A method for detecting ractopamine in livestock products and its application

Abstract

This invention belongs to the field of livestock product testing, specifically relating to the detection of ractopamine, and particularly to a method for detecting ractopamine in livestock products and its application. First, RAC nucleic acid aptamers and probes are annealed to form double strands, which are then bound to magnetic iron oxide. When the target analyte is present, the aptamer binds to it, and then binds to AuNPs modified with Hp-Sp probes. In the presence of streptavidin-horseradish peroxidase, the color changes from blue to green upon the addition of sodium azide solution, achieving visual qualitative detection. Quantitative detection is achieved by analyzing the relationship between the absorbance of the test solution at 414 nm and the concentration of ractopamine. The detection method of this application is simple, highly sensitive, has a short detection time, and achieves visual detection, showing high application potential in practical sample testing.

Description

Technical Field

[0001] This invention belongs to the field of livestock product testing, and relates to the detection of ractopamine, specifically a method for detecting ractopamine in livestock products and its application. Background Technology

[0002] Ractopamine is a beta-agonist drug. Consuming large amounts of meat or offal containing ractopamine residues may cause poisoning symptoms. Regulations regarding the applicability and safety of this drug in animal husbandry vary from country to country.

[0003] After using this drug, animals' metabolism is altered, prompting them to grow more muscle rather than fat, resulting in leaner pork. Because muscle is heavier than fat, it also makes the pigs heavier. On the surface, this means consumers can reduce fat in their diets, and producers can raise heavier pigs using less feed. However, ractopamine is far from an ideal additive because it is more likely to cause problems than other additives. A 2012 food and environment report stated that the effects of ractopamine include: "hyperactivity, tremors, weakness in the limbs, inability to walk, and death."

[0004] Currently, detection methods for ractopamine antibiotics include immunoassay, capillary electrophoresis, electrochemical methods, mass spectrometry, and high-performance liquid chromatography (HPLC). Immunoassay offers high specificity but suffers from drawbacks such as high background absorption, cumbersome operation (requiring numerous incubation and washing steps), and susceptibility of enzymes to sample influence. Capillary electrophoresis offers fast analysis speed, high separation efficiency, and low operating costs, but the technology is not yet mature and is unsuitable for small molecule detection. Mass spectrometry and HPLC require large, expensive instruments. Patent CN 103343126A discloses a ractopamine aptamer and an electrochemical biosensor for detecting ractopamine. This sensor primarily utilizes the DPV spectrum and the relationship between peak current and concentration, but the detection limit is only in the nM range. To further improve the sensitivity of ractopamine detection, this project has conducted in-depth research on the form of the ractopamine aptamer, probe, and sensor. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention proposes a method for detecting ractopamine in livestock products and its application.

[0006] The technical solution of this invention is implemented as follows:

[0007] A method for detecting ractopamine in livestock products, comprising the following steps:

[0008] (1) The biotin-labeled RAC nucleic acid aptamer and probe stock solution were annealed and diluted with Tris-HCl buffer. Then, magnetic beads washed with Tris-HCl buffer were added, incubated and washed again to obtain modified magnetic beads. The modified magnetic beads solution was resuspended with PBS buffer.

[0009] (2) The mixture of AuNPs solution, Hp probe solution, Sp probe solution and PBS buffer was incubated, centrifuged and the supernatant was discarded, and then resuspended in PBS buffer to obtain AuNPs solution modified with Hp-Sp probe.

[0010] (3) After mixing the magnetic bead solution from step (1) with the sample solution to be tested and PBS buffer, the mixture is incubated. After washing the resulting solution with PBS buffer, the AuNPs solution modified with Hp-Sp probe from step (2) is added, and the mixture is incubated again. After washing with PBS buffer, the resulting solid is resuspended in PBS buffer to obtain the sample detection solution.

[0011] (4) Add streptavidin-horseradish peroxidase to the sample detection solution in step (3), incubate, wash with PBS buffer, add acetate buffer solution containing ABTS and H2O2, incubate, add sodium azide solution to observe color change, and detect UV absorbance at 414nm.

[0012] (5) Substitute the absorbance value obtained in step (4) into the linear equation of absorbance and ractopamine concentration to obtain the amount of ractopamine in the livestock product sample.

[0013] Furthermore, in step (1), the sequence of the biotin-labeled RAC nucleic acid aptamer is shown in SEQ ID No. 1, and its 5' end is connected to biotin; the sequence of the probe is shown in SEQ ID No. 2; and the concentration of the probe reservoir is 10 μM.

[0014] Furthermore, the annealing conditions are 95℃ for 5 min and 0℃ for 10 min; the pH of the Tris-HCl buffer is 7, and the concentration of the modified magnetic bead solution is 3 mg / mL.

[0015] Furthermore, the preparation method of AuNPs solution in step (2) is as follows: quickly add 10 mL of 39 mM trisodium citrate to 100 mL of 1 mM chloroauric acid solution at 100 °C, continue boiling for 15 min, and then stir and cool to room temperature.

[0016] Furthermore, the sequence of the Hp probe is shown in SEQ ID No. 3, and its 5' end is provided with a thiol group; the sequence of the Sp probe is shown in SEQ ID No. 4, and its 5' end is provided with biotin.

[0017] Furthermore, the concentrations of the Hp probe solution and the Sp probe solution are 10 μM, the PBS buffer is a 10 mM PBS solution with pH 7.0 containing 0.3 M NaCl, and the volume ratio of AuNPs solution, Hp probe solution and Sp probe solution is 5:2:2; the total volume of the mixture is 500 μL.

[0018] Furthermore, in step (3), the sample solution to be tested is a solution obtained by crushing livestock products and passing them through a 100-mesh sieve; the volume ratio of the magnetic bead solution, the sample solution to be tested, and the AuNPs solution modified with Hp-Sp probe is 4:4:12.5, and the volume of the PBS buffer used for resuspension is 100 μL with pH 7.0.

[0019] Furthermore, in step (4), the concentration of streptavidin-horseradish peroxidase is 0.01 mg / mL, the acetate buffer solution is an acetate buffer solution with a concentration of 0.1 M and pH 4.2 containing 2 mM ABTS and 2.5 mM H2O2, and the concentration of sodium azide solution is 10 mM; the volume ratio of sample detection solution, streptavidin-horseradish peroxidase, acetate buffer solution containing ABTS and H2O2 and sodium azide solution is 100:0.5:200:2.

[0020] Furthermore, the linear equation between absorbance and ractopamine concentration is y = 0.002x + 0.337, R² = 0.964.

[0021] The above-mentioned detection methods are applied to the detection of pork, mutton, beef, and chicken.

[0022] The present invention has the following beneficial effects:

[0023] 1. The detection principle of this application is as follows: RAC nucleic acid aptamers and probes are first annealed to form double strands and then bound to magnetic iron oxide. When the target analyte is present, the aptamer binds to the target analyte and then binds to AuNPs modified with Hp-Sp probes. When streptavidin-horseradish peroxidase is present, the color changes from blue to green after the addition of sodium azide solution, thus achieving visual qualitative detection. The relationship between the wavelength of the test solution at 414 nm and the absorbance and the concentration of cladopamine is analyzed to achieve quantitative detection.

[0024] 2. Using the detection method of this application, the linear equation between absorbance and ractopamine concentration is y = 0.002x + 0.337, R² = 0.964, and the detection limit reaches the pM level. Within the target gene concentration range of 0.01 nM to 500 nM, there is a good linear relationship between the peak value of the ultraviolet absorption peak and the logarithm of the target gene concentration, with a detection limit as low as 3.2 pM. This invention improves the reaction rate and reduces the complexity of operation by introducing the streptavidin-horseradish peroxidase-catalyzed amplification reaction. Attached Figure Description

[0025] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0026] Figure 1 This is a schematic diagram of the detection principle in this application.

[0027] Figure 2 The absorbance curves are for different concentrations.

[0028] Figure 3 This represents a linear relationship between concentration and absorbance.

[0029] Figure 4 This is a diagram showing the specific experimental results of this application.

[0030] Figure 5 This is a secondary structure diagram of the aptamer and probe used in this application.

[0031] Figure 6 This is a diagram showing the results of a specific experiment. From left to right, the solutions are: control, clenbuterol hydrochloride, ractopamine, salbutamol, cimaterol, and phenylethanolamine A reaction termination solution. Detailed Implementation

[0032] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0033] The nucleotide sequences used in this application are shown in the table below:

[0034]

[0035] Example 1

[0036] A method for detecting ractopamine in livestock products, comprising the following steps:

[0037] (1) The biotin-labeled RAC nucleic acid aptamer and 10 μM probe stock solution were annealed and then diluted with Tris-HCl buffer at pH 7. Magnetic beads washed with Tris-HCl buffer were added, and the mixture was incubated and washed again to obtain modified magnetic beads. The modified magnetic beads solution was resuspended in PBS buffer to obtain a modified magnetic bead solution with a concentration of 3 mg / mL. The annealing conditions were 95℃ for 5 min and 0℃ for 10 min.

[0038] (2) The mixture of 50 μL AuNPs solution with 20 μL 10 μM Hp probe solution, 20 μL 10 μM Sp probe solution and PBS buffer was incubated. The total volume of the mixture was 500 μL. After centrifugation and discarding the supernatant, the mixture was resuspended in PBS buffer to obtain AuNPs solution modified with Hp-Sp probe. The AuNPs solution was prepared by rapidly adding 10 mL 39 mM trisodium citrate to 100 mL 1 mM chloroauric acid solution at 100 °C, boiling for 15 min, and then stirring and cooling to room temperature. The PBS buffer was a 10 mM PBS solution with pH 7.0 containing 0.3 M NaCl.

[0039] (3) Prepare ractopamine standards with concentrations of 0, 0.01, 0.05, 0.1, 0.5, 1, 5, 10, 50, 100, 300, and 500 nM using PBS buffer. Then, take 8 μL of the magnetic bead solution from step (1), mix it with 8 μL of the ractopamine standard solution and PBS buffer, and incubate it. After washing the resulting solution with PBS buffer, add 25 μL of AuNPs solution modified with Hp-Sp probe, incubate again, wash with PBS, and then resuspend the resulting solid in PBS buffer to obtain the sample detection solution. The volume of PBS buffer used for resuspension is 100 μL and the pH is 7.0.

[0040] (4) Take 100 μL of the sample detection solution treated in step (3) and add 0.5 μL of 0.01 mg / mL streptavidin-horseradish peroxidase. After incubation, wash with PBS buffer and then add 200 μL of 0.1 M, pH 4.2 acetate buffer solution containing 2 mM ABTS and 2.5 mM H2O2. After incubation, add 2 μL of 10 mM sodium azide solution and observe the color change. As the concentration increases, the color of the solution changes from blue to green. Detect the ultraviolet absorbance at 414 nm.

[0041] (5) A standard curve was established with the concentration of ractopamine standard as the x-axis and the ultraviolet absorbance at 414 nm as the y-axis. The results are as follows: Figure 3 As shown, the fitted standard curve is: y = 0.002x + 0.337, R² = 0.964.

[0042] Example 2

[0043] Specificity test:

[0044] The dopamine standard solution in Example 1 was replaced with blank PBS buffer, and PBS buffers containing 10 nM clenbuterol hydrochloride, ractopamine, salbutamol, cimaterol, and phenylethanolamine A, respectively, and the experimental procedure of Example 1 was repeated. After adding sodium azide solution, it was observed that only the color of the solution changed to green, as shown in the example. Figure 6 As shown, the colors of the other solutions did not change. The UV absorbance at 414 nm was measured, and the results are as follows. Figure 4 As shown, by Figure 4 It can be seen that the sensor in this application has specificity for recognizing ractopamine.

[0045] Application examples

[0046] Pork was purchased from the market, and 100g of pork was ground into a paste using a grinder. The paste was then squeezed through 100-mesh gauze to obtain the sample solution to be tested, replacing the dopamine standard solution in Example 1. The solution was tested, and it was observed that the color of the solution did not change. The absorbance value at 414nm was 0 after correction with PBS buffer, which further confirmed that the pork did not contain ractopamine.

[0047] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for detecting ractopamine in livestock products, characterized in that, Here are the steps: (1) The biotin-labeled RAC nucleic acid aptamer and probe stock solution were annealed and diluted with Tris-HCl buffer. Then, magnetic beads washed with Tris-HCl buffer were added, incubated and washed again to obtain modified magnetic beads. The modified magnetic beads solution was resuspended with PBS buffer solution. (2) The mixture of AuNPs solution with Hp probe solution, Sp probe solution and PBS buffer solution was incubated, centrifuged and the supernatant was discarded and then resuspended in PBS buffer solution to obtain AuNPs solution modified with Hp-Sp probe. (3) After mixing the magnetic bead solution from step (1) with the sample solution to be tested and PBS buffer solution, the resulting solution is washed with PBS buffer solution and then the AuNPs solution modified with Hp-Sp probe from step (2) is added, and the solution is incubated again. After washing with PBS buffer solution, the resulting solid is resuspended with PBS buffer solution to obtain the sample detection solution. (4) Add streptavidin-horseradish peroxidase to the sample detection solution in step (3), incubate, wash with PBS buffer solution, then add acetate buffer solution containing ABTS and H2O2, incubate, add sodium azide solution to observe color change, and detect UV absorbance at 414nm. (5) Substitute the absorbance value obtained in step (4) into the linear equation of absorbance and ractopamine concentration to obtain the amount of ractopamine in the livestock product sample. The sequence of the biotin-labeled RAC nucleic acid aptamer in step (1) is shown in SEQ ID No. 1, with biotin attached to its 5' end; the sequence of the probe is shown in SEQ ID No. 2; the concentration of the probe reservoir is 10 μM; The sequence of the Hp probe is shown in SEQ ID No. 3, and it has a thiol group at its 5' end. The sequence of the Sp probe is shown in SEQ ID No. 4, and it has a biotin group at its 5' end.

2. The method for detecting ractopamine in livestock products according to claim 1, characterized in that: The annealing conditions were 95℃ for 5 min and 0℃ for 10 min; the pH of the Tris-HCl buffer was 7, and the concentration of the modified magnetic bead solution was 3 mg / mL.

3. The method for detecting ractopamine in livestock products according to claim 1, characterized in that, The preparation method of AuNPs solution in step (2) is as follows: quickly add 10 mL of 39 mM trisodium citrate to 100 mL of 1 mM chloroauric acid solution at 100 °C, continue boiling for 15 min, and then stir and cool to room temperature.

4. The method for detecting ractopamine in livestock products according to claim 3, characterized in that: The concentrations of the Hp probe solution and Sp probe solution were 10 μM. The PBS buffer solution was a 10 mM PBS solution containing 0.3 M NaCl at pH 7.

0. The volume ratio of AuNPs solution, Hp probe solution and Sp probe solution was 5:2:

2. The total volume of the mixture was 500 μL.

5. The method for detecting ractopamine in livestock products according to claim 1, characterized in that: In step (3), the sample solution to be tested is a solution obtained by crushing livestock products and passing them through a 100-mesh sieve; the volume ratio of the magnetic bead solution, the sample solution to be tested, and the AuNPs solution modified with Hp-Sp probe is 4:4:12.5, and the volume of the PBS buffer solution used for resuspending is 100 μL with pH 7.

0.

6. The method for detecting ractopamine in livestock products according to claim 1, characterized in that: In step (4) The concentration of streptavidin-horseradish peroxidase was 0.01 mg / mL. The acetate buffer solution was a 0.1 M, pH 4.2 acetate buffer containing 2 mM ABTS and 2.5 mM H2O2. The concentration of sodium azide solution was 10 mM. The volume ratio of sample detection solution, streptavidin-horseradish peroxidase, acetate buffer solution containing ABTS and H2O2 and sodium azide solution was 100:0.5:200:

2.

7. The method for detecting ractopamine in livestock products according to claim 6, characterized in that: The linear equation between absorbance and ractopamine concentration is y = 0.002x + 0.337, R² = 0.

964.

8. The application of the detection method according to any one of claims 1-7 in the detection of pork, mutton, beef, and chicken.

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