bind novocalin in the sample. The surface of the reaction microplate is coated with nifedipine hapten for immobilizing antigen-antibody complexes.

[0032] The concentration of the fluorescently labeled streptavidin is 0.5 mg / mL, and the fluorescent label is fluorescein isothiocyanate, which is used to bind to biotin-labeled monoclonal antibodies to amplify the detection signal.

[0033] The washing solution is a Tris-HCl buffer solution (pH=7.4) containing 0.05% Tween-20, with a concentration of 0.02 mol / L, used to remove unbound impurities and excess reagents, and reduce non-specific reactions.

[0034] The colorimetric substrate solution is prepared by mixing substrate A and substrate B in a volume ratio of 1:1, and is used to catalyze the colorimetric reaction. Substrate A contains 3,3',5,5'-tetramethylbenzidine at a concentration of 0.5 mg / mL. Substrate B is a citrate buffer solution containing 0.02% hydrogen peroxide and has a pH of 4.0.

[0035] The terminating solution is a 2 mol / L sulfuric acid solution, used to terminate the colorimetric reaction.

[0036] The concentration gradient of the noraconazole standard is 0 ng / mL, 0.1 ng / mL, 0.5 ng / mL, 1 ng / mL, 5 ng / mL, and 10 ng / mL, which is used to plot a standard curve and achieve quantitative detection.

[0037] A detection method for a kit for norepinephrine includes the following specific steps: S1, Sample pretreatment: Collect and preprocess the sample, add general sample processing solution for extraction, centrifuge and take the supernatant, dilute with washing solution to obtain the sample solution to be tested; The specific steps of S1 are as follows: S11, Sample Collection and Preprocessing: Collect the corresponding samples according to the testing requirements. After crushing the feed samples, pass them through an 80-mesh sieve and weigh 2g of the sieved sample. Centrifuge the serum samples (3000r / min, 5min) to remove the precipitate and take the supernatant. After air-drying the soil samples, pass them through a 100-mesh sieve and weigh 3g of the sieved sample. Take the supernatant directly from the water samples. Place all types of samples into 50mL centrifuge tubes. S12, Extraction: Add 10 mL of general sample processing solution to a centrifuge tube and vortex for 5 min (oscillation frequency 2000 r / min) to fully mix the sample with the processing solution and achieve efficient extraction of nifedipine. S13, Centrifugation: Place the shaken centrifuge tube in a centrifuge and centrifuge at 10000 r / min for 10 min to obtain the supernatant containing benzodiazepines. S14, dilution and purification: Take 2 mL of supernatant, add 5 mL of washing solution for dilution, vortex for 1 min to obtain the sample solution to be tested, which will be used for subsequent immune reactions; S2, Kit preparation: Equilibrate the kit components at room temperature, dilute the novogene standard to obtain the standard working solution, and prepare the mixed reaction solution of novogene specific probe and fluorescently labeled streptavidin. The specific steps of S2 are as follows: S21, Kit component equilibration: Remove all components in the kit from the refrigerated environment (8℃) and place them at room temperature (27℃) for equilibration for 35 minutes to ensure the activity of each component is stable; S22, Standard dilution: Take the Norfucoagulant standard and dilute each concentration of the standard with washing buffer at a ratio of 1:10 to obtain standard working solutions with concentrations of 0 ng / mL, 0.01 ng / mL, 0.05 ng / mL, 0.1 ng / mL, 0.5 ng / mL, and 1 ng / mL, respectively. S23, Preparation of the mixed solution of probe and fluorescently labeled streptavidin: Take the noprigen-specific probe and fluorescently labeled streptavidin at a volume ratio of 1:2, add washing solution to dilute 10 times, vortex for 40s to prepare a mixed reaction solution; S3, immune competitive reaction: Add standard working solution, sample solution to be tested and blank control solution to reaction microplate, then add mixed reaction solution, incubate and wash; The specific steps of S3 are as follows: S31, Sample addition: Add 100 μL of standard working solution (3 parallel wells for each concentration) and 100 μL of sample solution to be tested (3 parallel wells for each sample) to different wells of the reaction microplate, and add 100 μL of washing solution to blank control wells. S32, Add the mixed reaction solution: Add 50 μL of the probe and fluorescently labeled streptavidin mixture to each microwell, and gently shake the microplate for 3 min to fully mix the liquid in the well; S33, Incubation reaction: Place the microplate in a constant temperature incubator and incubate at 40°C for 25 min. During this process, the norogenin in the sample competes with the hapten coated on the surface of the microplate for binding to the specific probe, forming an antigen-antibody complex. S34, Washing: After incubation, discard the liquid in the wells, add 200 μL of washing solution to each microwell, let stand for 40 seconds and then discard, repeat the washing 5 times, after the last wash, invert the microplate and use absorbent paper to dry the residual liquid to remove unbound probes and impurities. S4, Antigen-antibody complex enhancement and residual impurity shielding: Add complex enhancement solution and impurity shielding agent to the washed microwells, incubate briefly, and then wash simply. The specific steps of S4 are as follows: S41, Add complex enhancement solution: Add 30 μL of complex enhancement solution (Tris-HCl buffer containing 2% polyethylene glycol PEG 6000, concentration 0.02 mol / L, pH=7.4) to each washed microwell, and gently shake the microplate for 50 s to ensure that the enhancement solution is in full contact with the antigen-antibody complex in the microwell and enhance the stability of complex binding. S42, Add residual impurity shielding agent: Add 20 μL of impurity shielding agent (washing solution containing 1% bovine serum albumin BSA) to each microwell, vortex for 3 min, and utilize the specific binding of BSA to the trace amounts of sample matrix impurities (such as protein in feed and humus in soil) remaining in the microwell to avoid interference with the subsequent colorimetric reaction. S43, Short incubation: Place the microplate in a 30°C environment for 10 minutes to ensure that the reinforcing liquid and shielding agent play their full role; S44, Simple Washing: Add 200 μL of washing solution to each microwell, let stand for 30 seconds and then discard. Invert the container to absorb the residual liquid, removing only excess enhancement solution and shielding agent, while retaining stable antigen-antibody complexes and bound shielding impurities. S5, Color development and signal detection: Add the color development substrate solution and incubate in the dark. Terminate the reaction with the stop solution and read the absorbance value using an ELISA reader. The specific steps of S5 are as follows: S51, Add colorimetric substrate solution: Add 100μL of colorimetric substrate solution to each microplate, gently shake the microplate for 10-20s, and place it in the dark; S52, colorimetric reaction: Incubate at room temperature (27℃) in the dark for 17 min. During this process, the enzyme in the fluorescently labeled streptavidin catalyzes the substrate to produce a colorimetric reaction. The color intensity of the solution is negatively correlated with the concentration of nifedipine. S53, Termination of reaction: Add 50 μL of termination solution to each well, gently shake the microplate for 20 s to terminate the colorimetric reaction, at which point the solution color is stable; S54, Signal detection: Place the microplate into the microplate reader, set the detection wavelength to 450nm, read the absorbance value (OD value) of each microplate, and record the detection data; S6, Result Judgment and Quantitative Analysis: Preprocess the detection data, plot the standard curve, substitute the sample absorbance value to calculate the concentration of nifedipine in the original sample and determine the result; The specific steps of S6 are as follows: S61, Data preprocessing: Calculate the average absorbance values ​​of parallel wells for each concentration standard and parallel wells for the sample, subtract the absorbance values ​​of the blank control wells, and obtain the corrected absorbance values. S62, Plot the standard curve: Plot the standard working solution concentration as the x-axis and the corrected absorbance value as the y-axis. Use the linear regression method to plot the standard curve and obtain the regression equation y=ax+b, where a is the slope, b is the intercept, and the correlation coefficient R²≥0.99. S63, Sample concentration calculation: Substitute the corrected absorbance value of the sample solution to be tested into the standard curve regression equation to calculate the concentration of nifedipine in the sample solution, and then convert the actual concentration of nifedipine in the original sample according to the dilution factor of S1. S64. Result determination: If the actual concentration of nifedipine in the sample is ≥ the detection limit (0.01 ng / mL), it is determined to be positive; if it is < the detection limit, it is determined to be negative.

[0038] The detection kits prepared in Examples 1-3 above were compared, and the following data were obtained: Example 1 Example 2 Example 3 Storage stability It can be stably stored for 12 months when refrigerated at 2-8℃. It can be stably stored for 12 months when refrigerated at 2-8℃. It can be stably stored for 12 months when refrigerated at 2-8℃. Recovery rate 85% 90% 88% As shown in the table above, the test kits prepared in Examples 1-3 all showed good performance in terms of storage stability and recovery rate. After use, Example 2 showed the best results.

[0039] Although the present invention has been described above with reference to embodiments, various modifications can be made and components can be replaced with equivalents without departing from the scope of the invention. In particular, as long as there is no structural conflict, the features in the disclosed embodiments can be combined with each other in any manner. The lack of an exhaustive description of these combinations in this specification is merely for the sake of brevity and resource conservation. Therefore, the present invention is not limited to the specific embodiments disclosed herein, but includes all technical solutions falling within the scope of the claims.

Claims

1. A test kit for nictinamide, characterized by, The kit comprises a sample universal treatment solution, a nocardia specific probe, a fluorescently labeled streptavidin, a washing solution, a color developing substrate solution, a termination solution, a nocardia standard, and a reaction microplate. The sample universal treatment solution is prepared by mixing acetonitrile, potassium dihydrogen phosphate buffer and disodium ethylenediaminetetraacetate at a volume ratio of 3:6:1, wherein the concentration of the potassium dihydrogen phosphate buffer is 0.1 mol / L, and the concentration of the disodium ethylenediaminetetraacetate is 0.05 mol / L. The nocardia specific probe is a biotin-labeled nocardia monoclonal antibody, and the concentration is 1 mg / mL. The surface of the reaction microplate is coated with a nocardia hapten.

2. The test kit for nictinoid according to claim 1, characterized by, The concentration of the fluorescently labeled streptavidin is 0.5 mg / mL, and the fluorescent label is fluorescein isothiocyanate.

3. The test kit for nictinoid according to claim 1, characterized by, The washing solution is a Tris-HCl buffer solution containing 0.05% Tween-20, and the concentration is 0.02 mol / L.

4. The test kit for nictinoid according to claim 1, characterized by, The color developing substrate solution is prepared by mixing a substrate A solution and a substrate B solution at a volume ratio of 1:1, wherein the substrate A solution contains 3,3',5,5'-tetramethylbenzidine at a concentration of 0.5 mg / mL, and the substrate B solution is a citric acid buffer solution containing 0.02% hydrogen peroxide, and the pH is 4.

0.

5. The test kit for nictin according to claim 1, characterized by, The termination solution is a 2 mol / L sulfuric acid solution.

6. The test kit for nictin according to claim 1, wherein The concentration gradient of the nocardia standard is 0 ng / mL, 0.1 ng / mL, 0.5 ng / mL, 1 ng / mL, 5 ng / mL, and 10 ng / mL.

7. A detection method for a detection kit for nigrin, characterized by, The kit comprises the following specific steps: S1, sample pretreatment: collecting and pretreating the sample, adding the sample universal treatment solution for extraction, centrifuging to obtain the supernatant, diluting with the washing solution to obtain the sample solution to be detected; S2, reagent kit preparation: equilibrating the reagent kit components at room temperature, diluting the nocardia standard to obtain a standard working solution, and preparing a mixed reaction solution of the nocardia specific probe and the fluorescently labeled streptavidin; S3, immune competition reaction: adding the standard working solution, the sample solution to be detected, and a blank control solution to the reaction microplate, and then adding the mixed reaction solution, incubating, and washing; S4, antigen-antibody complex strengthening and residual impurity shielding: adding a complex strengthening solution and an impurity shielding agent to the washed microplate, incubating for a short time, and then washing simply; S5, color development and signal detection: adding the color developing substrate solution to incubate in the dark, adding the termination solution to terminate the reaction, and reading the absorbance value by an enzyme-labeled instrument; S6, result determination and quantitative analysis: preprocessing the detection data, drawing a standard curve, calculating the nocardia concentration in the original sample by substituting the sample absorbance value, and determining the result.

8. The detection method of a detection kit for nictinoid according to claim 7, characterized by, The specific steps of S1 are as follows: S11, sample collection and pretreatment: collecting the corresponding sample according to the detection requirement, crushing the feed sample through an 80-mesh screen, weighing 1-2 g of the screened sample, centrifuging the serum sample to remove the precipitate, taking the supernatant, naturally air-drying the soil sample, screening it through a 100-mesh screen, weighing 2-3 g of the screened sample, directly taking the supernatant of the water sample, and placing the various samples in a 50-mL centrifuge tube; S12, extraction: adding 10 mL of the sample universal treatment solution to the centrifuge tube, vortexing for 5 min to mix the sample and the treatment solution well, and achieving efficient extraction of nocardia. S13, Centrifugation: Place the shaken centrifuge tube in a centrifuge and centrifuge at 10000 r / min for 10 min to obtain the supernatant containing benzoin. S14, dilution and purification: Take 1-2 mL of supernatant, add 4-5 mL of washing buffer for dilution, vortex for 1 min to obtain the sample solution to be tested, which will be used for subsequent immune reactions; The specific steps of S2 are as follows: S21, Kit component equilibration: Remove all components in the kit from the refrigerated environment and place them at room temperature for 30-35 minutes to ensure the activity of each component is stable; S22, Standard dilution: Take the Norfucoagulant standard and dilute each concentration of the standard with washing buffer at a ratio of 1:10 to obtain standard working solutions with concentrations of 0 ng / mL, 0.01 ng / mL, 0.05 ng / mL, 0.1 ng / mL, 0.5 ng / mL, and 1 ng / mL, respectively. S23, Preparation of the mixed solution of probe and fluorescently labeled streptavidin: Take the noradrencin-specific probe and fluorescently labeled streptavidin at a volume ratio of 1:2, add washing solution to dilute 10 times, vortex for 30-40s to prepare a mixed reaction solution.

9. The detection method of a detection kit for nictinoid according to claim 7, characterized by, The specific steps of S3 are as follows: S31, Sample addition: Add 100 μL of standard working solution and 100 μL of sample solution to be tested to different wells of the reaction microplate, and set up blank control wells at the same time; S32, Add the mixed reaction solution: Add 50 μL of the probe and fluorescently labeled streptavidin mixture to each microwell, and gently shake the microplate for 1-3 min to fully mix the liquid in the well; S33, Incubation reaction: Place the microplate in a constant temperature incubator and incubate at 38-40℃ for 20-25 min. During this process, the norogenin in the sample competes with the hapten coated on the surface of the microplate for binding to the specific probe, forming an antigen-antibody complex. S34, Washing: After incubation, discard the liquid in the wells, add 200 μL of washing solution to each microwell, let stand for 30-40 seconds and then discard, repeat washing 3-5 times, after the last wash, invert the microplate and use absorbent paper to dry the residual liquid to remove unbound probes and impurities. The specific steps of S4 are as follows: S41, Add complex enhancement solution: Add 30 μL of complex enhancement solution to each washed microwell, gently shake the microplate for 30-50 seconds to ensure that the enhancement solution is in full contact with the antigen-antibody complex in the microwell and enhance the stability of complex binding. S42, Add residual impurity shielding agent: Add 20 μL of impurity shielding agent to each micropore and vortex for 1-3 min; S43, Short incubation: Place the microplate in an environment of 25-30℃ for 5-10 minutes to ensure that the strengthening liquid and the shielding agent can play their full role; S44, Simple Washing: Add 200 μL of washing solution to each microwell, let stand for 20-30 seconds and then discard. Invert the well to absorb any remaining liquid, removing only excess reinforcing solution and shielding agent, while retaining stable antigen-antibody complexes and bound shielding impurities.

10. The method according to claim 7, wherein the method is a method for detecting a test kit for nobelatins, characterized by, The specific steps of S5 are as follows: S51, Add colorimetric substrate solution: Add 100μL of colorimetric substrate solution to each microplate, gently shake the microplate for 10-20s, and place it in the dark; S52, colorimetric reaction: Incubate at room temperature in the dark for 15-17 min. During this process, the enzyme in the fluorescently labeled streptavidin catalyzes the substrate to produce a colorimetric reaction. The color intensity of the solution is negatively correlated with the concentration of nifedipine. S53, Termination of reaction: Add 50 μL of termination solution to each well, gently shake the microplate for 10-20 s to terminate the colorimetric reaction. At this time, the solution color is stable. S54, Signal detection: Place the microplate into the microplate reader, set the detection wavelength to 450nm, read the absorbance value of each microplate, and record the detection data; The specific steps of S6 are as follows: S61, Data preprocessing: Calculate the average absorbance values ​​of parallel wells for each concentration standard and parallel wells for the sample, subtract the absorbance values ​​of the blank control wells, and obtain the corrected absorbance values. S62, Plot the standard curve: Plot the standard working solution concentration as the abscissa and the corrected absorbance value as the ordinate using the linear regression method to obtain the regression equation y=ax+b, where a is the slope and b is the intercept. S63, Sample concentration calculation: Substitute the corrected absorbance value of the sample solution to be tested into the standard curve regression equation to calculate the concentration of nifedipine in the sample solution, and then convert the actual concentration of nifedipine in the original sample according to the dilution factor of S1. S64, Result Interpretation: If the actual concentration of nifedipine in the sample is ≥ the detection limit, it is judged as positive; If the result is less than the detection limit, the result is considered negative.