Method for rapidly detecting various veterinary drug residues in dairy product by adopting biochip

Abstract

The invention discloses a method for rapidly detecting various veterinary drug residues in a dairy product by adopting a biochip. The method comprises the following steps: (a) recovering a dairy product sample to be detected, a biochip detection card and micropores to room temperature; (b) selecting a biochip detection card and a micropore, marking the biochip detection card and the micropore, and preheating the micropore on a dry heater; (c) adding a to-be-detected sample into the micropores, repeatedly sucking, beating and uniformly mixing until a micropore solid is completely dissolved, and incubating; (d) transferring all the liquid in the micropores into a sample adding hole of the biochip detection card, uniformly mixing and reacting; and (e) interpreting a result. The method for detecting the chloramphenicol, the lincomycin, the tylosin and the tilmicosin in the dairy product is low in detection limit and can meet the dairy product limit requirement formulated in the national standard.

Description

Technical Field

[0001] This invention belongs to the field of biochip technology, specifically relating to a method for rapid detection of multiple veterinary drug residues in dairy products using biochips. Background Technology

[0002] Veterinary drug residues refer to the residues of parent compounds, metabolites, or conjugates of veterinary drugs remaining in food derived from treated animals. Veterinary drug residues in dairy products are mainly caused by the overuse of veterinary drugs during animal husbandry. This overuse not only affects human health, producing adverse effects such as toxicity, carcinogenicity, and teratogenicity, but also increases the body's resistance to microorganisms. Common symptoms include toxic reactions, allergic reactions, secondary infections, nausea, and vomiting. Some individuals may also experience urticaria, fever, or even anaphylactic shock. Veterinary drug residues in dairy products directly endanger consumers' health. Currently, the main detection methods for veterinary drug residues in dairy products include microbial detection methods, immunoassays, liquid chromatography, gas chromatography, liquid chromatography-mass spectrometry (LC-MS), and gas chromatography-mass spectrometry (GC-MS). Enzyme-linked immunosorbent assay (ELISA) and liquid chromatography-mass spectrometry (LC-MS) require complex pretreatment and can take several hours to days. Single-target test strips cannot meet the needs of simultaneous detection of multiple residues, resulting in low detection efficiency. With the acceleration of economic globalization and internationalization, the quality and safety of dairy products has become a global public health concern. Among these concerns, veterinary drug residues are a significant factor affecting dairy product safety. Therefore, establishing an efficient, accurate, and rapid method for detecting commonly used veterinary drug residues in dairy products is of paramount importance. Summary of the Invention

[0003] The purpose of this invention is to provide a method for rapid detection of multiple veterinary drug residues in dairy products using a biochip.

[0004] The above-mentioned objective of this invention can be achieved through the following technical solution: a method for rapid detection of multiple veterinary drug residues in dairy products using a biochip, comprising the following steps:

[0005] (a) Allow the dairy product sample to be tested, the biochip detection card, and the microwell to return to room temperature;

[0006] (b) Select the biochip detection card and microwell, mark them, and preheat the microwell on a dry heater;

[0007] (c) Take the dairy sample to be tested and add it into the microwell. Mix it repeatedly by suction and beating until the solid in the microwell is completely dissolved, and then incubate.

[0008] (d) Transfer all the liquid in the microwell to the sample well of the biochip detection card, mix well and react;

[0009] (e) Interpretation result.

[0010] In the above-mentioned method for rapid detection of multiple veterinary drug residues in dairy products using biochips:

[0011] Preferably, step (a) of restoring the dairy sample to be tested to room temperature includes: warming the frozen or refrigerated dairy product to room temperature of 20~25°C and mixing it thoroughly.

[0012] Preferably, the dry heater in step (b) is kept at a constant temperature of 40±1℃.

[0013] Preferably, in step (c), 300 μL of the dairy sample to be tested is added to the microwell.

[0014] Preferably, incubation in step (c) lasts for 5 minutes.

[0015] Preferably, in step (c), the repeated aspiration and mixing is performed using a pipette, and the number of aspirations is not less than 5.

[0016] Preferably, the liquid transfer in step (d) needs to be completed within 30 seconds.

[0017] Preferably, in step (d), the mixture is reacted for 10 minutes after mixing.

[0018] Preferably, the interpretation results in step (e) include negative: the sample does not contain the analyte or is below the detection limit; suspected positive: the sample contains the analyte near the detection limit; positive: the sample contains the analyte above the detection limit.

[0019] Preferably, the test substance in step (e) is chloramphenicol, lincomycin, tylosin, or tilmicosin.

[0020] The method of this invention utilizes a biochip detection method based on the antigen-antibody binding principle to rapidly determine the levels of chloramphenicol, lincomycin, tylosin, and tilmicosin in dairy products. For example, the surface of the biochip detection card is immobilized with a specific antibody probe array targeting these four veterinary drug residues; and / or the microwells are pre-coated with antigens for these four veterinary drug residues.

[0021] The present invention has the following advantages:

[0022] (1) The biochip method of the present invention is simple, fast and superior to the national standard method for sample pretreatment. The biochip method can simultaneously perform qualitative detection and analysis of four kinds of veterinary drug residues and does not have high requirements for detection equipment.

[0023] (2) The detection limits of the biochip detection card method for detecting chloramphenicol, lincomycin, tylosin and tilmicosin in dairy products in this invention are 0.3 μg / kg, 30 μg / kg, 10 μg / kg and 7 μg / kg, respectively; the limits in the national standards are: not detectable, 150 μg / kg, 100 μg / kg and 50 μg / kg. The biochip detection method of this invention can meet the limits for dairy products set in the national standards.

[0024] (3) The biochip detection method of the present invention is rapid, accurate, efficient and highly sensitive, and is suitable for the detection of chloramphenicol, lincomycin, tylosin and tilmicosin in dairy products. Detailed Implementation

[0025] To further illustrate the technical means and effects of this invention, the following embodiments are provided for further explanation. It should be understood that the specific embodiments described herein are merely illustrative of the invention and not intended to limit it.

[0026] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased from legitimate channels.

[0027] Example 1

[0028] The method for rapid detection of multiple veterinary drug residues in dairy products using a biochip provided in this embodiment includes the following steps:

[0029] (a) Sample pretreatment: Take 1 mL of refrigerated milk sample, let it stand at room temperature (23℃) for 15 minutes, and vortex to mix.

[0030] The biochip detection card and microwell were placed in an environment of 23°C for 10 minutes to equilibrate.

[0031] The surface of the biochip detection card is immobilized with a specific antibody probe array targeting four veterinary drug residues: chloramphenicol, lincomycin, tylosin, and tilmicosin.

[0032] The micropores are pre-coated with antigens containing four veterinary drug residues: chloramphenicol, lincomycin, tylosin, and tilmicosin.

[0033] The biochip detection card and microwell were provided by Meizheng Biotechnology Co., Ltd.

[0034] (b) Equipment preheating:

[0035] Select the biochip detection card and microwell, label them, and place the microwell on a 40℃ dry heater;

[0036] (c) Incubation reaction:

[0037] Use a pipette to add 300 μL of milk sample to the microwell, and aspirate repeatedly 8 times until the solid is completely dissolved; incubate at 40°C for 5 minutes (timing error ±5 seconds).

[0038] (d) Chip loading:

[0039] Transfer all the liquid in the microwell to the sample well of the biochip detection card within 20 seconds and shake gently to mix.

[0040] React at room temperature for 10 minutes (avoid direct sunlight);

[0041] (e) Result reading:

[0042] After the reaction is complete, remove the film covering the window of the biochip detection card and place it in the reader for interpretation.

[0043] Table 1 Result Interpretation

[0044]

[0045] .

[0046] Example 2

[0047] The detection limit of chloramphenicol was determined using the method described in Example 1.

[0048] Blank matrix milk samples (spiked at concentrations of 0.1 μg / kg, 0.5 μg / kg, and 1 μg / kg) were diluted and measured according to the biochip card detection method (double-well, double-parallel). Table 2-3 shows that the detection limit (0.3 μg / kg) of the biochip card detection method is negative when the concentration is higher than the spiked concentration (0.1 μg / kg), and positive when the concentration is lower than the spiked concentrations (0.5 μg / kg and 1 μg / kg).

[0049] Table 2. Detection limits of chloramphenicol (biochip method)

[0050]

[0051] Table 3. Detection results of chloramphenicol biochip

[0052]

[0053] The detection limit of lincomycin was determined using the method described in Example 1.

[0054] Blank matrix milk samples (spiked at concentrations of 10 μg / kg, 40 μg / kg, and 60 μg / kg) were diluted and measured using the biochip card detection method (double-drilled, double-parallel). Table 4-5 shows that the detection limit of the biochip card method (20-30 μg / kg) is negative at concentrations above the spiked concentration (10 μg / kg) and positive at concentrations below the spiked concentrations (40 μg / kg and 60 μg / kg).

[0055] Table 4. Limits of detection for lincomycin (biochip method)

[0056]

[0057] Table 5. Detection results of lincomycin biochip

[0058]

[0059] The detection limit of tylosin was determined using the method described in Example 1.

[0060] Blank matrix milk samples (spiked at concentrations of 0.5 μg / kg, 15 μg / kg, and 30 μg / kg) were diluted and measured according to the biochip card detection method (double-well, double-parallel). Table 6-7 shows that the detection limit of the biochip card method (5-10 μg / kg) is negative at concentrations above the spiked concentration (0.5 μg / kg) and positive at concentrations below the spiked concentrations (15 μg / kg, 30 μg / kg).

[0061] Table 6. Limits of detection for tylosin (biochip method)

[0062]

[0063] Table 7. Results of Tylosin Detection Using Biochip (Biochip Method)

[0064]

[0065] The detection limit of tilmicosin was detected using the method described in Example 1.

[0066] Blank matrix milk samples (spiked at concentrations of 4 μg / kg, 8 μg / kg, and 16 μg / kg) were diluted and measured according to the biochip card detection method (double-well, double-parallel). Table 8-9 shows that the detection limit of the biochip card method is 4-7 μg / kg. At concentrations above the spiked concentration (4 μg / kg), the detection result is negative; at concentrations below the spiked concentrations (8 μg / kg, 16 μg / kg), the detection result is positive.

[0067] Table 8. Limit of detection for tilmicosin (biochip method)

[0068]

[0069] Table 9. Results of Tilmicosin Biochip Detection (Biochip Method)

[0070]

[0071] The biochip method of this invention is simple, fast and superior to the national standard method for sample pretreatment. The biochip method can simultaneously perform qualitative detection and analysis of four veterinary drug residues and has low requirements for detection equipment. However, the biochip method cannot perform quantitative analysis of sample detection results at present and can only be used for rapid qualitative detection of samples, which still has certain limitations.

[0072] It should be noted that the above embodiments are merely further illustrations of the present invention and not limitations. Any adjustments or changes made by those skilled in the art within the equivalent meaning and scope of the technical solutions of the present invention should be considered as included within the protection scope of the present invention.

Claims

1. A method for rapid detection of multiple veterinary drug residues in dairy products by using biochip, characterized in that, The method comprises the following steps: (a) restoring the dairy sample to be tested, the biochip detection card and the micro-hole to room temperature; (b) selecting the biochip detection card and the micro-hole, marking them well, and preheating the micro-hole on a dry heater; (c) taking the dairy sample to be tested and adding it into the micro-hole, mixing it repeatedly by pipetting until the solids in the micro-hole are completely dissolved, and then incubating; (d) transferring all the liquid in the micro-hole to the sample addition hole of the biochip detection card, mixing it well, and then reacting; (e) judging the result.

2. The method for rapid detection of multiple veterinary drug residues in dairy products by using biochip according to claim 1, characterized in that, The step (a) of restoring the dairy sample to be tested to room temperature comprises: restoring the frozen or refrigerated dairy product to room temperature of 20-25℃ and mixing it well.

3. The method for rapid detection of multiple veterinary drug residues in dairy products by using biochip according to claim 1, characterized in that, The dry heater in the step (b) is controlled at a constant temperature of 40±1℃.

4. The method for rapid detection of multiple veterinary drug residues in dairy products by using biochip according to claim 1, characterized in that, In the step (c), 300 μL of the dairy sample to be tested is taken and added into the micro-hole.

5. The method for rapid detection of multiple veterinary drug residues in dairy products by using biochip according to claim 1, characterized in that, The step (c) of mixing by repeatedly pipetting is completed by using a pipettor, and the pipetting times are not less than 5 times.

6. The method for rapid detection of multiple veterinary drug residues in dairy products by using biochip according to claim 1, characterized in that, The step (c) of incubating is for 5 minutes.

7. The method for rapid detection of multiple veterinary drug residues in dairy products by using biochip according to claim 1, characterized in that, The step (d) of transferring the liquid needs to be completed within 30 seconds.

8. The method for rapid detection of multiple veterinary drug residues in dairy products by using biochip according to claim 1, characterized in that, The step (d) of mixing and then reacting is for 10 minutes.

9. The method for rapid detection of multiple veterinary drug residues in dairy products by using biochip according to claim 1, characterized in that, The step (e) of judging the result comprises: negative: the sample does not contain the substance to be tested or is lower than the detection limit; suspected positive: the sample contains the substance to be tested near the detection limit; and positive: the sample contains the substance to be tested higher than the detection limit. The step (e) of judging the result comprises: negative: the sample does not contain the substance to be tested or is lower than the detection limit; suspected positive: the sample contains the substance to be tested near the detection limit; and positive: the sample contains the substance to be tested higher than the detection limit.

10. The method for rapid detection of multiple veterinary drug residues in dairy products by using biochip according to claim 9, characterized in that, The step (e) of judging the result comprises: negative: the sample does not contain the substance to be tested or is lower than the detection limit; suspected positive: the sample contains the substance to be tested near the detection limit; and positive: the sample contains the substance to be tested higher than the detection limit. The step (e) of judging the result comprises: negative: the sample does not contain the substance to be tested or is lower than the detection limit; suspected positive: the sample contains the substance to be tested near the detection limit; and positive: the sample contains the substance to be tested higher than the detection limit.

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