Quinolone drug receptor detection method based on topoisomerase IV and application of quinolone drug receptor detection method in livestock and poultry products

Abstract

The invention belongs to the technical field of biology, and particularly relates to a topoisomerase IV-based quinolone drug receptor detection method and application of the topoisomerase IV-based quinolone drug receptor detection method in livestock and poultry products, and the method comprises the following steps: coating a high-adsorption elisa plate with topoisomerase IV as a quinolone drug receptor protein; horseradish peroxidase enzyme-labeled ciprofloxacin-bovine serum albumin is used as an enzyme marker and a tracer; the topoisomerase IV is a tetramer composed of two C subunits and two E subunits, the subunit ParC amino acid sequence is shown in SEQ ID NO. 1, and the subunit ParE amino acid sequence is shown in SEQ ID NO. 2. The method provided by the invention has high sensitivity and accuracy.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to a topoisomerase IV (ParC / ParE heterodimer) based on in vitro recombinant expression, a direct competitive enzyme-labeled receptor detection method is constructed by optimizing the corresponding reaction conditions, which is used for the synchronous screening of 13 quinolone drugs in livestock and poultry products. BACKGROUND

[0002] Quinolone drugs have 4-quinolone nucleus (containing fluorine substituent, piperazine ring and other modified groups) as the basic structure, and belong to pyrone acid derivatives in chemical structure. Commonly used quinolone drugs include enoxacin, ciprofloxacin, danofloxacin, lomefloxacin, ofloxacin, norfloxacin, perfloxacin, enoxacin, difloxacin, sarafloxacin, oxolinic acid, flurazolidone and gatifloxacin. Due to non-standard use, abuse or illegal addition, quinolone drug residues are often detected in livestock and poultry products (livestock and poultry meat, poultry eggs and milk) exceeding the standard. Excessive quinolone drug residues in livestock and poultry products can be enriched in the human body through the food chain, endangering human health, and also accelerating the generation and spread of drug-resistant strains, seriously threatening public health safety. Rapid detection technology is an important technical means to ensure the safety of animal-derived food, and the development of rapid detection technology is the basis for implementing relevant laws and regulations and achieving scientific supervision. The commonly used recognition elements in the current rapid detection technology include receptors, antibodies, molecularly imprinted polymers and aptamers. The rapid detection method with receptor as the recognition element is the receptor detection method.

[0003] The core of the receptor binding detection method is to fix the purified drug target receptor protein on the carrier (such as a microplate), and when the drug to be detected binds to the receptor, a detectable signal can be generated through competitive or non-competitive mode. In competitive detection, the pre-labeled ligand competes with the drug to be detected for binding to the receptor, and the increase of the drug concentration will lead to the decrease of the binding amount of the labeled ligand, and the signal intensity will decrease accordingly. The receptor detection method has the advantages of strong specificity, high sensitivity, quantitative ability, convenient operation and controllable cost, and has become an important scheme in the field of drug rapid detection. Compared with the microbial method, the receptor detection method does not require live culture, and can be quantitative. Compared with the immunodetection method based on antigen-antibody binding, the natural high affinity of the receptor protein is strong and the anti-matrix interference ability is strong. The same receptor protein can detect all drugs in the same category, effectively reducing the cost. Especially in the analysis of trace residues in complex matrix, multi-target screening and on-site batch detection, the comprehensive performance of the receptor detection method is significantly better than that of the microbial method and part of the immunodetection method, and the quinolone drug receptor detection method has not been reported so far.

[0004] The target of quinolones is two enzymes of the type II topoisomerase family, DNA gyrase and topoisomerase IV. In gram-negative bacteria, quinolones bind to DNA gyrase, while in gram-positive bacteria, quinolones preferentially bind to topoisomerase IV. The action site of quinolones is the subunit ParC of topoisomerase IV and the subunit GyrA of DNA gyrase. The blockage of DNA replication is not by removing the enzyme, but by forming a drug-enzyme-DNA triad, which converts the enzyme into a toxic agent for DNA replication. Therefore, the subunits ParC and ParE of topoisomerase IV can be prepared and expressed and purified. The purified subunits ParC and ParE self-assemble into a tetrameric protease-topoisomerase IV. The topoisomerase IV is used as a receptor to develop a rapid detection method for quinolones in livestock and poultry products (livestock and poultry meat, poultry eggs and cow milk).

[0005] Like antibody-based immunoassays, receptor assays can also be combined with various techniques, such as enzyme-labeled receptor assays. Enzyme-labeled receptor assays use receptors instead of antibodies to bind to the small molecules of the antibiotics to be detected. Enzyme-labeled receptor assays have the advantages of recognizing only active molecules and wide-spectrum recognition, and are suitable for simultaneous detection of multiple drugs in the same category. Compared with other forms of receptor assays (radioactive receptor assays, gold-labeled receptor assays and receptor biosensors, etc.), enzyme-labeled receptor assays have the advantages of safety, high sensitivity, low cost and being less susceptible to matrix effect interference. The enzyme-labeled system (horseradish peroxidase) can generate a large amount of colored product through substrate reaction, and convert the binding of trace amounts of drugs and specific receptor proteins into a strong signal (OD450 nm value) that can be detected, with high sensitivity. By fitting the four-parameter logistic standard curve of the logarithmic concentration and the signal value (B / B0), the target drug can be quantified in a wide concentration range (0.1-1 000 ng / mL). SUMMARY

[0006] In order to solve the problems of establishing a quinolone receptor detection method based on topoisomerase IV and its application in livestock and poultry products (livestock and poultry meat, poultry eggs and cow milk), the present application is based on in vitro recombinant expression of receptor protein topoisomerase IV. The optimal receptor coating concentration and the optimal enzyme label dilution multiple, the receptor protein coating buffer and the coating conditions, the blocking buffer and the blocking time, the competition reaction time, the standard and sample diluent, the sample extraction liquid dilution multiple and other conditions are optimized. A direct competitive enzyme-labeled receptor detection method for simultaneously detecting 13 kinds of quinolones (enrofloxacin, ciprofloxacin, danofloxacin, lomefloxacin, ofloxacin, norfloxacin, perfloxacin, enoxacin, difloxacin, sarafloxacin, oxolinic acid, flumequine and gatifloxacin) is established.

[0007] The technical solutions provided by the present application are as follows:

[0008] A topoisomerase IV-based quinolone drug receptor detection method, comprising the following steps: topoisomerase IV is coated as a quinolone drug receptor protein on a high-adsorption enzyme-labeled plate, and horseradish peroxidase enzyme-labeled ciprofloxacin-bovine serum albumin is used as an enzyme label and a tracer; the topoisomerase IV is a tetramer composed of 2 C subunits and 2 E subunits, wherein the amino acid sequence of the subunit ParC is shown in SEQ ID NO. 1, and the amino acid sequence of the subunit ParE is shown in SEQ ID NO. 2.

[0009] Further, the quinolone drug receptor protein is diluted and coated on the enzyme-labeled plate using a coating buffer; the coating buffer is a PBS buffer.

[0010] Further, the coating concentration of the topoisomerase IV is 1-1.5 μg / mL, and the dilution multiple of the enzyme label is 200-240.

[0011] Further, the quinolone drugs include enrofloxacin, ciprofloxacin, danofloxacin, lomefloxacin, ofloxacin, norfloxacin, perfloxacin, enoxacin, difloxacin, sarafloxacin, oxolinic acid, flumequine and gatifloxacin.

[0012] The application further provides application of the above-mentioned topoisomerase IV-based quinolone drug receptor detection method in detection of quinolone drugs in livestock and poultry products; the livestock and poultry products include livestock meat, poultry meat, poultry eggs and cow milk.

[0013] Further, 8-12% FBS buffer is used as a blocking buffer, and the blocking time is 2 h.

[0014] Further, the competition reaction time is 20-40 min.

[0015] Further, PBS buffer containing 4-6% acetonitrile is used as a standard sample and sample diluent.

[0016] Further, when the livestock and poultry products are livestock meat or poultry meat, the detection method comprises the following steps: the livestock and poultry product sample is added to ultrapure water containing a β-glucuronidase / sulfatase mixed solution, mixed, and then water bath incubated, acetonitrile is added after incubation, mixed at room temperature for extraction, diluted with a sample diluent, centrifuged, and then subjected to direct competition receptor detection analysis;

[0017] When the livestock and poultry products are poultry eggs, the detection method comprises the following steps: the livestock and poultry product sample is added to ultrapure water containing a β-glucuronidase / sulfatase mixed solution, mixed, and then water bath incubated, acetonitrile aqueous solution is added after incubation, mixed at room temperature for extraction, diluted with a sample diluent, centrifuged, and then subjected to direct competition receptor detection analysis.

[0018] When the livestock product is cow milk, the detection method comprises: centrifuging the cow milk to precipitate particulate matter and a lipid layer; taking the middle layer, diluting with a sample diluent, and then performing direct competitive receptor detection analysis.

[0019] Further, the sample diluent is a PBS buffer containing 5% acetonitrile.

[0020] The application provides a topoisomerase IV-based quinolone drug receptor detection method, and the steps are as follows:

[0021] 1. The checkerboard method is used to optimize the coating concentration of the receptor protein and the dilution multiple of the enzyme marker, the receptor protein (topoisomerase IV) is coated on a high-absorption enzyme-labeled plate, and horseradish peroxidase enzyme-labeled ciprofloxacin-bovine serum albumin (HRP-CFLX-BSA) is used as an enzyme marker and tracer. The CFLX-BSA conjugated antigen is purchased from Suzhou Boaolong Technology Co., Ltd., and the Boaolong HRP enzyme labeling kit is used to label the CFLX-BSA.

[0022] (1) Coating: The quinolone drug receptor protein (topoisomerase IV) is gradient-diluted using a coating buffer to obtain gradient concentrations of 10, 5, 2.5000, 1.2500, 0.6250 and 0.3125 μg / mL, one concentration per column. Vertically add 100 μL of sample to each well to avoid liquid splashing or adding on the well wall, and do not generate bubbles. Place in a 4 °C refrigerator for 16 h to allow the protein to fully adsorb to the plate. Set 3 parallels for each plate.

[0023] (2) Washing and blocking: tap the plate, add 250 μL of washing solution to each well, and wash the plate 3 times, then tap the plate. Add sufficient (250 μL) blocking solution to each well, and incubate the microplate in a 37 °C constant-temperature and constant-humidity incubator for 2 h. Vertically and quickly pour the liquid onto a dust-free paper or filter paper and tap the plate. Do not overflow the washing solution to contaminate the adjacent wells. Wash the plate 6 times with the washing solution, and tap the plate. Add 150 μL of TE buffer to each well and equilibrate for 15 min.

[0024] (3) Incubate the enzyme-DNA complex: dilute the supercoiled plasmid to a concentration of 100 ng / μL using a TE buffer, and add 100 μL to each well. Incubate in a 37 °C constant-temperature and constant-humidity incubator for 1 h. Immediately after incubation, place on ice and wash the plate 5 times with pre-cooled 4 °C washing solution (250 μL), and tap the plate.

[0025] (4) Competition reaction: 50 μL of the gradient-diluted quinolone standard or sample was added to each well, one concentration per row, and then 50 μL of HRP-CFLX-BSA diluted according to the optimal dilution multiple was added to each well of the row, and the plate was incubated in a 37 °C constant-temperature and constant-humidity incubator for 30 min, the plate was tapped, and the plate was washed with washing solution for 3 times, and the plate was tapped.

[0026] (5) Color development and termination: 100 μL of single-component TMB color developing solution was added to each well, and the plate was incubated in a 37 °C constant-temperature and constant-humidity incubator for 20 min in the dark. 100 μL of termination solution was added to each well to terminate the reaction. The order of adding each step was consistent, especially the order of color developing solution and termination solution was consistent to ensure that the color development time of each well was the same. The color developing solution and the termination solution were taken out 30 min in advance and restored to room temperature.

[0027] (6) Detection: The OD value of each well was determined within 15 min using an enzyme-labeled instrument at a wavelength of 450 nm.

[0028] 2. Checkerboard method for titer determination

[0029] (1) Concentration gradient setting: column gradient (topoisomerase IV coating concentration): 10, 5, 2.5, 1.25, 0.625, 0.3125 μg / mL. The 7th column is a negative control (no topoisomerase IV). Row gradient (HRP-CFLX-BSA dilution multiple): 1:50, 1:100, 1:200, 1:400, 1:800, 1:1600. Each row and column intersection represents one combination.

[0030] (2) Coating: according to the column gradient concentration, 100 μL of the same concentration was added to each column. The others are the same as 1.1.

[0031] (3) Washing and blocking: the same as 1.1.

[0032] (4) Incubation of enzyme-DNA complex: the same as 1.1.

[0033] (5) Competition reaction: 50 μL of PBS buffer was added to each well. According to the row gradient dilution multiple, the same dilution multiple was added to each row, and 50 μL was added to each well. The others are the same as 1.1. Drug standards were added first, and then HRP-CFLX-BSA was added to avoid pre-reaction.

[0034] (6) Color development and termination: the same as 1.1.

[0035] (7) Detection: the same as 1.1.

[0036] 3. Checkerboard method for inhibition and standard inhibition curve establishment

[0037] (1) Concentration gradient setting: row gradient (topoisomerase IV coating concentration): 10, 5, 2.5, 1.25, 0.625, 0.3125 μg / mL. Column gradient (drug standard concentration): 5000, 1000, 500, 100, 50, 10, 5, 1, 0.5, 0.1 ng / mL.

[0038] (2) Coating: according to the row gradient concentration, the same concentration in each row, 100 μL was added to each well. The others are the same as 1.1.

[0039] (3) Washing and blocking: the blocking solution is 250 μL of 10% fetal bovine serum (FBS) PBST buffer containing 2 mmol / L DTT. The others are the same as 1.1.

[0040] (4) Incubation of enzyme-DNA complex: the same as 1.1.

[0041] (5) Competition reaction: 50 μL of PBS buffer was added to the 11th well of each row. According to the column gradient drug standard concentration, the same concentration in each column, 50 μL was added to each well. According to the dilution multiple of the enzyme marker when the OD450 nm value of each coating concentration is about 1.1, HRP-CFLX-BSA was diluted, 50 μL per well in each row. The others are the same as 1.1.

[0042] (6) Color development and termination: the same as 1.1.

[0043] (7) Detection: the same as 1.1.

[0044] Taking the logarithmic value of the drug standard concentration as the abscissa and the B / B0 value as the ordinate (B represents the OD450 nm value corresponding to different drug standard concentration wells, and B0 represents the OD450 nm value corresponding to the 11th well when the drug standard concentration is 0), the four-parameter logistic model of GraphPad Prism 8 software is used for data fitting, the standard inhibition curve is established, and the corresponding half inhibition concentration (IC50) value is calculated.

[0045] The application provides an application of a quinolone drug receptor detection method in livestock and poultry products (livestock and poultry meat, poultry eggs and cow milk), and the steps are as follows:

[0046] 1. Sample collection and pretreatment

[0047] Each 5 g (accurate to ±0.01 g) of blank poultry egg (chicken egg, duck egg and goose egg) sample was placed in a 50 mL centrifuge tube, 4 mL of ultrapure water containing 16 μL of β-glucuronidase / sulfatase mixed solution was added, mixed and then incubated in a 37 °C water bath for 3 h. 8 mL of 90% acetonitrile aqueous solution was added to each centrifuge tube, and the mixture was placed in a rotary mixer and mixed at room temperature for 1 h. Then centrifugation was performed at 5,000 rpm for 10 min, and the supernatant was combined after centrifugation for 3 times. After dilution with sample diluent, direct competitive receptor detection analysis was performed.

[0048] Each 5 g (accurate to ±0.01 g) of blank poultry egg (chicken egg, duck egg and goose egg) sample was placed in a 50 mL centrifuge tube, 4 mL of ultrapure water containing 16 μL of β-glucuronidase / sulfatase mixed solution was added, mixed and then incubated in a 37 °C water bath for 3 h. 8 mL of 90% acetonitrile aqueous solution was added to each centrifuge tube, and the mixture was placed in a rotary mixer and mixed at room temperature for 1 h. Then centrifugation was performed at 5,000 rpm for 10 min, and the supernatant was combined after centrifugation for 3 times. After dilution with sample diluent, direct competitive receptor detection analysis was performed.

[0049] 5 g (accurate to ±0.01 g) of blank milk was measured and placed in a 50 mL centrifuge tube, and centrifugation was performed at 10,000 rpm for 15 min at 4 °C to precipitate particulate matter and lipid layers. The middle layer was diluted with sample diluent and then subjected to direct competitive receptor detection analysis.

[0050] 2. Method detection limit, limit of quantification, standard addition recovery rate and coefficient of variation

[0051] The sensitivity of the method was evaluated by LOD and LOQ. 20 negative samples were pretreated, and the sample extract was optimally diluted with diluent (5% acetonitrile in PBS buffer). The established direct competitive enzyme-labeled receptor detection method was used for detection analysis, and the average OD450 nm (M) and standard deviation (SD) were calculated. LOD is defined as the analyte concentration corresponding to OD450 nm (M+3×SD), and LOQ is defined as the analyte concentration corresponding to OD450 nm (M+10×SD). A certain amount of CFLX standard was added to the negative sample, the sample was pretreated, and then the sample extract was diluted by an appropriate multiple to make the CFLX concentration in the test sample LOQ, 3 LOQ and 10 LOQ. Each concentration was measured 3 times, and the recovery rate and coefficient of variation (CV) were calculated.

[0052] Beneficial effects

[0053] The present application is based on the natural broad-spectrum affinity of topoisomerase IV to quinolones, and a single detection can simultaneously screen 13 common quinolone drugs (enrofloxacin, ciprofloxacin, danofloxacin, lomefloxacin, ofloxacin, norfloxacin, perfloxacin, enoxacin, difloxacin, sarafloxacin, oxolinic acid, flumequine and gatifloxacin), which is significantly better than the traditional immunization method (single antibody only detects one or a few drugs), and greatly improves the high-throughput detection efficiency. The development cost is low, and there is no need to develop antibodies for each drug, and the recombinant enzyme (topoisomerase IV) can be mass-produced. By optimizing the reaction conditions and sample pretreatment process, high sensitivity and accuracy are still maintained in complex matrices such as livestock and poultry meat (high fat, high protein), poultry eggs (high egg white protein, high fat) and cow milk (high milk fat, high casein). The method LOD range is 0.06-0.11 ng / mL, the LOQ range is 0.19-0.36 ng / mL, and in the method validation, ciprofloxacin is used as a representative to optimize the method, the extraction solvent in the sample pretreatment is 12 mL, the supernatant is combined after centrifugation for 3 times, the total supernatant volume is assumed to be 12 mL, the dilution ratio is 8 times, the final detection liquid volume is 64 mL, and the sample mass is 5 g, so the method LOD range of 0.06-0.11 ng / mL corresponds to the residual concentration of 1.2-2.1 μg / kg in the original sample. In actual operation, the volume loss will occur in the centrifugation step, the total supernatant volume is <12 mL, and the extraction efficiency is not 100%, so the corrected method LOD corresponds to a residual concentration of less than 2.1 μg / kg in the original sample, which is much lower than the minimum value (10 μg / kg) of the maximum residue limit (MRLs) of ciprofloxacin in the European Union and China for livestock and poultry meat (beef, mutton, pork, chicken, duck and goose), poultry eggs (chicken eggs, duck eggs and goose eggs) and cow milk, meeting the requirements of trace residue screening, and combining with enzyme labeling signal amplification technology, precise quantification in a wide linear range is realized. BRIEF DESCRIPTION OF DRAWINGS

[0054] Figure 1 Enrofloxacin standard inhibition curve in the present application.

[0055] Figure 2 Ciprofloxacin standard inhibition curve in the present application.

[0056] Figure 3 Danofloxacin standard inhibition curve in the present application.

[0057] Figure 4 Lomefloxacin standard inhibition curve in the present application.

[0058] Figure 5 Ofloxacin standard inhibition curve in the present application.

[0059] Figure 6Standard inhibition curve of norfloxacin in the present application.

[0060] Figure 7 Standard inhibition curve of perfloxacin in the present application.

[0061] Figure 8 Standard inhibition curve of enoxacin in the present application.

[0062] Figure 9 Standard inhibition curve of difloxacin in the present application.

[0063] Figure 10 Standard inhibition curve of sarafloxacin in the present application.

[0064] Figure 11 Standard inhibition curve of oxolinic acid in the present application.

[0065] Figure 12 Standard inhibition curve of flurroxacin in the present application.

[0066] Figure 13 Standard inhibition curve of gatifloxacin in the present application.

[0067] Figure 14 Standard inhibition curve of CFLX in beef sample extract of different dilution multiples in the present application.

[0068] Figure 15 Standard inhibition curve of CFLX in chicken sample extract of different dilution multiples in the present application.

[0069] Figure 16 Standard inhibition curve of CFLX in egg sample extract of different dilution multiples in the present application.

[0070] Figure 17 Standard inhibition curve of CFLX in milk sample extract of different dilution multiples in the present application. DETAILED DESCRIPTION

[0071] Example 1

[0072] The present embodiment provides a topoisomerase IV, which is a tetramer composed of 2 C subunits and 2 E subunits, wherein the amino acid sequence of the subunit ParC is shown in SEQ ID NO. 1, and the amino acid sequence of the subunit ParE is shown in SEQ ID NO. 2. The preparation method of the topoisomerase IV comprises: constructing a prokaryotic expression vector, transforming into an engineering bacterium to induce expression, exploring expression conditions, then carrying out mass expression and protein purification, combining the purified subunits ParC and ParE into topoisomerase IV, and then carrying out purification and activity verification.

[0073] MSDMAERLALHEFTENAYLNYSMYVIMDRALPFIGDGLKPVQRRIVYAMSELGLN ASAKFKKSARTVGDVLGKYHPHGDSACYEAMVLMAQPFSYRYPLVDGQGNWGAP DDPKSFAAMRYTESRLSKYSELLLSELGQGTADWVPNFDGTLQEPKMLPARLPNI LLNGTTGIAVGMATDIPPHNLREVAQAAIALIDQPKTTLDQLLDIVQGPDYPTEAE IITSRAEIRKIYENGRGSVRMRAVWKKEDGAVVISALPHQVSGARVLEQIAAQMR NKKLPMVDDLRDESDHENPTRLVIVPRSNRVDMDQVMNHLFATTDLEKSYRINL NMIGLDGRPAVKNLLEILSEWLVFRRDTVRRRLNYRLEKVLKRLHILEGLLVAFL NIDEVIEIIRNEDEPKPALMSRFGLTETQAEAILELKLRHLAKLEEMKIRGEQSE LEKERDQLQGILASERKMNNLLKKELQADAQAYGDDRRSPLQEREEAKAMSEHD MLPEPVTIVLSQMGWVRSAKGHDIDAPGLNYKAGDSFKAAVKGKSNQPVVFVDS TGRSYAIDPITLPSARGQGEPLTGKLTLPPGATVDHMLMESDDQKLLMASDAGY GFVCTFNDLVARNRAGKALITLPENAHVMPPVVIEDASDMLLAITQAGRMLMFP VSDLPQLSKGKGNKIINIPSAEAARGEDGLAQLYVLPPQSTLTIHVGKRKIKLR PEELQKVTGERGRRGTLMRGLQRIDRVEIDSPRRASSGDSEE (SEQ ID NO. 1);

[0074] The amino acid sequence of the subunit ParE is: MTQTYNADAIEVLTGLEPVRRRPGMYTDTTRPNHLGQEVIDNSVDEALAGHAKRVDVILHADQSLEVIDDGRGMPVDIHPEEGVPAVELILCRLHAGGKFSNKNYQFSGGLHGVGISVVNALSKRVEVNVRRDGQVYNIAFENGEKVQDLQVVGTCGKRNTGTSVHFWPDETFFDSPRFSVSRLTHVLKAKAVLCPGVEITFKDEINNTEQRWCYQDGLNDYLAEAVNGLPTLPEKPFIGNFAGDTEAVDWALLWLPEGGELLTESYVNLIPTMQGGTHVNGLRQGLLDAMREFCEYRNILPRGVKLSAEDIWDRCAYVLSVKMQDPQFAGQTKERLSSRQCAAFVSGVVKDAFILWLNQNVQAAELLAEMAISSAQRRMRAAKKVVRKKLTSGPALPGKLADCTAQDLNRTELFLVEGDSAGGSAKQARDREYQAIMPLKGKILNTWEVSSDEVLASQEVHDISVAIGIDPDSDDLSQLRYGKICILADADSDGLHIATLLCALFVKHFRALVKHGHVYVALPPLYRIDLGKEVYYALTEEEKEGVLEQLKRKKGKPNVQRFKGLGEMNPMQLRETTLDPNTRRLVQLTIDDEDDQRTDAMMDMLLAKKRSEDRRNWLQEKGDMAEIEV (SEQ ID NO. 2).

[0075] Example 2

[0076] Establishment of a detection method for quinolone receptor based on topoisomerase IV

[0077] 2.1 Checkerboard method to determine titer

[0078] (1) Concentration gradient setting: column gradient (topoisomerase IV coating concentration): 10, 5, 2.5, 1.25, 0.625, 0.3125 µg / mL. The 7th column is negative control (no topoisomerase IV). Row gradient (HRP-CFLX-BSA dilution multiple): 1:50, 1:100, 1:200, 1:400, 1:800, 1:1600. Each row and column intersection represents one combination.

[0079] (2) Coating: according to column gradient concentration, the same concentration in each column, 100 μL was added to each well. Incubate in 4 °C refrigerator for 16 h to allow the protein to be fully absorbed on the well plate.

[0080] (3) Washing and blocking: tap the plate, wash the plate 3 times with washing solution (250 μL) per well, and tap the plate. Add enough blocking solution (250 μL) to each well, and incubate the microplate in a 37 °C constant temperature and humidity incubator for 2 h. After vertically and quickly pouring the liquid, tap the plate on the dust-free paper or filter paper. Do not let the washing solution overflow and contaminate the adjacent wells. Wash the plate 6 times with washing solution, and tap the plate. Add TE buffer 150 μL to each well and equilibrate for 15 min.

[0081] (4) Incubate enzyme-DNA complex: add 50 μL of gradient-diluted quinolone standard or sample to be tested to each well, one concentration per row, then add 50 μL of HRP-CFLX-BSA diluted according to the optimal dilution factor to each well of the row, and incubate in a 37 °C constant temperature and humidity incubator for 30 min, tap the plate, and wash the plate 3 times with washing solution, and tap the plate.

[0082] (5) Competition reaction: add 50 μL of PBS buffer to each well. Dilute according to the row gradient dilution factor, and add 50 μL to each well of the row with the same dilution factor. Incubate in a 37 °C constant temperature and humidity incubator for 30 min, tap the plate, and wash the plate 3 times with washing solution, and tap the plate. Add the drug standard first, then add HRP-CFLX-BSA, and avoid pre-reaction.

[0083] (6) Color development and termination: add 100 μL of single-component TMB color developing solution to each well, and incubate in a 37 °C constant temperature and humidity incubator for 20 min. Add 100 μL of termination solution to each well to terminate the reaction. The order of adding samples at each step should be consistent, especially the order of color developing solution and termination solution, to ensure that each well has the same color development time. The color developing solution and termination solution should be taken out 30 min in advance and restored to room temperature.

[0084] (7) Detection: use an enzyme labeler to measure the OD value of each well at 450 nm within 15 min.

[0085] 2.2 Checkerboard method for establishing inhibition and standard inhibition curve

[0086] (1) Concentration gradient setting: row gradient (topoisomerase IV coating concentration): 10, 5, 2.5, 1.25, 0.625, 0.3125 μg / mL. Column gradient (drug standard concentration): 5000, 1000, 500, 100, 50, 10, 5, 1, 0.5, 0.1 ng / mL.

[0087] (2) Coating: according to row gradient concentration, the same concentration in each row, 100 μL was added to each well. The others are the same as 2.1.

[0088] (3) Wash and seal: The sealing liquid is 250 μL of 10% FBS buffer containing 2 mmol / L DTT. The other is the same as 2.1.

[0089] (4) Incubate enzyme-DNA complex: The same as 2.1.

[0090] (5) Competition reaction: Add 50 μL of PBS buffer to the 11th well of each row. According to the column gradient drug standard concentration, add 50 μL of each column with the same concentration. According to the dilution multiple of the enzyme label corresponding to the OD450 nm value of about 1.1 of each coating concentration, dilute HRP-CFLX-BSA, 50 μL per well per row. The other is the same as 2.1.

[0091] (6) Color development and termination: The same as 2.1.

[0092] (7) Detection: The same as 2.1.

[0093] Take the logarithmic value of the drug standard concentration as the abscissa, and take the B / B0 value as the ordinate (B represents the OD450 nm value corresponding to different drug standard concentration wells, and B0 represents the OD450 nm value corresponding to the 11th well when the drug standard concentration is 0), and use the four-parameter logistic model of GraphPad Prism 8 software to fit the data, establish a standard inhibition curve, and calculate the corresponding half-inhibitory concentration (IC50) value.

[0094] 2.3 Determination of the optimal receptor protein coating concentration and the optimal enzyme label dilution multiple

[0095] Determine the optimal HRP-CFLX-BSA dilution multiple corresponding to each coating concentration of topoisomerase IV. The experimental steps are the same as 2.1.

[0096] Determine the optimal combination of topoisomerase IV coating concentration and HRP-CFLX-BSA dilution multiple, and the experimental steps are as follows:

[0097] (1) Concentration gradient setting: row gradient (topoisomerase IV coating concentration): 10, 5, 2.5, 1.25, 0.625, 0.3125 μg / mL. Column gradient (CFLX standard concentration): 1000, 1000, 100, 100, 10, 10, 1, 1, 0.1, 0.1, 0 and 0 ng / mL. (HRP-CFLX-BSA dilution multiple).

[0098] (2) Coating: according to the row gradient concentration, the same concentration in each row, and add 100 μL to each well. The other is the same as 2.1.

[0099] (3) Wash and block: Blocking solution is 250 µL 10% FBS buffer. Others are the same as 5.1.4.1.1.

[0100] (4) Incubate enzyme-DNA complex: The same as 2.1.

[0101] (5) Competition reaction: Add 50 µL PBS buffer to each well, then add 50 µL HRP-CFLX-BSA diluted according to the optimal dilution factor to each well in each column. Others are the same as 2.1. Add PBS buffer first, then add HRP-CFLX-BSA to avoid pre-reaction.

[0102] (6) Color development and termination: The same as 2.1.

[0103] (7) Detection: The same as 2.1.

[0104] Take the logarithm value of the CFLX standard concentration as the abscissa, and the B / B0 value as the ordinate (B represents the OD450 nm value corresponding to different CFLX standard concentration wells, and B0 represents the OD450 nm value corresponding to the CFLX standard concentration of 0), and use the four-parameter logistic model of GraphPad Prism 8 software to fit the data, establish a standard curve, and calculate the corresponding IC50 value. The results are shown in Table 1. This research scheme can systematically determine the optimal combination of topoisomerase IV coating concentration and the optimal HRP-CFLX-BSA dilution factor, ensuring the high sensitivity (low IC50) and stability (OD value) of the detection system. The combination with the smallest IC50 value is selected as the optimal receptor coating concentration and the optimal enzyme marker dilution factor. As can be seen from Table 1, when the topoisomerase IV coating concentration is 1.25 µg / mL and the enzyme marker dilution factor is 220, the IC50 value is the smallest (2.47). Therefore, the optimal receptor protein coating concentration selected in this study is 1.25 µg / mL, and the optimal enzyme marker dilution factor is 220.

[0105] Table 1 Checkerboard method to evaluate the optimal receptor protein coating concentration and the optimal enzyme marker dilution factor

[0106]

[0107] 2.4 Optimization of coating conditions and coating buffer

[0108] (1) Concentration gradient setting: Each row: optimal coating concentration of topoisomerase IV and optimal dilution factor of HRP-CFLX-BSA. Column gradient (CFLX standard concentration): 1000, 1000, 100, 100, 10, 10, 1, 1, 0.1, 0.1, 0 and 0 ng / mL.

[0109] (2) Coating: 3 coating conditions were 37 °C for 2 h, room temperature for 2 h, and 4 °C for 16 h. Topoisomerase IV was coated with PBS buffer to the optimal coating concentration, 100 μL per well for each coating condition.

[0110] After determining the optimal coating condition, topoisomerase IV was coated with PBS buffer, Tris-HCl buffer, and carbonate buffer to the optimal coating concentration, 100 μL per well for each buffer. The other was the same as 2.1.

[0111] (3) Washing and blocking: The blocking solution was 250 μL of 10% FBS buffer. The other was the same as 2.1.

[0112] (4) Incubation of enzyme-DNA complex: The same as 2.1.

[0113] (5) Competition reaction: According to the Ladder gradient CFLX standard concentration, the same concentration was added to each column, and 50 μL was added to each well. Then 50 μL of HRP-CFLX-BSA diluted according to the optimal dilution multiple was added to each column. The other was the same as 2.1. CFLX standard was added first, and then HRP-CFLX-BSA was added to avoid pre-reaction.

[0114] (6) Color development and termination: The same as 2.1.

[0115] (7) Detection: The same as 2.1.

[0116] The logarithmic value of the CFLX standard concentration was used as the abscissa, and the B / B0 value was used as the ordinate (B represented the OD450 nm value of the hole corresponding to different CFLX standard concentrations, and B0 represented the OD450 nm value corresponding to the CFLX standard concentration of 0), and the four-parameter logistic model of GraphPad Prism 8 software was used for data fitting to establish a standard curve and calculate the corresponding IC50 value. The results are shown in Table 2. According to the B0 value and the IC50 value, the coating buffer and the coating condition with the maximum B0 value and the minimum IC50 value were evaluated as the best coating buffer and the best coating condition. As can be seen from Table 2, the best coating buffer is PBS buffer (pH 7.4), because its pH is close to the pi of topoisomerase IV, which can maximize the coating efficiency and maintain the protein activity, thereby obtaining high B0 value (signal intensity) and low IC50 value (high sensitivity).

[0117] Table 2 Optimization of coating conditions and coating buffers

[0118]

[0119] 2.5 Optimization of blocking buffer and blocking time

[0120] (1) Concentration gradient setting: Each row: optimal coating concentration of Topo IV and optimal dilution multiple of HRP-CFLX-BSA. Column gradient (CFLX standard concentration): 1000, 1000, 100, 100, 10, 10, 1, 1, 0.1, 0.1, 0, and 0 ng / mL.

[0121] (2) Coating: According to the optimal coating buffer and coating conditions. The rest is the same as 2.1.

[0122] (3) Washing and blocking: The blocking effects of different blocking buffers (10% FBS buffer, 5% FBS buffer, 5% skim milk powder buffer for blocking, 5% BSA buffer, and 1% chicken ovalbumin buffer) and blocking time (0.5 h, 1 h, and 2 h) were compared. When comparing blocking buffers, each row corresponds to one blocking buffer; when comparing blocking time, each plate corresponds to one blocking time. The washing steps are the same as 2.1.

[0123] (4) Incubation of enzyme-DNA complex: The same as 2.1.

[0124] (5) Competition reaction: According to the column gradient CFLX standard concentration, 50 μL of each column was added. Then 50 μL of HRP-CFLX-BSA diluted according to the optimal dilution multiple was added to each well of each column. The rest is the same as 2.1. CFLX standard was added first, and then HRP-CFLX-BSA was added to avoid pre-reaction.

[0125] (6) Color development and termination: The same as 2.1.

[0126] (7) Detection: The same as 2.1.

[0127] Taking the logarithmic value of the CFLX standard concentration as the abscissa and the B / B0 value as the ordinate (B represents the OD450 nm value corresponding to different CFLX standard concentrations, and B0 represents the OD450 nm value corresponding to the CFLX standard concentration of 0), the data were fitted using the four-parameter logistic model of GraphPad Prism 8 software, a standard curve was established, and the corresponding IC50 value was calculated. The results are shown in Table 3. According to the B0 value and the IC50 value, the blocking buffer and the blocking time with the maximum B0 value and the minimum IC50 value were selected as the optimal blocking buffer and the optimal blocking time. As can be seen from Table 3, the blocking effect of 10% FBS buffer as the blocking buffer and 2 h as the blocking time is the best.

[0128] Table 3 Optimization of blocking buffer and blocking time

[0129]

[0130] 2.6 Optimization of competition reaction time

[0131] (1) Concentration gradient setting: Each row: optimal coating concentration of topoisomerase IV and optimal dilution fold of HRP-CFLX-BSA. Column gradient (CFLX standard concentration): 1000, 1000, 100, 100, 10, 10, 1, 1, 0.1, 0.1, 0, and 0 ng / mL.

[0132] (2) Coating: according to the optimal coating buffer and coating conditions. The others are the same as 2.1.

[0133] (3) Washing and blocking: blocking according to the optimal blocking time and optimal blocking buffer. The washing steps are the same as 2.1.

[0134] (4) Incubation of enzyme-DNA complex: the same as 2.1.

[0135] (5) Competition reaction: according to the column gradient CFLX standard concentration, the same concentration in each column, and 50 μL was added to each well. Then 50 μL of HRP-CFLX-BSA diluted according to the optimal dilution fold was added to each well in each column, and incubated in a constant temperature and humidity incubator at 37 °C for 15, 30, 45, and 60 min, corresponding to one competition reaction time for each plate. The others are the same as 2.1. CFLX standard was added first, and then HRP-CFLX-BSA was added to avoid pre-reaction.

[0136] (6) Color development and termination: the same as 2.1.

[0137] (7) Detection: the same as 2.1.

[0138] Taking the logarithmic value of the CFLX standard concentration as the abscissa and the B / B0 value as the ordinate (B represents the OD450 nm value corresponding to the different CFLX standard concentration wells, and B0 represents the OD450 nm value corresponding to the CFLX standard concentration of 0), the data were fitted using the four-parameter logistic model of GraphPad Prism 8 software to establish the standard curve and calculate the corresponding IC50 value, as shown in Table 4. As can be seen from Table 4, with the increase of the competition reaction time, the IC50 value first decreased and then increased, and the B0 value continuously increased with the occurrence of non-specific binding. The competition reaction time when the IC50 value entered the plateau period and the B0 value was close to 1.1 was selected as the optimal competition reaction time, i.e. 30 min.

[0139] Table 4 Optimization of competition reaction time

[0140]

[0141] 2.7 Optimization of standard and sample diluent

[0142] Accurately weigh 1.02 mg of CFLX standard, dissolve in 0.03 mol / L NaOH solution, and then dilute with acetonitrile to 10 mL to prepare a 100 μg / mL standard stock solution. Dilute the standard stock solution with diluent to prepare standard working solutions of 1000, 100, 10, 1, and 0.1 ng / mL.

[0143] (1) Concentration gradient setting: each row: optimal coating concentration of topoisomerase IV and optimal dilution multiple of HRP-CFLX-BSA. Column gradient (CFLX standard concentration): 1000, 1000, 100, 100, 10, 10, 1, 1, 0.1, 0.1, 0, and 0 ng / mL. Mix acetonitrile solution and PBS buffer solution according to different volume ratios to prepare standard and sample diluents containing 0%, 5%, 10%, 20%, 50%, and 100.0% (V / V) acetonitrile, with one diluent per plate.

[0144] (2) Coating: perform according to the optimal coating buffer and coating conditions. The others are the same as 2.1.

[0145] (3) Washing and blocking: block according to the optimal blocking time and optimal blocking buffer. The washing steps are the same as 2.1.

[0146] (4) Incubation of enzyme-DNA complex: the same as 2.1.

[0147] (5) Competition reaction: add 50 μL of CFLX standard according to the column gradient CFLX standard concentration, with the same concentration in each column. Then add 50 μL of HRP-CFLX-BSA diluted according to the optimal dilution multiple to each well in each column. The others are the same as 2.1. Add CFLX standard first, and then add HRP-CFLX-BSA to avoid pre-reaction.

[0148] (6) Color development and termination: the same as 2.1.

[0149] (7) Detection: the same as 2.1.

[0150] The logarithmic value of the CFLX standard concentration was used as the abscissa, and the B / B0 value was used as the ordinate (B represented the OD450 nm value corresponding to the CFLX standard concentration hole, and B0 represented the OD450 nm value corresponding to the CFLX standard concentration of 0), and the four-parameter logistic model of the GraphPad Prism 8 software was used for data fitting to establish a standard curve and calculate the corresponding IC50 value, as shown in Table 5. The IC50 value was used to evaluate the sensitivity to determine the optimal content of acetonitrile in the standard and sample diluents. As shown in Table 5, the acetonitrile content in the diluent should be selected as 0% or 5%, the IC50 value is small, and the sensitivity of the established receptor detection method is high. Considering the solubility of the drug standard in PBS buffer, the PBS buffer containing 5% acetonitrile was selected as the standard and sample diluent.

[0151] Table 5 Optimization of diluent

[0152]

[0153] 2.8 Establishment of direct competitive enzyme-labeled receptor detection method

[0154] (1) Concentration gradient setting: Each row: optimal coating concentration of topoisomerase IV and optimal dilution multiple of HRP-CFLX-BSA. Column gradient (quinolone standard concentration): 1000, 1000, 100, 100, 10, 10, 1, 1, 0.1, 0.1, 0 and 0 ng / mL.

[0155] (2) Coating: according to the optimal coating buffer and coating conditions. Others are the same as 2.1.

[0156] (3) Washing and blocking: blocking according to the optimal blocking time and optimal blocking buffer. The washing steps are the same as 2.1.

[0157] (4) Incubation of enzyme-DNA complex: same as 2.1.

[0158] (5) Competition reaction: according to the column gradient quinolone standard concentration, 50 μL of each column with the same concentration was added to each well. Then 50 μL of HRP-CFLX-BSA diluted according to the optimal dilution multiple was added to each column. Others are the same as 2.1. Quinolone standard was added first, and then HRP-CFLX-BSA was added to avoid pre-reaction.

[0159] (6) Color development and termination: same as 2.1.

[0160] (7) Detection: same as 2.1.

[0161] The logarithm of the concentration of quinolone standard was used as the abscissa, and the B / B0 value was used as the ordinate (B represents the OD450 nm value corresponding to different quinolone standard concentrations, and B0 represents the OD450 nm value corresponding to the quinolone standard concentration of 0). The data were fitted using the four-parameter logistic model of GraphPad Prism 8 software to establish a standard curve and calculate the corresponding IC50 value to evaluate the affinity of topoisomerase IV and quinolones. The standard inhibition curve of quinolones is shown in Figures 1 to 13 The IC50 values of each standard inhibition curve are shown in Table 6, and the sensitivity of the established receptor detection method is evaluated by the IC50 value.

[0162] Table 6 Sensitivity of enzyme-labeled receptor detection method

[0163]

[0164] 2.9 Cross-reactivity test

[0165] (1) Concentration gradient setting: Each row: optimal coating concentration of topoisomerase IV and optimal dilution multiple of HRP-CFLX-BSA. Column gradient (CFLX standard concentration): 1000, 1000, 100, 100, 10, 10, 1, 1, 0.1, 0.1, 0 and 0 ng / mL; Column gradient (other quinolone standard concentration): 1000, 1000, 100, 100, 10, 10, 1, 1, 0.1, 0.1, 0 and 0 ng / mL; Column gradient (non-quinolone standard concentration): 1000, 1000, 100, 100, 10, 10, 1, 1, 0.1, 0.1, 0 and 0 ng / mL. Other quinolones are enrofloxacin, and non-quinolones include tetracyclines (tetracycline), β-lactams (amoxicillin), aminoglycosides (kanamycin), amphenicols (chloramphenicol), macrolides (erythromycin) and sulfonamides (sulfadiazine).

[0166] (2) Coating: according to the optimal coating buffer and coating conditions. Others are the same as 2.1.

[0167] (3) Washing and blocking: blocking according to the optimal blocking time and optimal blocking buffer. The washing steps are the same as 2.1.

[0168] (4) Incubation of enzyme-DNA complex: same as 2.1.

[0169] (5) Competition reaction: 50 μL of CFLX standard or other drug standards at column gradient concentration was added to each well. Or 50 μL of enrofloxacin, tetracycline, amoxicillin, kanamycin, chloramphenicol, erythromycin and sulfadiazine standards at column gradient concentration was added to each well. Then 50 μL of HRP-CFLX-BSA diluted according to the optimal dilution multiple was added to each well of each column. The rest was the same as 2.1. The drug standards were added first, and then HRP-CFLX-BSA was added to avoid pre-reaction.

[0170] (6) Color development and termination: the same as 2.1.

[0171] (7) Detection: the same as 2.1.

[0172] The logarithmic value of the concentration of CFLX standard or other drug standard was used as the abscissa, and the B / B0 value was used as the ordinate (B represented the OD450 nm value corresponding to the concentration of CFLX standard or other drug standard in the well, and B0 represented the OD450 nm value corresponding to the concentration of CFLX standard or other drug standard being 0), and the four-parameter logistic model of GraphPad Prism 8 software was used for data fitting to establish a standard curve and calculate the corresponding IC50 value. Then the cross-reactivity (CR) was calculated according to the IC50 value, CR (%) = IC50 of the tested substance / IC50 of CFLX, and the cross-reaction test was used to evaluate the response degree of the detection method to the same quinolone drug (enrofloxacin) and other types of drugs (tetracyclines, β-lactams, aminoglycosides, amphenicols, macrolides and sulfonamides), i.e. the specificity of the method, and the specificity verification results are shown in Table 7. As can be seen from the data in Table 7, the cross-reactivity of enrofloxacin is 92.4%, indicating that the method has broad-spectrum detection capability for the same drugs. The cross-reactivity of non-quinolone drugs is less than 1%, indicating that topoisomerase IV does not react with these non-quinolone drugs, proving that topoisomerase IV protein has high specificity, meeting the demand of receptor detection method for high specificity receptors.

[0173] Table 7 Specificity verification

[0174]

[0175] Example 3

[0176] Application of quinolone drug receptor detection method in livestock and poultry products (livestock and poultry meat, poultry eggs and cow milk)

[0177] 3.1 Sample collection and pretreatment

[0178] Each 5 g (accurate to ±0.01 g) of livestock meat (beef, mutton and pork), poultry meat (chicken, duck and goose) samples were placed in 50 mL centrifuge tubes, 4 mL of ultrapure water containing 16 μL of β-glucuronidase / sulfatase mixed solution (1:1 by volume) was added, mixed and incubated in a 37 °C water bath for 3 h. 8 mL of acetonitrile was added to each centrifuge tube, and placed in a rotary mixer for 1 h of extraction at room temperature. Then centrifuged at 5,000 rpm for 10 min, centrifuged 3 times, and the supernatant was combined, diluted with sample diluent, and then subjected to direct competitive receptor detection analysis.

[0179] Each 5 g (accurate to ±0.01 g) of blank poultry eggs (chicken eggs, duck eggs and goose eggs) samples were placed in 50 mL centrifuge tubes, 4 mL of ultrapure water containing 16 μL of β-glucuronidase / sulfatase mixed solution was added, mixed and incubated in a 37 °C water bath for 3 h. 8 mL of 90% acetonitrile aqueous solution was added to each centrifuge tube, and placed in a rotary mixer for 1 h of extraction at room temperature. Then centrifuged at 5,000 rpm for 10 min, centrifuged 3 times, and the supernatant was combined, diluted with sample diluent, and then subjected to direct competitive receptor detection analysis.

[0180] 5 g (accurate to ±0.01 g) of blank milk was measured and placed in a 50 mL centrifuge tube, centrifuged at 10,000 rpm for 15 min at 4 °C to precipitate particulate matter and lipid layer. The middle layer was taken and diluted with sample diluent, and then subjected to direct competitive receptor detection analysis.

[0181] 3.2 Optimization of sample extraction solution dilution factor

[0182] The CFLX standard stock solution (100 μg / mL) was diluted with diluent (5% acetonitrile in PBS buffer) to standard working solutions of 1000, 100, 10, 1 and 0.1 ng / mL in a stepwise gradient.

[0183] An appropriate amount of CFLX standard stock solution (100 μg / mL) was added to the sample extract to obtain gradient concentrations of 1000, 100, 10, 1 and 0.1 ng / mL. The sample extract obtained in 2.1 was diluted 2-fold, 4-fold, 6-fold, 8-fold and 10-fold with diluent (5% acetonitrile in PBS buffer), and an appropriate amount of CFLX standard stock solution (100 μg / mL) was added to obtain gradient concentrations of 1000, 100, 10, 1 and 0.1 ng / mL.

[0184] (1) Concentration gradient setting: Each row: Topoisomerase IV optimal coating concentration and optimal HRP-CFLX-BSA dilution multiple. Column gradient (CFLX standard concentration): 1000, 1000, 100, 100, 10, 10, 1, 1, 0.1, 0.1, 0 and 0 ng / mL.

[0185] (2) Coating: According to the optimal coating buffer and coating conditions. The rest is the same as 2.1.

[0186] (3) Washing and blocking: Blocking according to the optimal blocking time and optimal blocking buffer. The washing steps are the same as 2.1.

[0187] (4) Incubation of enzyme-DNA complex: The same as 2.1.

[0188] (5) Competition reaction: According to the column gradient CFLX standard concentration, add 50 μL of the same concentration to each column. Then add 50 μL of HRP-CFLX-BSA diluted according to the optimal dilution multiple to each well of each column, and incubate in a 37 °C constant temperature and humidity incubator for 30 min. Each plate corresponds to one sample extract dilution multiple. The rest is the same as 2.1. Add CFLX standard first, then add HRP-CFLX-BSA to avoid pre-reaction.

[0189] (6) Color development and termination: The same as 2.1.

[0190] (7) Detection: The same as 2.1.

[0191] Take the logarithmic value of the CFLX standard concentration as the abscissa and the B / B0 value as the ordinate (B represents the OD450 nm value corresponding to the CFLX standard concentration well, and B0 represents the OD450 nm value corresponding to the CFLX standard concentration of 0), use the four-parameter logistic model of GraphPad Prism 8 software to fit the data, establish a standard curve, and calculate the corresponding IC50 value. The dilution multiple close to the IC50 value obtained by using the diluent is used as the optimal dilution multiple of the sample extract. The CFLX standard curve in different dilution multiples of beef, chicken, egg and milk sample extracts is shown in Figures 14 to 17 , and the IC50 values corresponding to different dilution multiples of the sample extract are shown in Table 8. From Figures 14 to 17It can be seen that the dilution of 8 times of the sample extract solution of beef, chicken, egg and milk samples almost overlaps with the standard curve corresponding to the diluent. As shown in Table 8, with the increase of the dilution multiple of the sample extract solution, the IC50 value of the standard curve gradually approaches the IC50 value of the standard curve corresponding to the diluent. When the sample extract solution of beef, chicken, egg and milk is diluted by 8 times, the IC50 value of the standard curve corresponding to the dilution is similar to the IC50 value of the standard curve corresponding to the diluent, indicating that the matrix effect of the sample extract solution is basically eliminated. Therefore, when detecting livestock and poultry meat, poultry eggs and milk samples, the sample extract solution needs to be diluted by 8 times before the receptor detection analysis.

[0192] Table 8 Comparison of IC50 values corresponding to different dilution multiples of sample extract solution

[0193]

[0194] 3.3 Method detection limit, limit of quantification, recovery rate and coefficient of variation

[0195] The sensitivity of the method is evaluated by LOD and LOQ. 20 negative samples are pretreated, and the sample extract solution is diluted by the optimal multiple with the diluent (5% acetonitrile PBS buffer solution), and then detected and analyzed by the established direct competitive enzyme-labeled receptor detection method, to calculate the average OD450 nm (M) and standard deviation (SD). The LOD is defined as the analyte concentration corresponding to OD450 nm (M+3×SD), and the LOQ is defined as the analyte concentration corresponding to OD450 nm (M+10×SD). A certain amount of CFLX standard is added to the negative sample, the sample is pretreated, and then the sample extract solution is diluted by a suitable multiple, so that the CFLX concentration in the sample to be tested is LOQ, 3 LOQ and 10 LOQ, each concentration is repeated for 3 times, and the recovery rate and coefficient of variation (CV) are calculated.

[0196] CFLX is selected as a representative for the detection of blank samples, and the results are shown in Table 9. As shown in Table 9, the LOD and LOQ ranges of beef, mutton, pork, chicken, duck, goose, egg, duck egg, goose egg and milk samples are 0.06-0.11 ng / mL and 0.19-0.36 ng / mL, respectively, and the converted LOD and LOQ ranges of the original sample are 1.2-2.1 μg / kg and 5.6-6.9 μg / kg, respectively, the average recovery rate range is 75.3%-94.8%, and the coefficient of variation range is 5.6%-11.7%.

[0197] Table 9 Method detection limit, limit of quantification, recovery rate and coefficient of variation

[0198]

Claims

1. A method for detecting quinolone drug receptors based on topoisomerase IV, characterized in that: The method comprises the following steps: coating topoisomerase IV as a quinolone drug receptor protein on a high-adsorption ELISA plate, and using horseradish peroxidase-labeled ciprofloxacin-bovine serum albumin as an enzyme marker and tracer; the topoisomerase IV is a tetramer composed of two C subunits and two E subunits, wherein the amino acid sequence of the subunit ParC is shown in SEQ ID NO. 1, and the amino acid sequence of the subunit ParE is shown in SEQ ID NO.

2.

2. The method for detecting quinolone receptors based on topoisomerase IV according to claim 1, characterized in that: The quinolone drug receptor protein is diluted with a coating buffer and coated on an ELISA plate; the coating buffer is a PBS buffer.

3. The method for detecting quinolone receptors based on topoisomerase IV according to claim 2, characterized in that: The coating concentration of topoisomerase IV is 1-1.5 μg / mL, and the dilution factor of the enzyme marker is 200-240.

4. The method for detecting quinolone receptors based on topoisomerase IV according to claim 1, characterized in that: The quinolone drugs include enrofloxacin, ciprofloxacin, danofloxacin, lomefloxacin, ofloxacin, norfloxacin, pefloxacin, enoxacin, difloxacin, sarafloxacin, oxolinic acid, fleroxacin and gatifloxacin.

5. Use of the topoisomerase IV-based quinolone receptor detection method of claim 1 in the detection of quinolone drugs in livestock and poultry products; the livestock and poultry products include livestock meat, poultry meat, poultry eggs and cow's milk.

6. The use according to claim 5, characterized in that 8-12% FBS buffer was used as the blocking buffer, and the blocking time was 2 h.

7. The method for detecting quinolone receptors based on topoisomerase IV according to claim 5, characterized in that: The competitive reaction time is 20~40min.

8. The method for detecting quinolone receptors based on topoisomerase IV according to claim 5, characterized in that: PBS buffer containing 4-6% acetonitrile was used as the diluent for standards and samples.

9. The use according to claim 5, characterized in that When the livestock and poultry product is livestock meat or poultry meat, the detection method comprises: adding a livestock and poultry product sample to ultrapure water containing a β-glucuronidase / sulfatase mixed solution, mixing and incubating in a water bath, adding acetonitrile after incubation, mixing and extracting at room temperature, diluting 7 to 9 times with a sample diluent, centrifuging, and performing direct competitive receptor detection analysis; When the livestock and poultry product is an egg, the detection method comprises: adding a livestock and poultry product sample to ultrapure water containing a β-glucuronidase / sulfatase mixed solution, mixing and incubating in a water bath, adding an acetonitrile aqueous solution after incubation, mixing and extracting at room temperature, diluting 7 to 9 times with a sample diluent, centrifuging, and performing direct competitive receptor detection analysis; When the livestock and poultry product is cow's milk, the detection method includes: centrifuging the cow's milk to precipitate particulate matter and a lipid layer; taking the middle layer, diluting it 7 to 9 times with a sample diluent, and then performing direct competitive receptor detection analysis.

10. The use according to claim 9, characterized in that The sample diluent was PBS buffer containing 5% acetonitrile.

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