Universal pretreatment method based on immunoaffinity rapid detection

Abstract

The invention relates to the technical field of drug residue detection, in particular to a universal pretreatment method based on immunoaffinity rapid detection. A universal pretreatment method based on immunoaffinity rapid detection comprises the following steps: a reagent 1, a reagent 2, a reagent 3, a reagent 4 and a reagent 5 are used for treating a sample to be detected in sequence, and the reagent 1 is an ammonium citrate buffer solution; the reagent 2 is beta-glucuronidase; the reagent 3 is aryl sulfatase; the reagent 4 is a mixed solution of acetonitrile, methanol, ethanol and organic acid; the organic acid is citric acid or formic acid; and the reagent 5 is N-propylethylenediamine. The pretreatment method provided by the invention is suitable for residue detection pretreatment of various large drugs related to veterinary drug residues in animal food at present, and solves the problems that pretreatment operation needs to be carried out for many times when various drugs in the same sample need to be measured in food safety detection, and the time, equipment and reagent consumption is large.

Description

Technical Field

[0001] The present application relates to the technical field of drug residue detection, and in particular to a universal pretreatment method based on immunoaffinity rapid detection. Background Art

[0002] The level of veterinary drug residues in animal foods is an important part of food safety inspection. Excessive veterinary drug residues not only pose a serious threat to public health, but also affect the import and export trade of animal foods. Countries around the world attach great importance to it. Since drug residue detection belongs to the analysis of trace organic substances in complex mixtures, and the analysis volume is large, it is very necessary to adopt effective, fast and popular purification methods. Immunoaffinity technology has developed rapidly in recent years, and its application in veterinary drug residue detection has become more and more extensive. Immunoaffinity technology is a technical means to detect target compounds by using the specific reaction of antigens and antibodies. The commonly used methods are enzyme-linked immunosorbent assay (ELISA) and colloidal gold immunochromatography (GICA).

[0003] With the development of science and technology, convenient and high-precision rapid detection methods for veterinary drug residues have gradually become the main means of daily risk monitoring. At present, more and more rapid immunoaffinity detection kits have gradually emerged on the market, greatly improving the detection efficiency.

[0004] Regarding the above-mentioned related technologies, the applicant believes that the following problems exist: the sample pretreatment methods in the instructions of each test kit or test paper strip are different, and food safety testing usually requires the determination of multiple types of drugs on the same sample, which requires multiple pretreatment operations, consuming a lot of time, equipment and reagents.

[0005] To solve the above-mentioned problems, the constraints that need to be considered are: the physical and chemical properties of various types of veterinary drugs vary greatly, the polarity differences are obvious, and the solubility differences are large. A single extraction reagent cannot take into account the extraction efficiency requirements of multiple types of target analytes; the target analytes remaining in animal products exist in different forms and need to exist in the form of free pure compounds to be extracted and detected, and some target analytes have special residual forms, among which those containing phenolic hydroxyl structures, such as salbutamol, exist in animal tissues in a high proportion to form covalent conjugates with compounds in the animal body; some target analytes such as tetracyclines, fluoroquinolones and macrolides have the property of chelating with divalent metal ions to form insoluble precipitates.

[0006] Therefore, it is very necessary to overcome the above-mentioned constraints to quickly realize the pre-treatment of different drugs and improve the detection efficiency. Summary of the invention

[0007] In order to be applicable to the pretreatment of residue detection of various major categories of drugs involved in veterinary drug residues in animal foods, the present application provides a universal pretreatment method based on rapid immunoaffinity detection.

[0008] The above technical objectives of this application are achieved through the following technical solutions:

[0009] A universal pre-treatment method based on immunoaffinity rapid detection comprises the following steps:

[0010] S1. Take the animal product to be tested, homogenize it, and obtain the sample to be tested;

[0011] S2. Add reagent 1 to the sample to be tested obtained in S1 and mix well, then add reagent 2 and reagent 3 in sequence and mix well to obtain an initial mixed solution;

[0012] S3. The initial mixed solution is enzymatically hydrolyzed for 110-130 min to obtain an enzymatic hydrolyzate;

[0013] S4. Centrifuge the enzymatic hydrolyzate obtained in S3, and collect the supernatant and precipitate respectively;

[0014] S5. Add reagent 4 to the precipitate obtained in S4, mix, centrifuge, and collect the supernatant;

[0015] S6. combining the supernatant obtained in S4 with the supernatant obtained in S5 to obtain a mixed solution;

[0016] S7. Add reagent 5 to the mixture obtained in S6, mix well, oscillate, centrifuge, and retain the supernatant;

[0017] S8. The supernatant obtained in S7 is concentrated by nitrogen blowing, and then diluted and reconstituted with the sample diluent of the kit;

[0018] S9. Freeze and centrifuge the diluted and reconstituted solution obtained in S8, and take the supernatant for inspection;

[0019] The reagent 1 is ammonium citrate buffer;

[0020] The reagent 2 is β-glucuronidase;

[0021] The reagent 3 is arylsulfatase;

[0022] The reagent 4 is a mixture of acetonitrile, methanol, ethanol and an organic acid; the organic acid is citric acid or formic acid;

[0023] The reagent 5 is N-propylethylenediamine.

[0024] By adopting the above technical scheme, ammonium citrate has a strong divalent metal ion chelating ability. The sample to be tested is first treated with ammonium citrate, and ammonium citrate chelates divalent metal ions, and occupies the chelating sites of target analytes such as tetracyclines, fluoroquinolones and macrolides with divalent metal ions, thereby preventing the above target analytes such as tetracyclines, fluoroquinolones and macrolides from combining with divalent metal ions to form precipitation, thereby ensuring that target analytes such as tetracyclines, fluoroquinolones and macrolides are detected in a free state.

[0025] β-glucuronidase and arylsulfatase are used to hydrolyze the two common conjugated bonds formed by the target analyte in the animal body, so that the target analyte is freed from the conjugated form for extraction. There are also strong acid hydrolysis and strong base hydrolysis methods for hydrolyzing conjugated bonds, but the disadvantage is that they are not suitable for the pretreatment of drugs with poor stability under strong acid and base conditions. For example, tetracycline drugs are prone to isomerization or ring structure destruction under acid and base conditions. The enzymatic hydrolysis method has mild reaction conditions and avoids the problem of insufficient accuracy and recovery rate due to drug loss.

[0026] In addition, ammonium citrate buffer can provide a suitable pH environment for β-glucuronidase and arylsulfatase, making the enzyme catalytic activity of β-glucuronidase and arylsulfatase stronger.

[0027] After being treated with ammonium citrate, β-glucuronidase, and arylsulfatase successively and centrifuged, the solution contains some target analytes with strong polarity, and the precipitate contains most of the target analytes with weak polarity and some target analytes with strong polarity. Therefore, the precipitate needs to be treated later to dissolve the target analytes. Most target analytes are easily soluble in acetonitrile, which is also the most commonly used extraction solvent. It also has a strong precipitation effect to remove proteins in the sample. However, some target analytes require methanol and ethanol with stronger polarity to improve solubility. The three solvents are miscible and can meet the extraction requirements of various target analytes. Most target analytes are amine-containing compounds, which often form a certain strength of binding with proteins in the sample matrix, so organic acids are added to improve the extraction efficiency.

[0028] As a commonly used solid phase adsorbent in dispersed solid phase extraction, N-propylethylenediamine can effectively adsorb impurities in sample matrices such as small molecule organic acids, pigments and metal ions, reducing interference with detection.

[0029] In the present application, the sample to be tested is first treated with ammonium citrate to eliminate the interference of divalent metal ions on target analytes such as tetracyclines, fluoroquinolones and macrolides; then β-glucuronidase and arylsulfatase are added to hydrolyze the conjugated bonds, so that the target analytes are released from the conjugated form for extraction; then a solvent with strong solubility and a wide solubility range is used to dissolve the target analytes; finally, N-propylethylenediamine is used to adsorb impurities, and lipids are removed by freeze centrifugation, thereby achieving pretreatment of multiple target analytes and greatly improving the detection efficiency.

[0030] Furthermore, in S2, the ratio of the sample to be tested to reagent 1 is 1 g of the sample to be tested corresponding to 1-1.5 ml of reagent 1, the ratio of reagent 1 to reagent 2 is (45-55):1, and the ratio of reagent 1 to reagent 3 is (45-55):1.

[0031] Furthermore, the ammonium citrate buffer in the reagent 1 is an aqueous solution of ammonium citrate with a concentration of 1.20-1.25 g / ml.

[0032] By adopting the above technical solution, the chelating capacity of the ammonium citrate buffer at this concentration meets the chelating requirements of divalent metal ions, and at this concentration, the pH value of the ammonium citrate buffer is 5.2, which can provide a better pH environment for β-glucuronidase and arylsulfatase to enzymatically degrade the conjugated bond, thereby improving the enzymatic hydrolysis effect.

[0033] Furthermore, the enzymatic hydrolysis in S3 is carried out under shaking in a 45° C. water bath shaker.

[0034] By adopting the above technical solution, water bath shaking can prevent substrate precipitation, making the enzymatic hydrolysis reaction more thorough.

[0035] Furthermore, the enzymatic hydrolysate is cooled to 25-27° C. before centrifugation in S4, and the centrifugal parameters are: centrifugal force of 4000 g, centrifugal temperature of 4° C., and centrifugal time of 10 min.

[0036] Furthermore, the volume ratio of acetonitrile, methanol and ethanol in the reagent 4 is 2:2:1; the organic acid is citric acid, and the weight of the organic acid is 0.5-0.6% of the total weight of acetonitrile, methanol and ethanol.

[0037] By adopting the above technical solution, the precipitate contains most of the target analytes with weaker polarity and some of the target analytes with stronger polarity. The reagent 4 configured in the present application uses organic solvents with relatively different polarities in a specific ratio, which can take into account the extraction of multiple types of drugs with a wide range of polarities, and has a strong purification effect of removing proteins. At the same time, the addition of citric acid can not only improve the extraction efficiency, but also promote the release of drugs and precipitation of proteins, and further chelate the divalent metal ions.

[0038] Furthermore, the ratio of the reagent 4 in S5 to the sample to be tested in S2 is: 1 g of the sample to be tested corresponds to 6-7 ml of the reagent 4; the centrifugal parameters are: centrifugal force is 8000 g, centrifugal temperature is 4° C., and centrifugal time is 10 min.

[0039] Furthermore, the ratio of the reagent 5 in S7 to the sample to be tested in S2 is: 1 g of the sample to be tested corresponds to 9-12 mg of the reagent 5; the centrifugal parameters are: centrifugal force is 4000 g, centrifugal temperature is 4° C., and centrifugal time is 5 min.

[0040] Furthermore, the nitrogen blowing concentration in S8 is carried out in a 40° C. water bath, and the volume after nitrogen blowing concentration is 6.5-7.5% of the volume before concentration; the volume after dilution and re-dissolution is 2-2.5 times the volume before dilution.

[0041] By adopting the above technical solution, nitrogen blowing and concentration are performed to concentrate the volume of the mixed liquid, remove the organic solvent, increase the concentration of the target analyte, and meet the accuracy requirements.

[0042] Furthermore, the freezing parameters in S9 are: -20°C freezing for 20 min; the centrifugal parameters are: centrifugal force of 12000-15000g, centrifugal temperature of 4°C, and centrifugal time of 3 min.

[0043] By adopting the above technical solution, the freezing treatment causes the lipids to solidify, and then the lipids are removed by high-speed centrifugation to achieve purification.

[0044] In summary, this application has the following beneficial effects:

[0045] In the present application, the sample to be tested is first treated with ammonium citrate, which chelates divalent metal ions to prevent target analytes such as tetracyclines, fluoroquinolones and macrolides from chelating with divalent metal particles to form precipitation; then β-glucuronidase and arylsulfatase are added to hydrolyze the conjugated bonds, so that the target analytes are released from the conjugated form for extraction; then a solvent with strong solubility and a wide solubility range is used to dissolve the target analytes; finally, N-propylethylenediamine is used to adsorb impurities, and lipids are removed by freeze centrifugation, thereby achieving pre-treatment of multiple target analytes such as β2-receptor agonists, tetracyclines, anabolic hormones, macrolides, sulfonamides, amide alcohols, nitroimidazoles, fluoroquinolones, β-lactams, aminoglycosides, etc., which greatly simplifies the detection steps, reduces the consumption of reagents and equipment, and improves work efficiency. DETAILED DESCRIPTION

[0046] The present application is further described in detail below with reference to the embodiments.

[0047] Raw materials and intermediate preparation examples

[0048] raw material

[0049] The raw materials in the examples of this application can be obtained from the market:

[0050] Ammonium citrate, analytical grade;

[0051] β-Glucuronidase, activity 30 U / mL, purchased from Merck, Germany;

[0052] Arylsulfatase, activity 60 U / mL, purchased from Merck, Germany;

[0053] Acetonitrile, analytical grade;

[0054] Methanol, analytical grade;

[0055] Ethanol, analytical grade;

[0056] Citric acid, analytical grade.

[0057] Example

[0058] A universal pre-treatment method based on immunoaffinity rapid detection comprises the following steps:

[0059] S0. Reagent preparation:

[0060] Reagents 1. Ammonium citrate buffer: Add 1.22 g of ammonium citrate to 100 mL of water and stir to dissolve to obtain ammonium citrate buffer;

[0061] Reagent 2. β-glucuronidase;

[0062] Reagent 3. Arylsulfatase;

[0063] Reagent 4. Mix 200 ml of acetonitrile, 200 ml of methanol, and 100 ml of ethanol, then add 2.5 g of citric acid and stir to obtain reagent 4;

[0064] Reagent 5. N-propylethylenediamine;

[0065] S1. Take 10g of the animal tissue sample specified in the ELISA kit, homogenize it, and obtain the sample to be tested;

[0066] S2. Place 1 g of the sample obtained in S1 into a 50 mL centrifuge tube, add 1 ml of reagent 1, and vortex mix for 20 seconds; then add 20 μL of reagent 2 and reagent 3, vortex mix for 20 seconds to obtain a primary mixed solution;

[0067] S3. The initial mixed solution 3 obtained in S2 was placed in a 45°C water bath shaker and shaken at a speed of 200 rpm for 120 min of enzymatic hydrolysis to obtain an enzymatic hydrolyzate;

[0068] S4. The enzymatic hydrolysate obtained in S3 was cooled to 25°C, and then centrifuged at a centrifugal force of 4000g and a centrifugal temperature of 4°C for 10 min, and the supernatant and precipitate were collected respectively;

[0069] S5. Add 6 ml of reagent 4 to the precipitate obtained in S4, vortex mix, shake at 500 rpm for 10 min, and then centrifuge at a centrifugal force of 8000 g and a centrifugal temperature of 4°C for 10 min to collect the supernatant;

[0070] S6. combining the supernatant obtained in S4 with the supernatant obtained in S5 to obtain a mixed solution;

[0071] S7. Add 10 mg of reagent 5 to the mixture obtained in S6, vortex mix for 1 min, and then centrifuge at a centrifugal force of 4000 g and a centrifugal temperature of 4°C for 5 min, and retain the supernatant;

[0072] S8. The supernatant obtained in S7 was concentrated by nitrogen blowing in a 40°C water bath to 0.5 ml; then, the supernatant was diluted and re-dissolved with the sample diluent of the ELISA kit to a fixed volume of 1 mL;

[0073] S9. Freeze the diluted and reconstituted solution obtained in S8 at -20°C for 20 min, then centrifuge at a centrifugal force of 15,000 g for 3 min, and collect the supernatant for inspection.

[0074] Application Examples

[0075] Application Example 1

[0076] A method for detecting β2-receptor agonist drug residues comprises the following steps:

[0077] 1. Refer to the instructions of the ELISA commercial kit, select the animal tissue type specified in the kit as the test blank sample, and weigh the corresponding sample volume;

[0078] 2. Refer to the instructions of the ELISA commercial kit and set three concentrations of the test drug (detection limit, 2.5 times the detection limit, and 5 times the detection limit) according to the nominal detection accuracy value of the kit, and add them to the blank sample. Repeat 6 parallel samples for each added concentration;

[0079] 3. Obtain the supernatant to be tested according to the pretreatment method in the embodiment;

[0080] 4. Complete the ELISA test standard curve, blank sample and sample addition procedures according to the instructions of each commercial kit, measure the data with an ELISA reader, and calculate the drug measurement value from the standard curve.

[0081] The test values ​​were compared with the precision and accuracy indicated on the commercial kit, and the results are shown in Table 1. According to the requirements of industry standards such as Document No. 17 of Nongyifa

[2005] "Technical Data Requirements for the Registration and Review of Veterinary Drug Residue Enzyme-Linked Immunoassay Reagents (Kits)", the accuracy (recovery rate) of ELISA testing is required to be ≥40%, and the precision requires that the intra-batch coefficient of variation is less than or equal to 25%, and less than or equal to 30% for banned drugs. Generally, the lower the concentration, the lower the accuracy requirement, and sometimes it can be as low as 40%. The lowest concentration added in the verification experiment is consistent with the lower limit of the kit.

[0082] Table 1 β2-receptor agonist drug residue test results

[0083]

[0084] Application Example 2

[0085] A method for detecting tetracycline drug residues comprises the following steps:

[0086] 1. Refer to the instructions of the ELISA commercial kit, select the animal tissue type specified in the kit as the test blank sample, and weigh the corresponding sample volume;

[0087] 2. Refer to the instructions of the ELISA commercial kit and set three concentrations of the test drug (detection limit, 2.5 times the detection limit, and 5 times the detection limit) according to the nominal detection accuracy value of the kit, and add them to the blank sample. Repeat 6 parallel samples for each added concentration;

[0088] 3. Obtain the supernatant to be tested according to the pretreatment method in the embodiment;

[0089] 4. Complete the ELISA test standard curve, blank sample and sample addition procedures according to the instructions of each commercial kit, measure the data with an ELISA reader, and calculate the drug measurement value from the standard curve.

[0090] The test values ​​were compared with the precision and accuracy indicated on the commercial kit, and the results are shown in Table 2. According to the requirements of industry standards such as Document No. 17 of Nongyifa

[2005] "Technical Data Requirements for the Registration and Review of Veterinary Drug Residue Enzyme-Linked Immunoassay Reagents (Kits)", the accuracy (recovery rate) of ELISA testing is required to be ≥40%, and the precision requires that the intra-batch coefficient of variation is less than or equal to 25%, and less than or equal to 30% for banned drugs. Generally, the lower the concentration, the lower the accuracy requirement, and sometimes it can be allowed to be as low as 40%. The lowest concentration added in the verification experiment is consistent with the lower limit of the kit.

[0091] Table 2 Tetracycline drug residue detection results

[0092]

[0093] Application Example 3

[0094] A method for detecting dexamethasone drug residues comprises the following steps:

[0095] 1. Refer to the instructions of the ELISA commercial kit, select the animal tissue type specified in the kit as the test blank sample, and weigh the corresponding sample volume;

[0096] 2. Refer to the instructions of the ELISA commercial kit and set three concentrations of the test drug (detection limit, 2.5 times the detection limit, and 5 times the detection limit) according to the nominal detection accuracy value of the kit, and add them to the blank sample. Repeat 6 parallel samples for each added concentration;

[0097] 3. Obtain the supernatant to be tested according to the pretreatment method in the embodiment;

[0098] 4. Complete the ELISA test standard curve, blank sample and sample addition procedures according to the instructions of each commercial kit, measure the data with an ELISA reader, and calculate the drug measurement value from the standard curve.

[0099] The test values ​​were compared with the precision and accuracy indicated on the commercial kit, and the results are shown in Table 3. According to the requirements of industry standards such as Document No. 17 of Nongyifa

[2005] "Technical Data Requirements for the Registration and Review of Veterinary Drug Residue Enzyme-Linked Immunoassay Reagents (Kits)", the accuracy (recovery rate) of ELISA testing is required to be ≥40%, and the precision requires that the intra-batch coefficient of variation is less than or equal to 25%, and less than or equal to 30% for banned drugs. Generally, the lower the concentration, the lower the accuracy requirement, and sometimes it can be as low as 40%. The lowest concentration added in the verification experiment is consistent with the lower limit of the kit.

[0100] Table 3 Dexamethasone drug residue test results

[0101]

[0102]

[0103] Application Example 4

[0104] A method for detecting erythromycin drug residues comprises the following steps:

[0105] 1. Refer to the instructions of the ELISA commercial kit, select the animal tissue type specified in the kit as the test blank sample, and weigh the corresponding sample volume;

[0106] 2. Refer to the instructions of the ELISA commercial kit and set three concentrations of the test drug (detection limit, 2.5 times the detection limit, and 5 times the detection limit) according to the nominal detection accuracy value of the kit, and add them to the blank sample. Repeat 6 parallel samples for each added concentration;

[0107] 3. Obtain the supernatant to be tested according to the pretreatment method in the embodiment;

[0108] 4. Complete the ELISA test standard curve, blank sample and sample addition procedures according to the instructions of each commercial kit, measure the data with an ELISA reader, and calculate the drug measurement value from the standard curve.

[0109] The test values ​​were compared with the precision and accuracy indicated on the commercial kit, and the results are shown in Table 4. According to the requirements of the industry standards such as Document No. 17 of Nongyifa

[2005] "Technical Data Requirements for the Registration and Review of Veterinary Drug Residue Enzyme-Linked Immunoassay Reagents (Kits)", the accuracy (recovery rate) of ELISA testing is required to be ≥40%, and the precision requires that the intra-batch coefficient of variation is less than or equal to 25%, and less than or equal to 30% for banned drugs. Generally, the lower the concentration, the lower the accuracy requirement, and sometimes it can be as low as 40%. The lowest concentration added in the verification experiment is consistent with the lower limit of the kit.

[0110] Table 4 Erythromycin drug residue test results

[0111]

[0112] Application Example 5

[0113] A method for detecting sulfonamide drug residues comprises the following steps:

[0114] 1. Refer to the instructions of the ELISA commercial kit, select the animal tissue type specified in the kit as the test blank sample, and weigh the corresponding sample volume;

[0115] 2. Refer to the instructions of the ELISA commercial kit and set three concentrations of the test drug (detection limit, 2.5 times the detection limit, and 5 times the detection limit) according to the nominal detection accuracy value of the kit, and add them to the blank sample. Repeat 6 parallel samples for each added concentration;

[0116] 3. Obtain the supernatant to be tested according to the pretreatment method in the embodiment;

[0117] 4. Complete the ELISA test standard curve, blank sample and sample addition procedures according to the instructions of each commercial kit, measure the data with an ELISA reader, and calculate the drug measurement value from the standard curve.

[0118] The test values ​​were compared with the precision and accuracy indicated on the commercial kit, and the results are shown in Table 5. According to the requirements of the industry standards such as the document No. 17 of Nongyifa

[2005] "Technical Data Requirements for the Registration and Review of Veterinary Drug Residue Enzyme-Linked Immunoassay Reagents (Kits)", the accuracy (recovery rate) of ELISA testing is required to be ≥40%, and the precision requires that the intra-batch coefficient of variation is less than or equal to 25%, and less than or equal to 30% for banned drugs. Generally, the lower the concentration, the lower the accuracy requirement, and sometimes it can be allowed to be as low as 40%. The lowest concentration added in the verification experiment is consistent with the lower limit of the kit.

[0119] Table 5 Erythromycin drug residue test results

[0120]

[0121] Application Example 6

[0122] A method for detecting chloramphenicol drug residues comprises the following steps:

[0123] 1. Refer to the instructions of the ELISA commercial kit, select the animal tissue type specified in the kit as the test blank sample, and weigh the corresponding sample volume;

[0124] 2. Refer to the instructions of the ELISA commercial kit and set three concentrations of the test drug (detection limit, 2.5 times the detection limit, and 5 times the detection limit) according to the nominal detection accuracy value of the kit, and add them to the blank sample. Repeat 6 parallel samples for each added concentration;

[0125] 3. Obtain the supernatant to be tested according to the pretreatment method in the embodiment;

[0126] 4. Complete the ELISA test standard curve, blank sample and sample addition procedures according to the instructions of each commercial kit, measure the data with an ELISA reader, and calculate the drug measurement value from the standard curve.

[0127] The test values ​​were compared with the precision and accuracy indicated on the commercial kit, and the results are shown in Table 6. According to the requirements of the industry standards such as Document No. 17 of Nongyifa

[2005] "Technical Data Requirements for the Registration and Review of Veterinary Drug Residue Enzyme-Linked Immunoassay Reagents (Kits)", the accuracy (recovery rate) of ELISA testing is required to be ≥40%, and the precision requires that the intra-batch coefficient of variation is less than or equal to 25%, and less than or equal to 30% for banned drugs. Generally, the lower the concentration, the lower the accuracy requirement, and sometimes it can be allowed to be as low as 40%. The lowest concentration added in the verification experiment is consistent with the lower limit of the kit.

[0128] Table 6 Chloramphenicol drug residue test results

[0129]

[0130] Application Example 7

[0131] A method for detecting metronidazole drug residues comprises the following steps:

[0132] 1. Refer to the instructions of the ELISA commercial kit, select the animal tissue type specified in the kit as the test blank sample, and weigh the corresponding sample volume;

[0133] 2. Refer to the instructions of the ELISA commercial kit and set three concentrations of the test drug (detection limit, 2.5 times the detection limit, and 5 times the detection limit) according to the nominal detection accuracy value of the kit, and add them to the blank sample. Repeat 6 parallel samples for each added concentration;

[0134] 3. Obtain the supernatant to be tested according to the pretreatment method in the embodiment;

[0135] 4. Complete the ELISA test standard curve, blank sample and sample addition procedures according to the instructions of each commercial kit, measure the data with an ELISA reader, and calculate the drug measurement value from the standard curve.

[0136] The test values ​​were compared with the precision and accuracy indicated on the commercial kit, and the results are shown in Table 7. According to the requirements of industry standards such as Document No. 17 of Nongyifa

[2005] "Technical Data Requirements for the Registration and Review of Veterinary Drug Residue Enzyme-Linked Immunoassay Reagents (Kits)", the accuracy (recovery rate) of ELISA testing is required to be ≥40%, and the precision requires that the intra-batch coefficient of variation is less than or equal to 25%, and less than or equal to 30% for banned drugs. Generally, the lower the concentration, the lower the accuracy requirement, and sometimes it can be allowed to be as low as 40%. The lowest concentration added in the verification experiment is consistent with the lower limit of the kit.

[0137] Table 7 Metronidazole drug residue test results

[0138]

[0139] Application Example 8

[0140] A method for detecting fluoroquinolone drug residues comprises the following steps:

[0141] 1. Refer to the instructions of the ELISA commercial kit, select the animal tissue type specified in the kit as the test blank sample, and weigh the corresponding sample volume;

[0142] 2. Refer to the instructions of the ELISA commercial kit and set three concentrations of the test drug (detection limit, 2.5 times the detection limit, and 5 times the detection limit) according to the nominal detection accuracy value of the kit, and add them to the blank sample. Repeat 6 parallel samples for each added concentration;

[0143] 3. Obtain the supernatant to be tested according to the pretreatment method in the embodiment;

[0144] 4. Complete the ELISA test standard curve, blank sample and sample addition procedures according to the instructions of each commercial kit, measure the data with an ELISA reader, and calculate the drug measurement value from the standard curve.

[0145] The test values ​​were compared with the precision and accuracy indicated on the commercial kit, and the results are shown in Table 8. According to the requirements of the industry standards such as Document No. 17 of Nongyifa

[2005] "Technical Data Requirements for the Registration and Review of Veterinary Drug Residue Enzyme-Linked Immunoassay Reagents (Kits)", the accuracy (recovery rate) of ELISA testing is required to be ≥40%, and the precision requires that the intra-batch coefficient of variation is less than or equal to 25%, and less than or equal to 30% for banned drugs. Generally, the lower the concentration, the lower the accuracy requirement, and sometimes it can be allowed to be as low as 40%. The lowest concentration added in the verification experiment is consistent with the lower limit of the kit.

[0146] Table 8 Fluoroquinolone drug residue test results

[0147]

[0148] Application Example 9

[0149] A method for detecting ampicillin drug residues comprises the following steps:

[0150] 1. Refer to the instructions of the ELISA commercial kit, select the animal tissue type specified in the kit as the test blank sample, and weigh the corresponding sample volume;

[0151] 2. Refer to the instructions of the ELISA commercial kit and set three concentrations of the test drug (detection limit, 2.5 times the detection limit, and 5 times the detection limit) according to the nominal detection accuracy value of the kit, and add them to the blank sample. Repeat 6 parallel samples for each added concentration;

[0152] 3. Obtain the supernatant to be tested according to the pretreatment method in the embodiment;

[0153] 4. Complete the ELISA test standard curve, blank sample and sample addition procedures according to the instructions of each commercial kit, measure the data with an ELISA reader, and calculate the drug measurement value from the standard curve.

[0154] The test values ​​were compared with the precision and accuracy indicated on the commercial kit, and the results are shown in Table 9. According to the requirements of the industry standards such as Document No. 17 of Nongyifa

[2005] "Technical Data Requirements for the Registration and Review of Veterinary Drug Residue Enzyme-Linked Immunoassay Reagents (Kits)", the accuracy (recovery rate) of ELISA testing is required to be ≥40%, and the precision requires that the intra-batch coefficient of variation is less than or equal to 25%, and less than or equal to 30% for banned drugs. Generally, the lower the concentration, the lower the accuracy requirement, and sometimes it can be allowed to be as low as 40%. The lowest concentration added in the verification experiment is consistent with the lower limit of the kit.

[0155] Table 9 Ampicillin drug residue test results

[0156]

[0157] Application Example 10

[0158] A method for detecting gentamicin drug residues comprises the following steps:

[0159] 1. Refer to the instructions of the ELISA commercial kit, select the animal tissue type specified in the kit as the test blank sample, and weigh the corresponding sample volume;

[0160] 2. Refer to the instructions of the ELISA commercial kit and set three concentrations of the test drug (detection limit, 2.5 times the detection limit, and 5 times the detection limit) according to the nominal detection accuracy value of the kit, and add them to the blank sample. Repeat 6 parallel samples for each added concentration;

[0161] 3. Obtain the supernatant to be tested according to the pretreatment method in the embodiment;

[0162] 4. Complete the ELISA test standard curve, blank sample and sample addition procedures according to the instructions of each commercial kit, measure the data with an ELISA reader, and calculate the drug measurement value from the standard curve.

[0163] The test values ​​were compared with the precision and accuracy indicated on the commercial kit, and the results are shown in Table 10. According to the requirements of the industry standards such as Document No. 17 of Nongyifa

[2005] "Technical Data Requirements for the Registration and Review of Veterinary Drug Residue Enzyme-Linked Immunoassay Reagents (Kits)", the accuracy (recovery rate) of ELISA testing is required to be ≥40%, and the precision requires that the intra-batch coefficient of variation is less than or equal to 25%, and less than or equal to 30% for banned drugs. Generally, the lower the concentration, the lower the accuracy requirement, and sometimes it can be as low as 40%. The lowest concentration added in the verification experiment is consistent with the lower limit of the kit.

[0164] Table 10 Gentamicin drug residue test results

[0165]

[0166]

[0167] Combined with application examples 1-10 and Tables 1-10, it can be seen that the pretreatment method of the present application is used to pretreat the sample to be tested, and then the drug residue is determined. The test results of β2-receptor agonists, tetracyclines, anabolic hormones (dexamethasone), macrolides (erythromycin), sulfonamides, amides (chloramphenicol), nitroimidazoles (metronidazole), fluoroquinolones, β-lactams (ampicillin), and aminoglycosides (gentamicin) all meet the industry standard requirements of the document No. 17 of Nongyifa

[2005] "Technical Data Requirements for the Registration and Review of Veterinary Drug Residues Enzyme-Linked Immunoassay Reagents (Kits)". It is shown that the pretreatment method of the present application is suitable for the detection of multiple types of drug residues. By processing one sample, the residues of multiple drugs can be detected, which greatly simplifies the detection steps, reduces the consumption of reagents and equipment, and improves work efficiency.

[0168] This specific embodiment is merely an explanation of the present application and is not a limitation of the present application. After reading this specification, those skilled in the art may make modifications to the present embodiment without any creative contribution as needed, but such modifications are protected by the patent law as long as they are within the scope of the claims of the present application.

Claims

1. A universal pretreatment method based on immunoaffinity rapid detection, characterized in that: The following steps are involved: S1. Take the animal product to be tested, homogenize it, and obtain the sample to be tested; S2. Add reagent 1 to the sample to be tested obtained in S1 and mix well, then add reagent 2 and reagent 3 in sequence and mix well to obtain an initial mixed solution; S3. enzymatically hydrolyzing the initial mixed solution to obtain an enzymatic hydrolyzate; S4. Centrifuge the enzymatic hydrolyzate obtained in S3, and collect the supernatant and precipitate respectively; S5. Add reagent 4 to the precipitate obtained in S4, mix, centrifuge, and collect the supernatant; S6. combining the supernatant obtained in S4 with the supernatant obtained in S5 to obtain a mixed solution; S7. Add reagent 5 to the mixture obtained in S6, mix well, centrifuge, and retain the supernatant; S8. The supernatant obtained in S7 is concentrated by nitrogen blowing, and then diluted and reconstituted with the sample diluent of the kit; S9. Freeze and centrifuge the diluted and reconstituted solution obtained in S8, and take the supernatant for inspection; The reagent 1 is ammonium citrate buffer; The reagent 2 is β-glucuronidase; The reagent 3 is arylsulfatase; The reagent 4 is a mixture of acetonitrile, methanol, ethanol and an organic acid; the organic acid is citric acid or formic acid; The reagent 5 is N-propylethylenediamine.

2. A universal pretreatment method based on immunoaffinity rapid detection according to claim 1, characterized in that: The ratio of the sample to be tested to reagent 1 in S2 is 1 g of the sample to be tested corresponds to 1-1.5 ml of reagent 1, the ratio of reagent 1 to reagent 2 is (45-55):1, and the ratio of reagent 1 to reagent 3 is (45-55):

1.

3. A universal pretreatment method based on immunoaffinity rapid detection according to claim 1, characterized in that: The ammonium citrate buffer in the reagent 1 is an aqueous solution of ammonium citrate with a concentration of 1.20-1.25 g / ml.

4. A universal pretreatment method based on immunoaffinity rapid detection according to claim 1, characterized in that: The enzymatic hydrolysis in S3 was carried out under shaking in a 45°C water bath shaker.

5. A universal pretreatment method based on immunoaffinity rapid detection according to claim 1, characterized in that: In the S4, the enzymatic hydrolysate is cooled to 25-27° C. before centrifugation. The centrifugal parameters are: centrifugal force of 4000 g, centrifugal temperature of 4° C., and centrifugal time of 10 min.

6. A universal pretreatment method based on immunoaffinity rapid detection according to claim 1, characterized in that: The volume ratio of acetonitrile, methanol and ethanol in the reagent 4 is 2:2:1; the organic acid is citric acid, and the weight of the organic acid is 0.5-0.6% of the total weight of acetonitrile, methanol and ethanol.

7. A universal pretreatment method based on immunoaffinity rapid detection according to claim 1, characterized in that: The ratio of the reagent 4 in S5 to the sample to be tested in S2 is: 1 g of the sample to be tested corresponds to 6-7 ml of the reagent 4; the centrifugal parameters are: centrifugal force is 8000 g, centrifugal temperature is 4° C., and centrifugal time is 10 min.

8. A universal pretreatment method based on immunoaffinity rapid detection according to claim 1, characterized in that: The ratio of the reagent 5 in S7 to the sample to be tested in S2 is: 1 g of the sample to be tested corresponds to 9-12 mg of the reagent 5; the centrifugal parameters are: centrifugal force is 4000 g, centrifugal temperature is 4° C., and centrifugal time is 5 min.

9. A universal pretreatment method based on immunoaffinity rapid detection according to claim 1, characterized in that: The nitrogen blowing concentration in S8 is carried out in a 40° C. water bath, and the volume after nitrogen blowing concentration is 6.5-7.5% of the volume before concentration; the volume after dilution and re-dissolution is 2-2.5 times the volume before dilution.

10. A universal pretreatment method based on immunoaffinity rapid detection according to claim 1, characterized in that: The freezing parameters in S9 are: -20°C freezing for 20 min; the centrifugal parameters are: centrifugal force of 12000-15000 g, centrifugal temperature of 4°C, and centrifugal time of 3 min.

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