An antibiotic extractant

Abstract

The present application provides an antibiotic extraction agent and a method for using the same. The extraction agent or method can extract various antibiotics from various samples including feed. The extraction agent or method has high extraction efficiency and high accuracy.

Description

Technical Field

[0001] This application relates to an antibiotic extractant, which belongs to the field of biochemical analysis and detection. Background Technology

[0002] Antibiotics have been used in animal feed since the 1950s, primarily for treating and promoting livestock growth. Currently, approximately 60 antibiotics are used in clinical treatment and animal feed. These include sulfonamides, tetracyclines, macrolides, aminoglycosides, and others. However, research has shown that the widespread use of antibiotics as feed additives has caused environmental damage and impacted human health, even leading to a series of problems for livestock and other farmed animals. Therefore, in 1997, the World Health Organization stated that the use of antibiotics in animal husbandry is a public health issue. Consequently, since 2015, my country has successively issued a series of laws and regulations prohibiting the use of antibiotics in feed. Although most antibiotics are not currently on the domestic list of prohibited antibiotics, their original forms or metabolites may accumulate and store in animal cells, tissues, or organs, forming "veterinary drug residues." Therefore, it is necessary to test for antibiotics in feed. Sample extraction is a crucial step in the testing process, and the extraction quality can significantly affect the accuracy of the results.

[0003] Currently, the main methods for extracting antibiotics from feed are extraction with organic solvents such as methanol and acetonitrile, and extraction with PBS buffer. These methods are time-consuming, complex, and require different extraction methods for different types of antibiotics. Previously, the inventors discovered a strain called GS101 (see CN202110509283.2), which exhibits excellent characteristics in antibiotic detection. To further improve its detection accuracy and develop a large-scale product, it is essential to develop a highly efficient antibiotic extractant specifically adapted to the GS101 strain for the efficient and accurate detection of multiple antibiotics. Summary of the Invention

[0004] This application first provides an antibiotic extraction reagent comprising citric acid monohydrate, disodium hydrogen phosphate dodecahydrate, and sodium hypotriacetate.

[0005] In some embodiments, the preparation method of the antibiotic extraction reagent is as follows: weigh 12.0g-13.0g of citric acid monohydrate, 10.0g-11.0g of disodium hydrogen phosphate dodecahydrate, and 21.0g-22.0g of sodium hypotriacetate, mix well; dissolve in 1L of deionized water to obtain the extraction reagent before use.

[0006] This application also provides the use of the above-mentioned extraction reagent in extracting antibiotics from samples.

[0007] In some implementations, the samples include feed, beef, pig urine, chicken serum, or milk.

[0008] In some embodiments, the antibiotics include: β-lactams, sulfonamides, quinolones, tetracyclines, macrolides, aminoglycosides, amyl alcohols, lincosamides, coumarins, and / or peptides.

[0009] This application also provides a method for detecting antibiotics in a sample, comprising the step of extracting antibiotics from the sample using the above-mentioned extractant, and then performing detection.

[0010] In some embodiments, the detection includes using a test stick made of microorganisms, such as Bacillus stearothermophilus GS101, Bacillus stearothermophilus ATCC10149, and / or Bacillus stearothermophilus ATCC12980.

[0011] In some embodiments, the antibiotics include: β-lactams, sulfonamides, quinolones, tetracyclines, macrolides, aminoglycosides, amyl alcohols, lincosamides, coumarins, and / or peptides.

[0012] In some implementations, the samples include feed, beef, pig urine, chicken serum, or milk.

[0013] In some implementations, an extraction reagent is added to the sample, the sample is capped and allowed to stand, then vigorously shaken to extract antibiotics from the sample; precipitation is performed before detection, and then a detection rod is used for detection.

[0014] To achieve the above objectives, the feed antibiotic extractant described in this application is prepared by the following steps: Weigh 12.0g-13.0g of citric acid monohydrate, 10.0g-11.0g of disodium hydrogen phosphate dodecahydrate, and 21.0g-22.0g of sodium hypotriacetate, and mix well. Dissolve in 1L of deionized water before use to obtain the feed antibiotic extractant.

[0015] This application also provides a method for extracting antibiotics from feed, comprising the following steps: Weighing 1.0 ± 0.1 g of a representative powder sample into a disposable 50 mL centrifuge tube. Adding feed antibiotic extraction reagent to the 30 mL mark, capping and letting stand for 5 minutes. Vigorously shaking for 1 minute, and allowing to settle for 1 minute before detection.

[0016] This application also provides a method for rapidly detecting antibiotics in samples using the aforementioned feed antibiotic extraction reagent. The method involves adding the feed antibiotic extraction reagent to the sample (weighing a representative powder sample and placing it in a disposable centrifuge tube, then adding the feed antibiotic extraction reagent), capping the tube, allowing it to stand, vigorously shaking, and allowing precipitation before detection. Detection is performed using a test strip prepared from a microbial strain coated with one or more highly sensitive strains to broad-spectrum (multiple) antibiotics.

[0017] Suitable test strips may include a test tube, a swab, and a rod. For example, a test strip may consist of three parts from top to bottom: a rod, a swab, and a test tube, the test tube being composed of liquid and / or solid reagents. Preferably, the test tube contains both liquid and solid reagents, the solid reagents being coated with one or more microorganisms highly sensitive to broad-spectrum (multiple) antibiotics, preferably *Bacillus steatophilus* GS101 (the entire contents of CN202110509283.2 are also incorporated herein). The solid reagents may further include a conventional microbial culture medium and an indicator. In some embodiments, the liquid reagent comprises a salt solution. The presence of antibiotics in the sample is detected by causing a color change in an indicator in the culture medium.

[0018] Based on the above, the inventors developed a feed antibiotic extraction reagent for use in antibiotic detection sticks. Its advantages are: firstly, good compatibility with *Bacillus stearothermophilus* GS101; secondly, high detection accuracy; thirdly, extraction efficiency for tetracycline up to 99.5%; and fourthly, efficient extraction of multiple antibiotics from feed. Detailed Implementation

[0019] The embodiments of this application will now be described. Elements and features described in one embodiment of this application may be combined with elements and features shown in one or more embodiments. It should be noted that, for clarity, representations and descriptions of components or processes unrelated to this application and known to those skilled in the art have been omitted from the description.

[0020] Example 1 Experimental Procedure

[0021] 1. Preparation method of antibiotic extract:

[0022] Weigh 12.5g of citric acid monohydrate (C6H8O7·H2O), 10.5g of disodium hydrogen phosphate dodecahydrate (Na2HPO4·12H2O), and 10.5g of sodium hypotriacetate (C6H8O7·H2O). 10 Mix 21.5g of NaO6 (Na2O6) thoroughly. Dissolve in 1L of deionized water before use to obtain the antibiotic extraction reagent.

[0023] 2. Antibiotic extraction methods:

[0024] Weigh 1.0 ± 0.1 g of representative powder sample into a disposable 50 mL centrifuge tube. Add antibiotic extraction reagent to the 30 mL mark, cap and let stand for 5 minutes. Shake vigorously for 1 minute, allow to settle for 1 minute before detection, and form the test solution.

[0025] 3. Antibiotic detection methods:

[0026] The detection method utilizes a test strip prepared from *Bacillus steatophilus* GS101, which exhibits high sensitivity to one or more broad-spectrum (multiple) antibiotics. A swab is dipped into the test solution and brought into contact with the reagent in the test tube; the indicator in the culture medium changes color to detect the presence of antibiotics in the sample.

[0027] Example 2: The effect of extraction time on detection results

[0028] Feed samples with negative antibiotic residue results were selected and spiked with 48 different antibiotics at the limit of detection. Three samples were prepared for each spiked drug. 1g of each spiked sample was taken and 30mL of feed antibiotic extraction reagent was added. The samples were then covered, allowed to stand, and shaken. The standing and shaking times and the detection results are shown in Table 1.

[0029] Table 1. Impact of extraction time on results

[0030] 3 1 50 3 2 66.7 4 1 70.83 4 2 75 5 1 100 5 2 100 6 1 100 6 2 100

[0031] As shown in Table 1, the extraction effect is better and better as the standing time and shaking time are extended. When the standing time is 5 minutes and the shaking time is 1 minute, the positive rate reaches 100%. Therefore, standing for 5 minutes and shaking for 1 minute can be selected as the optimal extraction time.

[0032] Example 3: The effect of the extractant of this application on the detection results compared with traditional extractants.

[0033] Feed samples that tested negative for antibiotic residues were selected and spiked with multiple antibiotics at concentrations up to the detection limit. Sample extraction was performed using the antibiotic extraction reagent described in this application, PBS buffer (0.01M pH 7.4), methanol / acetonitrile (1:1, v / v), and Na2EDTA-McIlvaine, respectively. Detection was then performed using a test strip prepared from strain GS101. 144 samples tested negative, with three samples tested for each spiked drug. The types of veterinary drugs spiked and the test results are shown in Table 2. "+" indicates a positive result, and "-" indicates a negative result.

[0034] Table 2 Detection Results

[0035]

[0036]

[0037]

[0038]

[0039] Table 3. False positive rate and false negative rate of this extraction method

[0040]

[0041]

[0042] Table 4. False positive and false negative rates using the PBS method

[0043]

[0044] Table 5 False Positive Rate of Methanol / Acetonitrile Method

[0045]

[0046] Table 6. False positive rate of the Na2EDTA-McIlvaine method

[0047]

[0048] As shown in Tables 2-6, the false negative rate was 0% and the false positive rate was 0.69% when using the extraction reagent described in this application; the false negative rate was 0% and the false positive rate was 2.78% when using the PBS method; the false negative rate was 0% and the false positive rate was 85.42% when using the methanol / acetonitrile method; and the false negative rate was 50% and the false positive rate was 0.69% when using the Na2EDTA-McIlvaine method. Therefore, the extraction reagent described in this application has the highest accuracy.

[0049] Example 4: Extraction effect of the extraction reagent of this application in different matrices

[0050] Feed, beef, pig urine, chicken serum, and milk samples with negative antibiotic residues were selected and spiked with penicillin G, sulfadiazine, enrofloxacin, tetracycline, lincomycin, florfenicol, and gentamicin at a concentration of 1000 ppb. Extraction was performed using the extraction reagents and methods described in this application, and detection was performed using HPLC. The results are shown in Table 7.

[0051] Table 7. Extraction rates of antibiotics in different sample types

[0052]

[0053] As shown in Table 7, the extraction rates of the antibiotic extraction reagent described in this application in feed, beef, pig urine, chicken serum and milk were 91.6%, 66.2%, 64.1%, 57.8% and 62.8%, respectively. Therefore, the extraction effect of this extraction reagent in feed is the best.

[0054] Example 5: Extraction effect of the extractant of this application on different antibiotics.

[0055] Feed samples with negative antibiotic residues were selected and spiked with different antibiotics at a concentration of 1000 ppb. Extraction was performed using the extraction reagents and methods described in this application, and detection was performed using HPLC. The results are shown in Table 8.

[0056] Table 8 Extraction effects of different antibiotics

[0057]

[0058]

[0059] As shown in Table 8, the extraction effects on different types of antibiotics varied, with extraction rates ranging from 82.1% to 99.5%. The extraction effect on tetracycline was the best, with an extraction rate of 99.5%.

[0060] Example 6: Compatibility of the extraction reagent of this application with different bacterial strains.

[0061] Negative feed samples were selected, and representative antibiotics of each class were spiked at the method detection limit. Extraction was performed using the method described in this application to obtain the sample test solution. The sample test solution was then tested using antibiotic test strips prepared by Bacillus stearothermophilus GS101, Bacillus stearothermophilus ATCC 10149 (commercial), and Bacillus stearothermophilus ATCC 12980 (commercial). Five replicates were performed for each sample. The test results are shown in Table 9.

[0062] Table 9 Compatibility between bacterial strains and extraction reagents

[0063]

[0064]

[0065] As shown in Table 9, when samples were prepared using the antibiotic extract described in this application, and tested with test strips made from Bacillus stearothermophilus GS101, Bacillus stearothermophilus ATCC10149, and Bacillus stearothermophilus ATCC12980, the positive rates were 100%, 78%, and 64%, respectively. Therefore, the antibiotic extract described in this application has the best compatibility with Bacillus stearothermophilus GS101.

[0066] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this application, and not to limit them. Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. These modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of this application, and they should all be covered within the scope of the claims and specification of this application. This application is not limited to the specific embodiments disclosed herein, but includes all technical solutions falling within the scope of the claims.

Claims

1. A method for detecting antibiotics in feed, characterized in that, First, it was extracted with an antibiotic extractant, and then detected using a test strip prepared from Bacillus stearothermophilus strain GS101. The results showed that: The preparation method of the antibiotic extractant is as follows: Weigh 12.0g-13.0g of citric acid monohydrate, 10.0g-11.0g of disodium hydrogen phosphate dodecahydrate, and 21.0g-22.0g of sodium hypotriacetate, and mix well; dissolve in 1L of deionized water to obtain the extraction reagent before use; The antibiotics consist of the following 48 antibiotics: penicillin G, ampicillin, amoxicillin, cloxacillin, oxacillin, ceftiofur, cefepime, cefalexin, cefotaxime, trimethoprim, sulfadiazine, sulfadiazine, sulfamethoxazole, sulfamethoxazole, sulfamethoxazole, sulfamethoxazole, dafluxacin, diflufloxacin, enrofloxacin, flumethin, sarafloxacin, lomefloxacin, ofloxacin, norfloxacin, pefloxacin, oxytetracycline, tetracycline, chlortetracycline, doxycycline, tylosin, erythromycin, pirizine, tilmicosin, trachomatis, trachomatis, spiramycin, lincomycin, gentamicin, streptomycin, dihydrostreptomycin, kanamycin, neomycin, spectinomycin, neomycin, bacitracin, chloramphenicol, florfenicol, thiamphenicol, quinethol, and oxaquinic acid; The specific steps are as follows: add the antibiotic extractant to the feed, cover and let stand, shake vigorously to extract the antibiotic from the feed; precipitate before testing, and then use the test rod for testing.

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